Marie-Françoise Noirot
email: marie-francoise.gros@jouy.inra.fr

Je Remercie

Isabelle Martin-Verstraete

Susan Schlimpert

Marie-Françoise Noirot-Gros

Transito Garcia-Garcia

Sandrine Poncet

Abderahmane Derouiche

Lei Shi

Ivan Mijakovic

Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

Keywords: protein phosphorylation, signaling, DNA replication, bacterial cell cycle, bacteria cell division

This study was carry out at Micalis Institute, INRA, Jouy en Josas (France) as part of the European ITN AMBER project. This thesis has been developed under atypical conditions but with the support, encouragement and

1

Prologue

The focus of this thesis is the understanding of coordination between replication initiation and metabolism initiated by the observation of phosphorylation of proteins involved in DNA replication initiation control. Phosphorylation is a widespread strategy of post-translational modification that modulates protein activities in response to cellular signals. This study was initiated by the detection of YabA phosphorylation, the main replication controller in Bacillus subtilis, by a kinase that acts during stationary phase and leads to the discovery of new activities of YabA underlining the recruitment of proteins in the cell for diverse functions across the different lifestyles. The introduction will provide an overview of the physiological stage where YabA has a function, this includes: initiation control, sporulation and biofilm formation and ends with a chapter of protein phosphorylation in bacteria that will address the role of this posttranslational modification in sporulation, biofilm and DNA replication.

Bacillus subtilis, a model of Gram-positive bacteria

Bacillus subtilis is a member of the phylum Firmicutes (Bacillaceae family). It is a Gram-positive bacterium whose genome has low % GC content. It was originally named "Vibrio subtilis" after its discovery in 1835 by Christian Gottfried Ehrenberg, and renamed "Bacillus subtilis" in 1872 by Ferdinand Cohn. B. subtilis is considered as an obligate aerobe, but can also grow anaerobically by respiration on nitrate or by fermentation on glucose [START_REF] Nakano | Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth[END_REF][START_REF] Hoffmann | The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system[END_REF][START_REF] Lacelle | Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis[END_REF]. It is a rod shaped and ubiquitous bacteria, commonly found in soil. B. subtilis is not considered pathogenic or toxic and can be easily cultivated and handled in laboratory.

B. subtilis is able to survive or adapt in adverse conditions. Upon sensing unfavorable environmental signals, B. subtilis can engage in various life-styles by triggering different genetic programs [START_REF] Dubnau | Swim or chill: lifestyles of a bacillus[END_REF]. When the nutrients or the resources become scarce and cell density reaches a critical threshold, Bacillus cells can (i) enter into stationary phase, which is characterized by formation of motile cells, (ii) differentiate into enduring and metabolically inactive spores, highly resistant to a wide variety of extreme conditions such as heat, cold, drying and radiations (Piggot et al., 2004;[START_REF] Driks | Overview: Development in bacteria: spore formation in Bacillus subtilis[END_REF], (iii) become competent and take up DNA from environment for acquisition of new genetic material [START_REF] Msadek | When the going gets tough: survival and environmental signaling networks in Bacillus subtilis[END_REF][START_REF] Dubnau | Genetic competence in Bacillus subtilis[END_REF]. In all three cases, strikingly different genetic programs are turned on and can guide the cell through the differentiation processes. In addition, B. subtilis shows an interesting "social behavior", in that the cells communicate with each other. Cells can form multicellular structures able to adhere to surface, the biofilm [START_REF] Lemon | Biofilm development with an emphasis on Bacillus subtilis[END_REF]. Cells can also group and move through a phenomenon called "swarming" [START_REF] Kearns | Swarming motility in undomesticated Bacillus subtilis[END_REF][START_REF] Van Gestel | From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate[END_REF]. An intracellular communication system called "quorum sensing" is also present in B. subtilis [START_REF] Tran | Divergent structure of the ComQXPA quorum sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis[END_REF]. This mechanism allows the cells to react to the environment as a global bacterial population by coordinated modulation of gene expression in response to changes in cell density.

The use of B. subtilis as an experimental model system began with the "original strain" of Ferdinand Cohn described in 1872 and commonly referred to as the "Marburg strain". In 1958, John Spizizen showed for the first time that the strain B. subtilis 168, non-pathogenic and auxotrophic for tryptophan, is transformable by exogenous DNA [START_REF] Spizizen | Transformation of biochemically deficient strains of Bacillus-Subtilis by deoxyribonucleate[END_REF]. Three years later, Costa Anagnostopoulos defined the transformation conditions allowing the genetic competence in this bacterium [START_REF] Anagnostopoulos | Requirements for Transformation in Bacillus Subtilis[END_REF]. This discovery led the scientific community to adopt this strain as a genetic tool, which is one of the most studied I. INTRODUCTION 4 microorganisms after Escherichia coli. The sequence of its genome was published the same year [START_REF] Kunst | The complete genome sequence of the Gram-positive bacterium Bacillus subtilis[END_REF] as that of Escherichia coli K-12 [START_REF] Blattner | The complete genome sequence of Escherichia coli K-12[END_REF]. The B. subtilis genome is 4215 kb and 4406 ORF (Open Reading Frame) have been predicted. The sequencing of its genome has allowed more comprehensive and global experimental approaches such as transcriptomic, proteomics and interactomics (Nicolas et al. 2012;Hecker and Volker 2004;Voigt et al., 2004;[START_REF] Otto | Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis[END_REF]Marchadier et al. 2011) to the scientific community. Currently, SubtiWiki still collect all current knowledge on all genes and proteins of B. subtilis, and has incorporated modules and links that allow to graphically present gene expression, metabolic and regulatory pathways and protein-protein interactions (http://www.subtiwiki.uni-goettingen.de/v3/).

Furthermore, thanks to its easy cultivation at high densities and its great natural ability to secrete and export proteins directly into the extracellular medium, B. subtilis became a tool of interest for the industry. It is used in industry for the production of numerous proteins such as amylase (used in the bread industry), proteases, cellulases (used in the detergent industry) or antibiotics such as bacitracin and polymyxin. It is considered as a "cell factory" for the production of enzymes (Brockmerier et al., 2006). It is also used in organic farming as a stimulator of natural defenses for the prevention of many plant diseases caused by fungi or bacteria (Bayer, Serenade® Max), an increasingly as a probiotic [START_REF] Tompkins | Safety evaluation of two bacterial strains used in Asian probiotic products[END_REF]. A variety of B. subtilis (B. subtilis natto) is also used to make natto, a traditional Japanese dish based on fermented soybeans.

The richness and the amount of information collected make B. subtilis a widely used bacteria in both fundamental research and industry. Given its high genetic tractability and its ability to adopt different life-styles, B. subtilis has become the Gram-positive model organism to study bacterial cell cycle and differentiation, including vegetative growth, sporulation and multicellular behaviors. It also serves as a model system for the study of various aspects of cellular metabolism and processes such as DNA replication, repair and segregation, transcription, translation, cell division, morphogenesis and their regulatory mechanisms.

The different life-styles of B. subtilis

Bacteria have evolved to sense environmental signals and to adapt to stress conditions. B.

subtilis can adopt different life-styles in response to environmental signals such as nutrient conditions, molecule concentrations or density of cells and differentiate into different cellular form or adopt different behaviors. The best known is its ability to differentiate into metabolically inactive spores that are highly resistant to environmental stresses. In addition to forming spores, bacillus cells can exhibit several behaviors by modulating its motility to explore its environment or instead becoming sessile to form biofilms, by developing natural genetic competence for taking up DNA from the environment, by producing a variety of extracellular substances, degradative enzymes, or even toxins that allow them to cannibalize their neighbors. The ability to sense and respond to these environmental cues involves versatile sensors and global regulators (Figure 1). In B. subtilis as in other gram positive bacteria, sensor kinases affect the level of phosphorylated global regulator Spo0A which in response activates genes related to motility switch, biofilm formation, and sporulation (Kováck, 2016). B. subtilis is thus an ideal system model that illustrate how environmentally activated sensor proteins integrate the signals towards global regulators and direct bacterial behavior.

DNA replication

All organisms must duplicate their genome in an accurate manner prior to entering cell division.

DNA replication has been well studied in bacteria, primarily because of the small size of their genome, their short and simplier cell cycle and the early availability of genetically tractable model species. In bacteria DNA replication is carried out by a multicomponent protein machine, called replisome, which achieves the efficient and faithful duplication of their chromosome.

Extensive studies in model Gram-negative bacteria E. coli and Gram-positive B. subtilis have revealed that in addition to the replication core machinery, other proteins involved in building and maintaining a stable replication fork. Specific subsets of proteins mediate the assembly of the replisome at the chromosomal origin of replication (initiation), the progression of the replication forks along the chromosome (elongation) and the arrest of replication when the new chromosome is completed (termination). As part of the mechanisms that maintain chromosomal integrity, these three stages are tightly orchestrated, as well as there are coordinated with other cellular processes, to adapt the replication rate to cell elongation and division in response to physiological changes. An important part of the regulatory systems deployed by the bacteria is to control its DNA replication initiation frequency.

This chapter is focused on the mechanisms that conduct and control DNA replication in bacteria.

This chapter will give a brief overview of the components, functions and regulation of the replication machinery, but will mainly focus on the main regulatory processes at play during the replication initiation step, with an emphasis on the similarities and differences between B.

subtilis and E. coli.

Initiation of DNA replication

DNA replication proceeds through three distinctive phases identified as initiation, elongation and termination. The initiation phase is characterized by the localized unwinding of the DNA mediated by a nucleoprotein complex at the origin of replication. This step directs the loading of a helicase and the assembly of the replisome machinery to compose two functional replication forks. In the elongation phase, the replication machineries achieve template-directed DNA synthesis on both stands in a highly processive and, as recently evidenced, discontinuous manner [START_REF] Beattie | Frequent exchange of the DNA polymerase during bacterial chromosome replication[END_REF]. Finally, during termination, DNA polymerization is arrested at a specific termination site.

Bacterial replication origins

DNA replication starts at specific chromosomal sequences called "origins", where initiator proteins launch the process of replication. In bacteria, DNA replication is initiated by the binding of the wildly conserved initiator protein DnaA. All origins contain DNA sequence motifs that position the replication initiator DnaA and direct the local opening of the DNA duplex. Bacterial replication origins exhibit conserved sequences that include several specific binding sites for DnaA (DnaA-box clusters), an AT-rich DNA Unwinding Element (DUE) as well as trinucleotide motif repeats termed DnaA-trio [START_REF] Richardson | The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding[END_REF]. However across species, the organizational architecture of the origins vary significantly regarding the number, spacing and location of the DnaA-boxes with respect to the DUEs (Figure 2).

The mechanism of DnaA-mediated initiation at oriC has been extensively studied in E. coli [START_REF] Messer | Functional domains of DnaA proteins[END_REF][START_REF] Messer | The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication[END_REF]. DnaA binds to its DnaA-boxes within oriC and forms a right-handed helical oligomer on the DNA [START_REF] Zorman | Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation[END_REF][START_REF] Erzberger | Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling[END_REF] [START_REF] Fuller | The DnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites[END_REF][START_REF] Marczynski | Redefining bacterial origins of replication as centralized information processors[END_REF][START_REF] Wolanski | oriC-encoded instructions for the initiation of bacterial chromosome replication[END_REF]. DnaA oligomerisation induces the local unwinding and

The DNA initiator protein, DnaA

As addressed above, DnaA is a conserved key regulator of replication initiation in bacteria. DnaA binds specifically double-stranded DNA at a 9 bp consensus region 5'-TTATC[CA]A[CA]A-3'

A B

(DnaA-boxes) at the origin region of replication on the bacterial chromosome, triggers the unwinding of the DNA duplex nearby, and additionally supports the loading of the DNA helicase, which in turn leads to the establishment of the DNA replication machinery. DnaA is a member of the AAA+ ATPase family (ATPases Associated with diverse cellular Activities) and contains four distinct domains: 1) domain I (N-terminal) is an "interaction domain", which binds the replicative helicase DnaB (DnaC in B. subtilis) and has been suggested to play a role in the selfassembly of DnaA at the origin; 2) a linker domain II, which is not essential for cell viability and is highly variable in length. Domain II may play a role in controlling replication efficiency; 3) Domain III, a highly conserved ATP-binding domain, homologous to AAA+ superfamily proteins, that comprises a large and a small subdomain (IIIa and IIIb, respectively). Domain III plays a role in DnaA self-interaction/oligomerisation and in ssDNA binding; 4) Domain IV (C-terminal), a highly conserved dsDNA binding domain that specifically recognizes DnaA-box sequences (Figure 4) [START_REF] Erzberger | The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation[END_REF]. et al., 1994). The fourth level is the repression of dnaA transcription immediately after replication initiation. DnaA binds directly to the dnaA promoter region and regulates its own expression (Katayama et al., 2010). There are also positive regulations. A mechanism of reactivation of DnaA involves two chromosomal regions called DARS (DNA reactivation sequences) which promote dissociation of ADP from DnaA, which becomes available for activation by ATP binding. Finally, DiaA (DnaA Initiator Associated protein) enhances ATP-DnaA binding to low-affinity sites. DiaA forms a tetramer that binds multiple DnaA molecules and stimulates DnaA assembly at oriC (Katayama et al., 2010).

Factors Strategy

Escherichia coli

Hda and DnaN Hydrolysis of DnaA-bound ATP by the ADP-Hda-DnaN complex datA Binding to DnaA molecules, reducing the number available for initiation SeqA Direct binding to hemimethylated GATC sites in oriC, inhibiting binding by DnaA.

Binding to hemimethylated GATC sites in the dnaA promoter region DnaA DnaA box binding in the dnaA promoter region YabA prevents over-initiation by inhibiting the cooperative binding of DnaA to oriC (Merrikh and Grossman, 2011) and down regulates replication initiation through interaction with the initiator protein DnaA and the clamp polymerase DnaN, which is associated with the replication machinery (Noirot-Gros et al., 2002, Noirot-Gros et al., 2006, Soufo et al., 2008). YabA, like Hda, regulates initiation through coupling to the elongation factor; however, the mechanism seems to be different since YabA shares no structural homology with the AAA+ family of ATPases, including DnaA, and lacks a DnaN-binding consensus motif (cf chapter 2.2.4). In vegetative cells, the primosomal DnaD and the ParA-like protein Soj were recently found to down regulate replication initiation by preventing the formation of a DnaA nucleofilament at oriC (Scholefield and Murray, 2013[START_REF] Scholefield | Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation[END_REF][START_REF] Murray | Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA[END_REF]. In cells committed to sporulation, the protein SirA prevents replication reinitiation by antagonizing DnaA binding to oriC [START_REF] Jamenson | Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA[END_REF], Rahn-Lee et al., 2011, Wagner et al., 2009). The replication initiation is also controlled by the master regulator for sporulation Spo0A. Spo0A-P (Spo0A-phosphate)

Bacillus subtilis

has been shown to bind the Spo0A-boxes at replication origin and inhibit DNA replication (Boonstra et al., 2013, Castilla-Llorente et al., 2006). Finally, like in E. coli, dnaA is also transcriptionally autoregulated in B. subtilis [START_REF] Ogura | Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis[END_REF]. The main negative regulatory systems that control replication initiation in E. coli and B. subtilis are listed in Table 2.

The main initiation controller YabA

YabA is the main regulator of DNA replication initiation in B. subtilis and was first identified in a genome-wide yeast two-hybrid screen looking for factors that interact directly with known replication proteins. In this study, YabA was shown to form a 'protein bridge' between the initiator DnaA and the DNA polymerase sliding clamp processivity factor DnaN (Noirot-Gros et al., 2002). YabA also interacts specifically with TlpA, a chemotactic transducer and with AcuB, an enzyme involved in protein post-traductional modification pathway, suggesting that YabA is potentially a multifunctional protein (Noirot-Gros et al., 2002). Deletion of yabA causes an increased frequency of initiation events and asynchronous DNA replication (Hayashi et al., 2005) as well as a growth phenotype associated with increased initiation events (Noirot-Gros et al., 2002;Noirot-Gros et al., 2006).

B. subtilis

YabA is a small protein (119 amino acids) organized in two putative structural domains.

The N-terminal domain of YabA contains a canonical leucine zipper (LZ) domain and the Cterminal contains cysteine residues correctly positioned to form a potential zinc cluster (ZC) (Noirot-Gros et al., 2006). This structural organization is unique, and not found in E. coli Hda, Despite numerous studies, the mechanism of YabA action remains elusive. YabA is not able to promote DnaA-ATP hydrolysis in vitro (Scholefield and Murray, 2013); however, it has been shown to affect the cooperative binding of DnaA to oriC (Merrikh and Grossman, 2011), and it is capable of disrupting DnaA oligomerisation in vitro (Scholefield and Murray, 2013). During the cell cycle, YabA is mostly present at the replication forks (Noirot-Gros et al., 2006, Hayashi et al., 2005). Therefore, it is not clear how and where YabA exerts its function in restricting the initiation activity of DnaA in vivo. Two different models for YabA function have been proposed, each predicting different mechanisms. One model proposes that YabA tethers DnaA to DnaN at the replisome during most of the cell cycle (Figure 6.A) (Noirot-Gros et al., 2006, Soufo et al., 2008, Katayama et al., 2010). Trapping of DnaA by YabA was corroborated by a study showing that a functional GFP-DnaA fusion colocalizes with the origin of replication only at the initiation time but mainly stays associated with the DNA replication machinery during most of the cell cycle (Soufo et al., 2008). DnaA localizes at the origin in small cells (which are at early points in their cell cycle) and at mid-cell coincident with DnaX, an essential component of the replication machinery, in large cells (in later stages of the cell cycle). In ∆yabA cells, by contrast, DnaA is localized with the origin throughout the cell cycle.

A B

The alternative model proposes that YabA acts directly on DnaA to affect its activity at oriC (Hayashi et al., 2005, Cho et al., 2008, Goranov et al., 2009). YabA binds to DnaA at oriC so as to inhibit its cooperative binding to further DnaA molecules throughout the cell cycle, up to the point where DNA replication is completed and the replisome disassembles. At this point, free

DnaN competitively titrates YabA away from its complex with DnaA, allowing the latter to bind cooperatively at the origin and to initiate a new round of replication (Figure 6.B) (Merrikh and Grossman, 2011). This model is consistent with the evidence that the frequency of replication initiation correlates with the cellular level of DnaN; with increased DnaN levels increasing replication initiation frequency, and decreased levels decreasing initiation frequency. An increase in the amount of DnaN causes a decrease in the association of YabA with DnaA, therefore increasing the association of DnaA with chromosomal regions. These experiments were done in a strain initiating replication from oriN, where over-production of DnaN does not have any detectable effect on replication (Goranov et al., 2009). Over-production of DnaN caused decreased association of YabA with the oriC region and increased association of DnaA with oriC and other DnaA targets in vivo, indicating that the role of DnaN could be controlling the binding of YabA at the origin (Merrikh and Grossman, 2011). Further studies to establish the dynamics and stoichiometry or the interactions between YabA, DnaA and DnaN are required to further refine and reconcile these models. [START_REF] Jameson | Control of initiation of DNA replication in Bacillus subtilis and Escherichia coli[END_REF] YabA is visually absent from oriC during most of the cell cycle (Noirot-Gros et al., 2006, Hayashi et al., 2005). Recently it has been reported that the absence of YabA increases the half time binding of DnaA at oriC, showing that YabA plays a dual role in the regulation of DnaA, as a tether at the replication forks, and as a chaser at origin regions. Using FRAP analysis of a functional YFP-DnaA allele in B. subtilis, it has been showed that DnaA is bound to oriC with a half-time of 2.5 seconds. In the absence of YabA, the half-time recovery was extended to 3.8 seconds.

B A

Additionally, the YFP-YabA fusion was used to visualize throughout the cell cycle the colocalization of YabA with both the oriC region marked with CFP, and with DnaX-mcherry as a marker for the replication machinery (Figure 7.A).

B Small cells Medium cells Large cells

A

I. INTRODUCTION 18 YabA co-localizes with the central DnaX focus in 54% of cells, and is not visible at bipolar oriC regions. In 10% of cells, all three markers co-localize (initiation) and in 9% of cells, YabA colocalizes with one of the two oriC regions. In the remaining cells (27%), a YabA focus is present, but does not co-localize with either oriC or DnaX. Therefore, YabA can also visibly accumulate at oriC during ongoing replication [START_REF] Schenk | Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication[END_REF]. Localization pattern of YFP-YabA was analyzed more precisely, grouping cells according to cell length, which correlates with the age of the cell. Figure 7.B shows that in small, medium sized and large cells, YabA is present at the replication forks and not at oriC regions in about 50% of cells (panels B, D and G), and co-localizes with oriC in 10% of young cells, in 6% of medium-age cells, and in 13% of old cells (panels C, F and H). Interestingly, YabA does not co-localize with either oriC or DnaX in about 25% of cells, possibly interacting with DnaA that is bound at promoter regions on the chromosome [START_REF] Schenk | Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication[END_REF].YabA directly alters cooperative binding of DnaA to DNA, independently of nucleotide hydrolysis and irrespective of the nucleotide bound to DnaA. In addition, the absence of YabA did not increase the binding efficiency of DnaA at dnaA boxes along the chromosome.

Yeast-two-hybrid analysis using a DnaA domain library revealed that YabA interacts with DnaA domain IIIa, on the opposite surface of the ATP-binding pocket of domain IIIa, further supporting the conclusion that YabA does not regulate the nucleotide-bound state of DnaA (Cho et al., 2008, Scholefield andMurray, 2013).

Another role of YabA related to the interaction between DnaA and DnaD has been proposed.

DnaA mutants which lost their interaction with YabA also showed a reduced or disabled interaction with DnaD (Cho et al., 2008), suggesting that YabA and DnaD recognize overlapping regions in DnaA. DnaD also inhibits the cooperative binding of DnaA to DNA without affecting binding to ATP or hydrolysis activity (Ishigo-Oka et al., 2001, Bonilla andGrossman, 2012). In vitro, DnaD inhibits DnaA helix formation. DnaD and YabA may therefore antagonize one another during DnaA assembly or disassembly (Scholefield and Murray, 2013).

Finally, YabA is an attractive candidate for regulating the activity of DnaA, both as replication initiator and transcriptional factor (see chapter 2.3.1), in response to alterations in replication status (Noirot-Gros et al., 2006, Soufo et al., 2008). Nevertheless, analysis of the global gene expression in a yabA null mutant revealed that the expression of genes controlled by DnaA was not altered during exponential growth or after a replication stress, indicating that YabA is not required for modulating DnaA transcriptional activity (Goranov et al, 2009). DnaA activity as a transcriptional factor is probably affected by other factors, including the need for new synthesis to generate DnaA-ATP [START_REF] Goranov | A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA[END_REF]Grossman, 2009) and perhaps the presence of DnaD and DnaB [START_REF] Smits | Primosomal proteins DnaD and DnaB are recruited to chromosomal regions bound by DnaA in Bacillus subtilis[END_REF]. Effects of these other factors might be partially redundant with YabA, helping to mask effects of loss of yabA.

The parABS system (Soj/Spo0J)

The parABS system was originally identified in low copy-number plasmids of E. coli and was found to be essential for accurate plasmid partitioning [START_REF] Abeles | Partition of unit-copy miniplasmids to daughter cells. The DNA sequence and functional organization of the P1 partition region[END_REF]. The system has three components: a Walker Box ATPase (ParA), a DNA-binding protein (ParB) and a ParBbinding sequence that acts as a centromere (parS). Interestingly, orthologs of ParA, ParB, and also parS sites, have been identified on the chromosome of many prokaryotic species. Further, the majority of these loci are present in origin-proximal regions, suggesting that the parABS system is primarily involved in the regulation of processes that involve the origins of bacterial chromosomes.

In B. subtilis, Soj and Spo0J are orthologs of ParA and ParB, respectively. Eight of the ten parS sites of the B. subtilis chromosome are located close to the origin of DNA replication and the sites closest to oriC are most frequently bound by Spo0J. parS nucleates the spreading of Spo0J into flanking regions of DNA to create large nucleoproteic structures that extend for several kbp around the parS site [START_REF] Breier | Whole-genome analysis of the chromosome partitioning and sporulation protein Spo0J (ParB) reveals spreading and origin-distal sites on the Bacillus subtilis chromosome[END_REF]. The involvement of Soj and Spo0J in the control of the initiation of replication [START_REF] Murray | Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA[END_REF] and in the segregation of the chromosome via recruitment of SMC (Structural Maintenance of Chromososme) proteins at oriC (Graham et al., 2014, Gruber andErrington, 2009) has also been shown.

Biochemical studies showed that Soj is a Walker-type ATPase able to form dimers in an ATPdependent manner. ATP-bound Soj forms dimers and cooperatively interact with DNA, while ADP-bound Soj is monomeric [START_REF] Leonard | Bacterial chromosome segregation: structure and DNA binding of the Soj dimer-a conserved biological switch[END_REF]. The monomeric Soj protein inhibits DnaA, while dimerization of Soj switches the protein into an activator of DnaA [START_REF] Scholefield | Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation[END_REF].

These results indicate that Soj acts as a molecular switch to control DnaA activity, with its opposing regulatory activities being dictated by its quaternary state. Spo0J regulates Soj activity by stimulating its ATPase activity, thus converting the dimer back to the monomeric form (Figure 8.A) [START_REF] Scholefield | Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA[END_REF][START_REF] Scholefield | Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation[END_REF]. In addition, Murray and Errington showed that Soj functions as a spatially regulated molecular switch, capable of either inhibiting or activating DnaA, depending on Soj subcellular localization [START_REF] Murray | Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA[END_REF]. Both localization and activity of Soj are controlled by Spo0J. The cited authors clearly showed that a monomer of Soj directly interact with DnaA and inhibit DnaA activity. Moreover, in a spo0J deletion mutant or in a mutant expressing an ATP hydrolysis-deficient Soj version (SojD40A), Soj fails to bind DnaA; DNA replication is therefore stimulated.

Soj appears to interact with the ATPase domain (IIIB) of DnaA, although it does not affect ATP binding or hydrolysis by DnaA [START_REF] Scholefield | Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA[END_REF]. Instead, it acts by inhibiting DnaA oligomerisation at oriC. A Soj mutant trapped in the monomeric state has been shown to inhibit

DnaA oligomer formation both in vitro and in vivo. Curiously, Soj trapped in the dimeric state is also able to interact with DnaA on a similar overlapping surface, without inhibiting DnaA oligomerisation. Thus, it has been suggested that monomeric Soj inhibits conformational changes in DnaA that are needed to form an active initiation complex, while dimeric Soj may stabilize DnaA in this oligomerisation-competent conformation [START_REF] Scholefield | Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA[END_REF]. DnaA is stably bound to its high-affinity sites during most of the cell cycle. At initiation, additional DnaA molecules are recruited through a domain I interaction (step 1). At this point, Soj could interact with DnaA to prevent the structural transition necessary for it to form a helix on doublestranded DNA (step 2). This nucleoprotein complex facilitates the unwinding of the DUE and the loading of a compact helix onto ssDNA (step 3). Soj could interact with this newly recruited DnaA preventing the bending of the α-helix linking domains III and IV [START_REF] Scholefield | Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA[END_REF].

Two potential mechanisms by which Soj could act to prevent DnaA helix formation has been proposed (Figure 8.B). First, Soj could bind to domain IIIB of DnaA and sterically inhibit helix assembly. Second, Soj could bind to domain IIIB and prevent DnaA undergoing the conformational changes required for it to assemble into a helix [START_REF] Scholefield | Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA[END_REF].

The primosomal DnaD

DnaD is an essential primosomal protein in B. subtilis. DnaD is needed during replication initiation for assembly of the replicative helicase at oriC and during replication restart at stalled replication forks. DnaD associates with DnaA at oriC and this association is necessary for the binding of DnaB; additionally, DnaI-mediated assembly of the helicase at oriC also depends on these events. Moreover, DnaD and DnaB associated with DnaA are also found associated with secondary DnaA binding regions outside oriC. In contrast to oriC, these secondary DnaA binding regions do not function as origins of replication, and there is no indication that they are capable of loading the replicative helicase [START_REF] Smits | Primosomal proteins DnaD and DnaB are recruited to chromosomal regions bound by DnaA in Bacillus subtilis[END_REF]. DnaD has been shown to inhibit the ATPdependent cooperative binding of DnaA to oriC [START_REF] Bonilla | The primosomal protein DnaD inhibits cooperative DNA binding by the replication initiator DnaA in Bacillus subtilis[END_REF] and to affect the formation of helical DnaA filaments in vitro (Scholefield and Murray, 2013). It remains unclear however how these activities can be reconciled with the role of DnaD in vivo, where it is essential for DNA replication initiation.

Titration of DnaA-boxes

In E. coli, dat sequences (DnaA Titration) of high affinity for DnaA binding sequester DnaA away from the origin of replication during the cell cycle. This sequestration regulates the amount of free DnaA molecules in the cell and avoids over-initiation. In exponentially growing B. subtilis cells, DnaA stably binds not only to the oriC region (region located upstream of dnaA [oriC1] and intergenic region between dnaA and dnaN [oriC2]), but also to six DnaA-box clusters (DBCs) located in the intergenic regions of gcp-ydiF, yqeG-sda, ywlC-ywlB, ywcI-vpr, yydA-yycS and thdFjag. Binding of DnaA to DBCs modulate the transcription of corresponding neighbouring genes [START_REF] Ishikawa | Distribution of stable DnaA-binding sites on the Bacillus subtilis genome detected using a modified ChIP-chip method[END_REF]. A deletion mutant lacking the six DBCs (Δ6) initiated replication early. An over-initiation due to a cumulative effect of DBCs deletions was also observed, mainly when two

DBCs ([yydA-yycS] and [ywcI-vpr]) were deleted (Figure 9) [START_REF] Okumura | Regulation of chromosomal replication initiation by oriC-proximal DnaA-box clusters in Bacillus subtilis[END_REF].

The reintroduction of a single DBC partly complemented these defects, even when the DBC was inserted into the replication-terminus region. These data indicate that DBCs function not only in transcriptional control of neighboring genes but also in negative regulation of replication initiation. Further, inactivation of Spo0J (cf next chapter) in Δ6 mutant cells yielded a pleiotropic phenotype, accompanied by severe growth inhibition. However, these defects were not observed in a mutant background in which replication initiation was driven by a DnaAindependent replication origin, suggesting that over-initiation is due to too much DnaA activity.

Moreover, introduction of a single DBC at various ectopic positions within the Δ6 chromosome partly suppressed the early-initiation phenotype; this effect was dependent on insertion location. Thus DBCs negatively regulate replication initiation by interacting with DnaA molecules, to sequestrate active ATP-DnaA and/or inactivate DnaA through promotion of the ATP-hydrolysis. DBCs therefore play a major role, together with Spo0J/Soj, in regulating the activity of DnaA to ensure correct and tight regulation of the initiation of chromosome replication [START_REF] Okumura | Regulation of chromosomal replication initiation by oriC-proximal DnaA-box clusters in Bacillus subtilis[END_REF].

Integration of DNA replication with other cellular processes

DNA replication is tightly coordinated with other cellular processes; all cells must replicate and segregate their chromosome to ensure faithful inheritance of their genetic information. It is therefore not surprising that proteins involved in chromosome replication also have additional roles in cell life. Recent works from a range of organisms have demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities.

DnaA, a global/multi-processes transcriptional factor

In addition to its essential role in replication initiation, DnaA is also a transcription factor. DnaA activates or represses transcription of various genes, including its own. It is thought that like the case for replication initiation (in E. coli), DnaA-ATP is the active form for transcriptional regulation. However, its ability to function as a transcription factor appears to increase when replication initiation or elongation is inhibited [START_REF] Goranov | A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA[END_REF]. Thus, accumulation of DnaA-ATP during replication stress may result in activation of the transcriptional regulatory activity of DnaA as well as its initiator activity. In B. subtilis, DnaA appears to control the expression of >50 genes organized in ∼20 operons in response to perturbations in replication [START_REF] Goranov | A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA[END_REF]. Genes controlled by DnaA are involved in many processes, including: replication (dnaA, dnaN, and dnaB), cell-division (yllB-ylxA-ftsL-pbpB), amino acid and nucleotide biosynthesis (lysC, proBA, pur and pyr operons, and nrdEF), perhaps translation and ribosome assembly (yqeH and ywlC) and other unknown functions. Transcription of these operons changes in response to inhibition of either replication elongation or replication initiation, independently of the well-characterized RecA-mediated SOS response (Breier andGrossman, 2009, Goranov et al., 2005). Furthermore, these operons contain putative DnaA binding sites in their regulatory regions, and DnaA appears to associate with at least eight of these in vivo, based on results from chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP-chip) experiments. Five of these associations appear to regulate gene expression [START_REF] Goranov | A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA[END_REF][START_REF] Ishikawa | Distribution of stable DnaA-binding sites on the Bacillus subtilis genome detected using a modified ChIP-chip method[END_REF]: DnaA directly activates the transcription of checkpoint gene sda (Breier andGrossman, 2009, Burkholder et al., 2001) and appears to directly repress the transcription of ywlC, vpr, and the yyzF-yydD, trmE-noc, and ywcI-sacT operons (Breier andGrossman, 2009, Ishikawa et al., 2007). Using in vitro DNA affinity purification and deep sequencing (IDAP-Seq) 269 DnaA binding regions were identified. Most sites had a slightly higher affinity for DnaA-ATP than DnaA-ADP, but few had a strong preference for binding DnaA-ATP such as sda promoter region and the region between the 3' ends of gcp and ydiF (Smith and Grossman, 2015). The difference in affinity for nucleotide bounds forms was fairly in the oriC region, including the dnaA promoter region and other between dnaA and dnaN, suggesting that DnaA binding to the oriC region is not particularly sensitive to the nucleotide bound state of DnaA. Thus, the activity of DnaA during replication initiation appears to be regulated primarily at the level of oligomerisation (rather than nucleotide binding). However, the observation that ATP-bound DnaA binds to the sda promoter region much more strongly than the ADP-bound form may points to an important role for bound nucleotide in transcriptional regulation at this locus, and perhaps other loci where DnaA functions as a transcriptional regulator (Smith and Grossman, 2015). Furthermore, of the 269 DnaA binding regions identified in vitro, only eight strongest binding ones have been identified in vivo (Breier andGrossman, 2009, Ishikawa et al., 2007), suggesting that other cellular factors and/or the amount of available DnaA in vivo restrict DnaA binding to these additional sites. Interestingly, it was identified several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo to some of these regions (Smith and Grossman, 2015).

In a recent study to evaluate the effects of dnaA on global gene expression in B. subtilis (separate from its role in replication initiation) the changes in mRNA levels were analyzed in cells with and without dnaA, using engineered strains in which dnaA and oriC were non-essential [START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF]. This study reveals that dnaA is required for many of the changes in gene expression in response to replication stress and also dnaA affects expression of a large network of genes during exponential growth. Some of the changes in gene expression were most likely due to DnaA directly regulating target genes. However, most of the effects were indirect affecting the expression of several regulons due to the effects of DnaA on the gene sda. DnaA activates transcription of sda and Sda inhibits the histidine protein kinases KinA and KinB required for activation of the master regulator of stationary phase and sporulation transcription factor Spo0A (see chapter 3.2.1). Furthermore, dnaA is needed for normal competence development and loss of dnaA causes a decrease in competence. This finding suggest that DnaA plays an important role in modulating cell physiology [START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF].

Cell growth and DNA metabolism

To proliferate, bacteria must duplicate their genetic material so that it can be passed to their progeny. This requires that DNA replication is coordinated with cell growth and division. In the natural environment, bacterial growth is dynamic and strongly influenced by changes in nutrient availability. Recent studies showed that bacteria utilize a range of regulatory systems, many of them species-specific, to coordinate DNA replication with cell growth.

In many bacteria, particularly those utilized by biotechnology industries (e.g. Bacillus,

Corynebacterium, Escherichia, Lactococcus), cell growth rate during the exponential phase is dictated by the abundance and quality of nutrients in the environment, and accordingly can vary from less than 20 minutes up to several hours. To ensure inheritance of genetic material over this wide range of potential doubling times, the rates of DNA synthesis and cell division in these organisms are tightly linked with the rate of cell growth. It has been shown that both E. coli and B. subtilis coordinate their DNA synthesis rate with their steady-state growth rate by regulating the frequency of DNA replication initiation [START_REF] Helmstetter | DNA synthesis during the division cycle of rapidly growing Escherichia coli B/r[END_REF][START_REF] Sharpe | Bacillus subtilis cell cycle as studied by fluorescence microscopy: constancy of the cell length at initiation of DNA replication and evidence for active nucleoid partitioning[END_REF].

Although this phenomenon was originally documented over forty years ago, the molecular mechanisms that connect bacterial metabolism with DNA replication initiation remain a mystery. A direct link between central carbon metabolism and DNA replication has been demonstrated in B. subtilis [START_REF] Jannière | Genetic evidence for a link between glycolysis and DNA replication[END_REF] and E. coli (Maciag et al., 2011). For B. subtilis, the target of the regulation by metabolic-related signals was shown to be mostly the elongation of the DNA replication process [START_REF] Jannière | Genetic evidence for a link between glycolysis and DNA replication[END_REF]. However in E. coli, the link exists at the steps of both initiation and elongation of DNA replication (Maciag et al., 2011), indicating the important global correlation between metabolic status of the cell and the events leading to cell reproduction.

DnaA is the master bacterial DNA replication initiator protein and is a candidate factor linking cell growth with DNA synthesis. DnaA directly stimulates DNA replication initiation from oriC once per cell cycle (Katayama et al., 2010). It has been observed that the concentration of DnaA is positively correlated with cell growth rate and size, suggesting that the amount of DnaA could dictate the frequency of DNA replication initiation [START_REF] Hill | Cell size and the initiation of replication in bacteria[END_REF]. Recently, the model for growth rate control of DNA replication initiation was rigorously evaluated in E. coli and B. subtilis [START_REF] Flatten | The DnaA protein is not the limiting factor for initiation of replication in Escherichia coli[END_REF]Murray and Koh, 2014). In both organisms, the amount of DnaA within cells was artificially increased and in neither situation was the frequency of DNA replication initiation significantly affected. These results indicate that the accumulation of DnaA alone does not control the time of initiation. However, it was shown in B. subtilis that nutrient-mediated growth rate regulation of DNA replication initiation requires DnaA and oriC, indicating that the target of this control system is DnaA activity localized at the endogenous replication origin (Murray and Koh, 2014). Thus, the key questions are (i) how does a bacterial cell detect its growth rate and

(ii) how does it transmit this information to the DNA replication initiation complex.

To investigate the connections between cell growth and chromosome replication in B. subtilis, molecular and chemical genetic approaches were used to limit essential cellular activities (e.g. carbon metabolism, respiration, fatty acid synthesis, protein synthesis, phospholipid synthesis and cell wall synthesis), followed by analysis of both growth rate and DNA replication initiation frequency (Murray and Koh, 2014). In all cases, the decrease in growth rate matched for a decrease in DNA replication initiation. Surprisingly however, for several perturbations it was found that inhibition of DNA replication occurred independently of DnaA (i.e. in a ∆dnaA mutant strain, which initiates chromosome replication using an integrated plasmid origin, regulation of DNA replication was retained when cell growth slowed). This unanticipated result suggests that multiple regulatory pathways are involved in coordinating DNA replication initiation with the rate of bacterial cell growth, with different regulators responding to distinct physiological and chemical changes.

When bacteria become starved for essential cellular building blocks such as amino acids and fatty acids, they induce a stress protocol termed the stringent response. Under these conditions, the small molecule alarmone (p)ppGpp is synthesized and acts by altering cellular physiology from catabolism to anabolism. One of the main targets for (p)ppGpp in many organisms is the RNA polymerase, where ligand binding leads to differential transcription of hundreds of genes [START_REF] Srivatsan | Control of bacterial transcription, translation and replication by (p)ppGpp[END_REF]. In addition, (p)ppGpp inhibits DNA synthesis and interestingly, it appears to affect distinct steps of the replication cycle in different organisms. In B. subtilis, (p)ppGpp targets DNA replication elongation by directly inhibiting the DNA primase (DnaG), which is responsible for synthesizing short oligoribonucleotides required for replication of the lagging-strand [START_REF] Wang | Nutritional control of elongation of DNA replication by (p)ppGpp[END_REF]. Using DNA microarrays to monitor DNA fork progression in synchronized cells, scientists have shown that replication arrest upon starvation occurs regardless of where the replication fork is located on the chromosome. It coul be concluded that in B. subtilis, arrested forks were not disrupted, as they did not recruit the recombination protein RecA, thereby allowing DNA synthesis to rapidly resume when starvation is alleviated [START_REF] Wang | Nutritional control of elongation of DNA replication by (p)ppGpp[END_REF]. This is contrasting with E. coli, where the stringent response was shown to trigger replication arrest at the initiation level [START_REF] Ferullo | The stringent response and cell cycle arrest in Escherichia coli[END_REF], although a most recent study also reported replication elongation arrest mediated by (p)ppGpp [START_REF] Maciag | ppGpp inhibits the activity of Escherichia coli DnaG primase[END_REF]. Thus, in B. subtilis, the nucleotide (p)ppGpp act as a sensor mechanism that prevents replication-fork progression as well as disruption in starving cells.

A protein network of high biological relevance furthermore supports the notion that DNA replication is coordinated with other cellular pathways; indeed, the subunits of the B. subtilis replication machinery not only interact between themselves but also interact specifically with proteins involved in many other aspects of bacterial physiology (Figure 10) (Noirot-Gros et al., 2002). Biochemical and cytolocalization studies suggest that the assembly of the replication machinery is mediated in part by protein interactions stable enough to promote the colocalization of individual components at midcell. In agreement with this hypothesis, the functional co-localization of the initiation regulator YabA with the replisome requires a physical interaction between YabA and both the initiator DnaA and the sliding clamp DnaN (Figure 5) (Noirot-Gros et al., 2006). YabA localizes with DnaX (replisome) during most of the cell cycle and also is localized at oriC during initiation; however in about 25% of cells, YabA focus is present but does not co-localize with either oriC or DnaX, possibly interacting with DnaA that is bound at promoter regions on the chromosome and playing another role [START_REF] Schenk | Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication[END_REF].

Furthermore, most of the protein partners that are not part of the replisome do not have a replisome-like subcellular localization. In particular, this is the case for the primosomal proteins DnaB and DnaD, which localize approximately to the membrane with some foci at the edge of the nucleoid and some close to the cell center [START_REF] Imai | Subcellular localization of DNAinitiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids[END_REF][START_REF] Meile | Systematic localisation of proteins fused to the green fluorescent protein in Bacillus subtilis: identification of new proteins at the DNA replication factory[END_REF]. Based on the analysis of the functional interplay between DnaB and DnaD, it is proposed that B. subtilis regulates the loading of the replicative helicase onto DNA by spatially controlling the physical interaction between DnaB and DnaD [START_REF] Bruand | Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication[END_REF].

Therefore, proteins with distinct subcellular localizations can functionally interact, suggesting that the biologically relevant interactions are transient or involve only a small fraction of these proteins. Altogether, these findings suggest that the interactions between the replisome subunits and proteins acting in various other pathways may have a regulatory role, such as the coordination of the activity of the replication machinery with various cellular pathways.

Cell fate in adverse time: Sporulation of B. subtilis

A significant characteristic of B. subtilis cells is their ability to sporulate under nutrient limiting conditions. This process of cellular differentiation allows the cell to form a dormant spore. The spore is metabolically inactive and environmentally highly resistant, capable of surviving in extreme conditions of temperature, desiccation and ionizing radiations. When the nutrients become available again, the spore can germinate, returning the cell to vegetative growth, even after thousands of years.

Sporulation cycle

The process of sporulation initiates after complete DNA replication with an asymmetric division near to one pole of the cell, resulting in the formation of a smaller cell, the forespore and a larger cell, the mother cell (Figure 11). These two cells each contain an identical copy of the genome, but differential pathways of gene expression lead to dramatically different cell fates. The transition from vegetative growth to entry into sporulation is controlled by a complex regulatory circuit, in which the most important event is activation of the master transcriptional regulator Spo0A. Spo0A is activated by a "phosphorelay system" governed by five sensor histidine kinases (KinA to KinE) that respond to different environmental stresses. The phosphoryl group from the histidine kinases is transferred to Spo0A via the phosphotranferases Spo0F and Spo0B which culminates in an active phosphorylated Spo0A (Spo0A~P) (Figure 12). Spo0A∼P acts as transcriptional regulator, controlling directly or indirectly the expression of over 500 genes (Molle et al., 2003). Indeed, during the transition phase, the intracellular concentration of (Fujita et al., 2005).

Sporulation is a very complex developmental process that requires plenty of energy and needs several hours to be completed. Several steps are necessary from the formation of a forespore to the release of a mature spore after lysis of the mother cell. These include engulfment, cortex synthesis and coat formation in order to confer to the spore the resistance properties required to survive to extreme conditions. The sporulation process is described in seven stages (from 0 to VII) and is driven by a cascade of 4 compartment-specific sigma factors that is initiated by the asymmetric division of the cell (Figure 11 and12). These sigma factors control expression of specific genes of the forespore and the mother cell at the onset of sporulation (σ F and σ E , respectively) and at late stage (σ G and σ K , respectively) [START_REF] Hilbert | Compartmentalization of gene expression during Bacillus subtilis spore formation[END_REF].

The vegetative cells are considered to be in Stage 0. The next phase, Stage I, is characterized by the condensation of two chromosomes (one for the mother cell and one for the forespore) of the vegetative cell, resulting in the formation of an axial filament of chromatin on either side of the cell. The RacA protein is necessary for anchoring the two chromosomes to the cell poles to promote proper chromosome segregation (Ben-Yehuda et al., 2003). RacA binds to GC-rich inverted repeats located around the origin of replication (Ben-Yehuda et al., 2005) and localizes to the cell poles through its interaction with the cell division protein DivIVA, which localizes to the two poles of the cell, through the recognition of highly negatively curved membranes [START_REF] Ramamurthi | Negative membrane curvature as a cue for subcellular localization of a bacterial protein[END_REF]. In addition to proper chromosome segregation, proper chromosome number is also necessary for robust sporulation and has been found to be tightly regulated by at least three proteins: SirA (sporulation inhibitor of replication A), Sda (suppressor of dnaA1) and Spo0A~P (see next chapter). The major event of sporulation after the formation of the axial filament is the asymmetric division which requires relocation of the division apparatus (Stage II). This process is crucial for the compartmentalization of gene expression.

Cells will then become genetically and morphologically different; one, the largest, the "mother cell," and the second, much smaller, the "prespore." This switch to a polar septum is dependent on two factors: an increase in levels of the cell division protein FtsZ and by the activation of the gene for the sporulation protein SpoIIE (Carniol et al., 2005). As soon as the division septum is formed, FtsZ disappears, while SpoIIE persists at the site until the activation of the first sporulation-specific sigma factor, the forespore-specific σ F . Spo0A~P and σ H positively regulate the transcription of the spoIIA (spoIIAA-AB-sigF) operon, but the activity of the σ F factor is inhibited by an anti-sigma factor SpoIIAB, which sequesters it in the form of the SpoIIAB-σF complex [START_REF] Duncan | SpoIIAB is an anti-sigma factor that binds to and inhibits transcription by regulatory protein sigma F from Bacillus subtilis[END_REF]. After completion of the asymmetric division; SpoIIE (which localizes to the polar septum) performs a second function, wherein it dephosphorylates SpoIIAA, which binds then and sequesters SpoIIAB, thereby relieving the inhibition of σ F (Figure 12) [START_REF] Duncan | Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division[END_REF]. Similar to σ F , σ E is produced prior to asymmetric division under the control of Spo0A~P and is found in both compartments after septation [START_REF] Fujita | An investigation into the compartmentalization of the sporulation transcription factor sigmaE in Bacillus subtilis[END_REF].

However, σ E is produced initially as an inactive pro-σ E precursor and is specifically activated only in the mother cell. The activation of σ E depends on SpoIIR and SpoIIGA. SpoIIR is produced in the forespore under σ F control and is then secreted into the intermembrane space of the septum that separates the mother cell and forespore, where it activates SpoIIGA [START_REF] Hofmeister | Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis[END_REF][START_REF] Imamura | Evidence that the Bacillus subtilis SpoIIGA protein is a novel type of signal-transducing aspartic protease[END_REF]. SpoIIGA then cleaves pro-σ E and a functional σ E transcription factor is released into the cytosol of the mother cell. The activation of σ E then allows transcription of the σ E regulon, which includes genes necessary for engulfment.

After asymmetric division, the polar septum curves around the forespore as the mother cell engulfs the forespore (stage III), resulting in the double membrane enveloped spore structure.

During engulfment, a peptidoglycan degradation machinery composed of SpoIID, SpoIIM and SpoIIP is initially needed for septal wall thinning and subsequently for movement of the engulfing membranes [START_REF] Bibliography Abanes-De Mello | A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore[END_REF]. Recent evidence has indicated that in addition to peptidoglycan degradation, membrane movement during engulfment also relies on active peptidoglycan synthesis [START_REF] Meyer | Cell wall synthesis is necessary for membrane dynamics during sporulation of Bacillus subtilis[END_REF]. At the end of engulfment, the forespore is a free floating cell in the mother cell cytosol. At this time the SpoIIIAA-SpoIIIAH proteins produced under the control of σ E and the SpoIIQ protein under the control of σ F are required for the activation of the forespore-specific σ G [START_REF] Camp | A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis[END_REF]. The gene encoding the forespore-specific σ G is under the transcriptional control of σ F , which ensures that it is only expressed in the forespore. Similar to σ E , the subsequent mother-cell specific transcription

I. INTRODUCTION

32 factor σ K is produced as an inactive pro-σ K protein. Pro-σ K is cleaved by SpoIVFB (Figure 12) [START_REF] Lu | Sporulation protein SpoIVFB from Bacillus subtilis enhances processing of the sigma factor precursor Pro-sigma K in the absence of other sporulation gene products[END_REF], which is an intermembrane protease that is initially held inactive in a complex along with SpoIVFA and BofA [START_REF] Resnekov | Negative regulation of the proteolytic activation of a developmental transcription factor in Bacillus subtilis[END_REF].

During the stage IV, two layers of peptidoglycan are synthesized between the two membranes:

a thick layer corresponding to the cortex and the wall of the future cell. This synthesis results from the action of genes expressed in the mother cell, notably under the control of σ E , but also of σ K [START_REF] Errington | Regulation of endospore formation in Bacillus subtilis[END_REF]. This step is followed by stage V which sees the synthesis of the spore coat around the cortex. The coat has been described as having four distinct layers: the basement layer, inner coat, outer coat, and cortex [START_REF] Mckenney | The Bacillus subtilis endospore: assembly and functions of the multilayered coat[END_REF]. Coat assembly begins immediately after invagination and continues throughout the sporulation. This is composed of at least 70 individual proteins that are produced by the mother cell and localize at the surface of the spore during invagination. The peptidoglycan precursors are also synthesized in the mother cell and are routed through the prespore outer membrane [START_REF] Mckenney | Dynamics of spore coat morphogenesis in Bacillus subtilis[END_REF]. At stage VI, maturation of the spores take place, upon which the spore develops its characteristic resistance properties. Finally during Stage VII, the mother cell lyses and releases the completed spore (Piggot and Hilbert, 2004). After the release, the spore undergoes further maturation with a final increase in (thermal) stress resistance (Abhyankar et al., 2014).

Control of replication initiation during sporulation

The activation of sporulation is closely coordinated with DNA replication to ensure that the cell contains two copies of the chromosome (one destined for the mother cell and the other for the forespore). More than one chromosome in the forespore compartment could be lethal to the cell or could lead to a reduced viability of the spore. In B. subtilis, three proteins control the replication at the point of entry into sporulation: Sda, SirA and Spo0A itself.

Sda

The source of coupling between the B. subtilis replication cycle and its entry into sporulation is sda (Supressor of dnaA). Sda is a small protein (46 aa) that inhibits KinA, one of the twocomponent sensor kinases involved in the initiation of sporulation (Burkholder et al., 2001).

However, sda transcription is positively regulated by DnaA (Breier and Grossman, 2009). Sda acts by binding directly to KinA (and likely KinB as well), thereby inhibiting the autophosphorylation of the histidine kinase, resulting in reduced levels of Spo0A∼P [START_REF] Cunningham | The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F[END_REF]; Figure 14B). The control of sda expression by DnaA constitutes a checkpoint that allows the cell to monitor initiation of chromosome replication, so as to prevent attempts to sporulate while cells are initiating DNA replication. Time-lapse microscopy revealed the relationship between sporulation and the replication cycle. The activity of the promoter sda was measured over the cell cycle using GFP protein as a reporter, revealing that expression of sda occurs in a pulsatile manner correlated with initiation of replication (Figure 13). At the onset of DNA replication, a burst of sda transiently inhibits initiation of sporulation, as shown by the reciprocal relationship between the expression of sda and that of spoIIA, a marker for early sporulation since spoIIA promoter is directly regulated by Spo0A∼P (Veening et al., 2009).

The sda promoter contains 5 dnaA boxes that allow positive regulation of sda transcription at the beginning of each cell cycle through binding of DnaA (Burkholder et al., 2001). Reductions in Sda levels correlate with the end of DNA replication, ensuring that two copies of the chromosome are present before sporulation initiates, helping the formation of viable spores.

However, to enter into the sporulation program, the cell needs to reduce rapidly the amount of Sda, in order to allow the accumulation of activated Spo0A at the appropriate time. This process is performed by ClpXP-mediated proteolysis of Sda [START_REF] Ruvolo | Proteolysis of the replication checkpoint protein Sda is necessary for the efficient initiation of sporulation after transient replication stress in Bacillus subtilis[END_REF].

In addition to dnaA boxes, sequence analysis of the promoter region of sda revealed the presence of two consensus Spo0A-binding sites and a binding site for the DNA damage protein LexA, a transcriptional repressor of the SOS regulon involved in regulation of DNA damage repair (Figure 14.A) (Veening et al., 2009). After cell division, one of the two resulting siblings is arbitrarily selected for further analysis. The increasing cell cycle length as time progresses is likely due to reduced nutrient availability (Veening et al., 2009) Transcription of sda is activated as soon as low levels of Spo0A∼P are accumulated (Fujita et al., 2005). Thus, Spo0A~P probably acts directly as a positive regulator of sda transcription. While the idea that Spo0A activates sda expression might seem counterintuitive, it could be important for preventing the Spo0A~P concentration from overshooting and for keeping Spo0A∼P levels low in cells that have just initiated a new round of replication. In addition, the sda promoter contains a putative LexA binding site. In response to UV damage and HPURa treatments, LexA is degraded, leading to the derepression of the target gene. The SOS response can therefore also mediate an increase in Sda levels in response to DNA damage or stalled replication forks. DNA damage-dependent up-regulation of sda prevents entry into sporulation when chromosomal DNA is damaged (Figure 14.B) (Veening et al., 2009). In conclusion, the checkpoint protein Sda serves as a genetic timer that allows a window of opportunity toward the end of each replication cycle for Spo0A~P to reach the levels required to initiate sporulation, thus ensuring that a cell enter the sporulation pathway when it contains the correct chromosomal copy number and has completed DNA replication and repair.

SirA

As another way to control chromosome number during sporulation, B. subtilis possess a checkpoint trigger to inhibit DNA replication initiation through the action of the protein SirA (sporulation inhibitor of replication A) (originally annotated yneE). The transcription of sirA is activated by Spo0A~P (Molle et al., 2003[START_REF] Fawcett | The transcriptional profile of early to middle sporulation in Bacillus subtilis[END_REF]. While cells are able to maintain correct chromosome copy number when artificially induced to sporulate under conditions of rapid growth, a deletion of sirA results in loss of chromosome (Rahn- [START_REF] Rahn-Lee | The conserved sporulation protein YneE inhibits DNA replication in Bacillus subtilis[END_REF].

Meanwhile, cells overproducing SirA are elongated and anucleated, some containing nucleoids which have been severed by division septa, a phenotype found in cells where DnaA is absent (Figure 15) (Wagner et al., 2009[START_REF] Rahn-Lee | The conserved sporulation protein YneE inhibits DNA replication in Bacillus subtilis[END_REF]. (Wagner et al., 2009) In addition, cells that lack oriC and initiate replication through an ectopically integrated origin (oriN) in a DnaA-independent manner are not affected by SirA over-expression (Wagner et al., 2009). Depletion of DnaA prevents the formation of GFP-SirA focus, and expression of SirA causes DnaA-GFP foci to disperse (Wagner et al., 2009). It was further shown that SirA inhibits DNA replication through a direct interaction with DnaA. A genetic screen indicated that SirA binds to the N-terminal domain I of DnaA (Rahn- Lee et al., 2011) and the binding surface was later identified on the structure of a complex of SirA with DnaA domain I (DnaA DI ) (Figure 16) (Jameson et al., 2014). Cells harbouring alleles with sirA point mutations within the DnaA domain I interacting surface, exhibit a similar phenotype to ∆sirA cells. Moreover, these mutations prevent the formation of SirA foci normally observed in sporulating cells. This data suggest that SirA localizes to the replisome via an interaction with DnaA during sporulation (Jameson et al., 2014). SirA could interact with replisome-bound DnaA to stabilize replisome-DnaA complexes, thereby inhibiting DnaA rebinding at the origin, like YabA. Alternatively, a strain encoding a DnaA mutant unable to interact with SirA is not viable, suggesting that SirA could act by inhibiting an essential interaction of DnaA with other components of the initiation complex. Another hypothesis is that SirA may inhibit the interaction between DnaA-DnaD, aborting the assembly of the replication initiation complex (Jameson et al., 2014) Together, Sda and SirA maintain the two chromosomal copies in a cell which enters sporulation.

Sda prevents sporulation from occurring too early in the replication cycle. Conversely, SirA expression is activated by Spo0A~P upon entrance of B. subtilis into sporulation, thus preventing over-replication during development and preventing an increase in chromosomal copy number, which can reduce spore viability. Recently, SirA was shown to facilitate chromosome segregation during sporulation, independently of its role in DNA replication regulation (Duan et al., 2016). Newly synthesized bacterial origins are localized to a cell pole (or future pole) with high fidelity. In sporulating cells, oriC must be segregated or "captured" at the respective poles in the future forespore and the mother cell compartments following the onset of DNA replication. In a ∆sirA mutant strain, 10%

of sporulating cells fail to capture oriC. This activity is distinct from the role of SirA in DNA replication regulation, as sirA mutants deficient in DNA replication inhibition are able to facilitate normal oriC capture. Soj has been implicated in oriC capture during sporulation, with 20% of cells failing to capture oriC in a ∆soj mutant strain (Duan et al., 2016[START_REF] Sullivan | Recruitment of SMC by ParB-parS Organizes the Origin Region and Promotes Efficient Chromosome Segregation[END_REF]. There is no further increase in the failure of cells to capture oriC in a ∆soj∆sirA double mutant, implying Soj and SirA are acting in the same pathway to facilitate oriC segregation during sporulation.

Using a gain of interaction bacterial-two-hybrid screen, a potential interaction site between the C-terminal of SirA and domain III of DnaA was identified. This site overlaps with residues previously identified in the Soj-interaction site on DnaA, suggesting this interaction may facilitate oriC capture (Duan et al., 2016).

Spo0A

Replication initiation is also controlled by the major transcriptional regulator of sporulation, Spo0A. It has long been recognized that there is a "sensitive period" in the cell cycle when the cell can enter into the sporulation pathway. The critical determinant is the concentration of Spo0A~P, which fluctuates over the course of the cell cycle and is at its highest immediately after DNA replication is completed (Veening et al., 2009). A threshold level of Spo0A~P must be reached for sporulation to be triggered. As Spo0A~P levels increase, low-threshold target genes are turned on; however, a higher threshold Spo0A~P concentration must be achieved in order to trigger the sporulation process (Fujita et al., 2005). It is, at that point, important for a cell to have completely copied its DNA, so that the spore can receive an intact chromosome and therefore further proliferate (Figure 17). A number of studies showed that B. subtilis realizes the necessary coordination of sporulation activation with the cell cycle by producing a single sharp pulse of active Spo0A~P every cycle, shortly after termination of DNA replication. Thereby, whenever the conditions are such that the threshold level of Spo0A~P is reached, this will occur at a phase of the cell cycle in which two complete copies of the chromosome are present. The cellular Spo0A~P concentration is namely controlled by Sda (see Figure 14B). Sda production is positively controlled at the transcriptional level by DnaA. Thus, Sda levels spike at the same time as, or just after, the replisome forms and then is rapidly proteolysed. This provides a feedback mechanism whereby Sda blocks phosphorylation of Spo0A and entry into sporulation during ongoing rounds of DNA replication. However, other factors than Sda influence Spo0A~P pulsing: deletion of Sda does not prevent the pulsing of Spo0F levels (spo0F is a "low-threshold" gene under the positive control of Spo0A~P), suggesting that Spo0A~P pulsing still occurs [START_REF] Levine | Pulsed feedback defers cellular differentiation[END_REF].

Narula and coworkers showed that the pulse-like dynamics of Spo0A activation largely rely on two requirements. The first one is the presence of a negative feedback involving Spo0A∼P and one of the upstream phosphotransferases, Spo0F [START_REF] Narula | Chromosomal arragement of phosphorelay genes couples sporulation and DNA replication[END_REF]. Crucially, this feedback is not instantaneous, but delayed since Spo0A∼P transcriptionally increases levels of Spo0F, which in turn is thought to interfere with substrate binding by KinA, the major sporulation kinase, lowering the phosphate flux towards Spo0A. The second requirement is that, during the replication cycle, there is a temporary imbalance between the copy numbers of spo0F and kinA (Figure 18).This imbalance is due to their different locations on the genome: since spo0F is located close to the origin, while kinA is located closer to ter, the kinA:spo0F ratio will temporarily drop after replication of spo0F, leading to a gradual decrease in Spo0A∼P. After kinA is also replicated, the Spo0A∼P level increases again, then overshoots (i.e., pulses), due to the aforementioned delayed negative feedback loop, and only then falls back to its equilibrium level.

In accordance with this model, the researchers showed that translocating kinA and/or spo0F led Sporulation is initiated by high levels of Spo0A~P, leading to a block of matrix formation. After asymmetric septation, transcriptional differentiation is directed by dedicated sigma-factors, eventually leading to spore maturation and mother cell lysis [START_REF] Slager | Hard-Wired Control of Bacterial Processes by Chromosomal Gene Location[END_REF] to sporulation defects, stressing the importance of their chromosomal location for successful endospore formation [START_REF] Narula | Chromosomal arragement of phosphorelay genes couples sporulation and DNA replication[END_REF].

In addition, B. subtilis replication origin oriC contains a number of Spo0A-boxes which partially overlap with DnaA-boxes (see Figure 2). Indeed, the consensus Spo0A-box sequence 5'-TGTTCGAA-3' is similar to the DnaA-box consensus sequence 5'-TGTGNATAA-3'. Spo0A~P has been shown to bind these DnaA-boxes in vitro (Castilla-Llorente et al., 2006); moreover, sequence changes that alter the resemblance to the Spo0A-box consensus, without affecting that to the DnaA-box, affect Spo0A~P, but not DnaA binding to the origin. The binding of Spo0A~P to oriC appears to play a role in chromosome copy number control in a ∆sda/∆sirA mutant strain when sporulation is induced by starvation, or in cells induced to sporulate during rapid growth (Boonstra et al., 2013). ∆sda and ∆sirA strains each show a more significant loss of copy number control than upon deletion of the Spo0A boxes, with the phenotype being more profound for ∆sda than ∆sirA.

From motile to sessile state: B. subtilis biofilm formation

B. subtilis exhibits a number of lifestyles, each characterized by a distinguishing pattern of gene expression. Upon starvation or under other harsh conditions, cells differentiate to produce an endospore that functions to preserve DNA and essential proteins ultimately needed for germination when conditions become favorable (Chapter 3). Under some conditions, before sporulation, cells form biofilms. Biofilms are multicellular communities including motile cells and matrix-producing cells with highly complex structures and distinct morphological features. One of the most important characteristics of biofilms is the presence of a self-produced extracellular matrix, which allows individual cells to stick to each other's within the biofilm (Branda et al., 2001;[START_REF] Lemon | Biofilm development with an emphasis on Bacillus subtilis[END_REF]. Cells within biofilms are more resistant to biocides and antibiotics and part of this resistance is attributed to protection provided by the extracellular matrix.

The five stages of biofilm development

Bacterial aggregation and subsequent biofilm maturation consists of five different stages and involves numerous conserved and/or species-specific factors. This process comprises the development, maturation and disassembly of the community (Figure 19). Within the biofilm, genetically identical cells express different genes and produce subpopulations of functionally distinct co-existing cell types. The first step involves the introduction of bacteria to a surface.

Bacteria encounter attractive or repelling forces that vary depending on nutrient levels, pH, ionic strength, and temperature. Motile bacteria have a competitive advantage, utilizing flagella to overcome hydrodynamic and repulsive forces. In a second step, upon intercepting the surface, motile cells differentiate into non-motile that adhere to each other and the surface by secreting an extracellular matrix (Branda et al., 2006). This substance is essential to the integrity of the biofilm as it holds the community together. Thirdly, biofilm expansion is mediated by the action of the motile cells and the release of surfactant molecules. As the biofilm expands and matures, the production of extracellular matrix continues, cells form multi-layered clusters and the community is protected and organized by the extracellular matrix. Fourthly, in addition to matrix producers, motile cells and spores are also present and are spatially organized within the maturing biofilm. Finally, the biofilm ages and aerial projections develop at the surface, which serves as preferential sites of sporulation and the spread of spores. Some cells secrete small molecules such as D-amino acids and polyamines that break down the extracellular matrix and lead the cells to disperse in the environment (Vlamakis et al., 2013).

The development of the B. subtilis biofilm is tightly controlled and requires the activation of three transcriptional regulators: ComA (Lopez et al., 2009b), Spo0A (Branda et al., 2001;Hamon and Lazazzera, 2001) and DegU [START_REF] Stanley | Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-dl-glutamic acid production and biofilm formation[END_REF].

The cell decision making pathways

In order to generate a biofilm, cells switch from a planktonic to a sessile state by down-regulating the expression of flagellar genes and inducing the expression of genes required for matrix production, led by the reinforcement of external cues (e.g. nutrient depletion, low oxygen levels or surface adherence). During exponential growth, one sub-population of B. subtilis cells can synthesize flagella and grow as individual motile cells. The motile state of B. subtilis populations is determined by the alternative sigma factor SigD, which drives the expression of genes essential for the synthesis and regulation of the flagellar apparatus [START_REF] Mukherjee | The structure and regulation of flagella in Bacillus subtilis[END_REF]. The transition from motility to an alternative type of cellular growth within surfaceassociated communities, involves the repression of flagellar genes and activation of genes essential for the production of biofilm extracellular matrix composed of exopolysaccharides (EPS), protein fibers and nucleic acids (Vlamakis et al., 2013). The respective gene networks controlling these mutually exclusive developmental programs are interconnected and share common regulators and regulatory feedback loops, which prevent their simultaneous activation within a cell [START_REF] Kovács | Bacterial differentiation via gradual activation of global regulators[END_REF]. The key determinant in the regulation of motility, biofilm formation and sporulation is the master regulator Spo0A.

The regulatory circuit that controls the expression of the matrix-encoding genes has been well studied (Figure 20) (Vlamakis et al., 2013). Phosphorylation, and thus activation, of the transcription factor Spo0A is central to biofilm initiation. Spo0A can be activated by various environmental signals that allow the cell to tune its behavior to the local environment. The concentration of Spo0A~P is determined by the activity of at least four kinases (KinA, KinB, KinC and KinD). KinE can also feed into this pathway but does not appear to play a role in matrix gene expression. When a threshold level of Spo0A~P/Spo0A is reached, two parallel pathways of antirepression are triggered to allow transcription of operons critical for biofilm matrix production.

The first anti-repression pathway ends with removal of the transition state regulator AbrB from DNA. AbrB directly binds to DNA to repress transcription from promoters involved in a multitude of cellular processes, including those needed for biofilm formation [START_REF] Hamon | Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis[END_REF]. AbrB itself is controlled by the Spo0A pathway by two distinct means: (i) Spo0A~P directly represses transcription of abrB [START_REF] Strauch | The Spo0A protein of Bacillus subtilis is a repressor of the abrB gene[END_REF] and (ii) Spo0A~P promotes the expression of abbA, which encodes an AbrB anti-repressor [START_REF] Banse | Parallel pathways of repression and antirepression governing the transition to stationary phase in Bacillus subtilis[END_REF]. Phosphorylation of AbrB constitute an additional input for regulating its activity. In vitro findings suggest that AbrB phosphorylation impedes its DNA binding. Moreover, in vivo results showed that phosphorylation leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation (Kobir et al., 2014).

The second anti-repression pathway revolves around the transcriptional repressor SinR, which directly inhibits transcription from the 15-genes eps operon (required for biosynthesis of the extracellular polysaccharide) and the tapA-sipW-tasA operon, required for the production of amyloid fibers (Kearns et al., 2005). Repression by SinR is relieved when, under biofilm induction, a small anti-repressor, SinI, is expressed, which antagonizes SinR through direct protein-protein interactions [START_REF] Newman | Molecular basis of the activity of SinR protein, the master regulator of biofilm formation in Bacillus subtilis[END_REF]. The sinI gene is activated by the master regulator Spo0A~P in response to environmental and cellular signals (Fujita et al., 2005). A second anti-repressor that binds to SinR, called SlrA, has also been discovered [START_REF] Kobayashi | SlrR/SlrA controls the initiation of biofilm formation in Bacillus subtilis[END_REF]. Transcription of slrA is under the control of the transcriptional repressor YwcC, although the signal that relieves this repression is unknown.

The action of SinR during biofilm formation is further complicated by SlrR (Chu et al., 2008). SlrR is essential for the control of biofilm formation in two ways. First, SlrR binds to SinR (to form a SinR-SlrR complex), titrating SinR away and thus preventing it from repressing matrix gene promoters (epsA-O, tapA-sipW-tasA and slrR itself). Second, the complex of SinR-SlrR directly represses the promoters of the hag gene (which encodes flagellin and is required for motility), as well as genes involved in cell separation (lytABC and lytF, which encode autolysins required for flagellar function). However, removal of SinR from the eps, tapA and slrR promoter regions is not sufficient to allow transcription of these operons to proceed. RemA and RemB are also essential for biofilm formation [START_REF] Winkelman | RemA is a DNA-binding protein that activates biofilm matrix gene expression in Bacillus subtilis[END_REF]. RemA has recently been identified as a DNA binding protein that directly activates gene expression from the eps and tapA operons, and also promotes transcription of the slrR gene.

A B 4.2.1. Quorum sensing

The mechanism of cell-cell communication in bacteria, termed quorum sensing (QS) controls a large number of developmental processes. QS relies on the secretion of small signaling molecules, called auto-inducers, which accumulate in the extracellular space in a densitydependent manner. QS cascades are essential for the formation of bacterial multicellular communities and complex biofilms. Once informed via QS cascades, motile cells often organize themselves into conspicuous multicellular structures that carry out specialized tasks.

The production and secretion of the quorum-sensing molecule surfactin is important for biofilm formation in B. subtilis (Lopez et al., 2009). Surfactin has been studied for its potent surfactant activity that shows a wide range of biological activities such as antibacterial, antiviral and antifungal actions as well as for its role in surface motility. The production of surfactin is sensed together with additional environmental signals, by a portion of the biofilm population that then produces the extracellular matrix (Vlamakis et al., 2013;López et al., 2009a). Surfactin is a lipopeptide that inserts into the membrane and causes potassium leakage from the cytoplasm.

Potassium leakage stimulates the activity of a membrane protein kinase, KinC, which is part of the Spo0A activation phosphorelay and governs the expression of genes involved in biofilm formation. However, surfactin is only produced by a sub-population of cells, and cells that produce surfactin are not the same cells that respond to the molecule. This particular mechanism represents a new way of thinking about self-generated quorum sensing signals in bacteria and has been referred as paracrine signaling (López et al., 2009b), in contrast to previously described QS systems where every cell in a population could respond and produce the signalling molecule. Other compounds with different structures than surfactin that cause potassium leakage, such as the fungicide nystatin and the antibiotic valinomycic also induce matrix gene expression via KinC (López et al., 2009a).

The machinery for surfactin synthesis is encoded within the srfAA-AB-AC-AD operon and is directly controlled by a regulator termed ComA. Activation of ComA by phosphorylation (ComA∼P) is driven by another quorum-sensing system, named ComQXP. The ComQXP is a major quorum response pathway in B. subtilis which also regulates the development of genetic competence [START_REF] Grossman | Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis[END_REF]. It senses the presence of the extracellular pheromone ComX via the membrane-associated sensor kinase ComP. ComP in turn phosphorylates the transcriptional factor ComA, resulting in the transcription of a regulon that includes the srf operon, responsible for the synthesis of a surfactin (Figure 21) (López et al., 2009b).

Stimulation of ComA activity also can be triggers by the pheromone CSF (also known as PhrC).

CSF is imported into the cell by the oligopeptide permease Opp (also known as Spo0K) and inhibits the activity of the regulator RapC which negatively regulates ComA [START_REF] Core | TPR-mediated interaction of RapC with ComA inhibits response regulator-DNA binding for competence development in Bacillus subtilis[END_REF]. et al., 1999). Finally, RapG represses phosphorylated DegU (DegU∼P) [START_REF] Ogura | Binding of response regulator DegU to the aprE promoter is inhibited by RapG, which is counteracted by extracellular PhrG in Bacillus subtilis[END_REF], whose main functions are to promote the secretion of multiple enzymes and repress social motility (next chapter).

The two component signaling pathways

Two-component regulatory systems regulate diverse metabolic processes, the bacterial cell cycle, cell-cell communication, and virulence by sensing changes in the environment and modulating gene expression in response to a variety of stimuli [START_REF] Hoch | Two-component and phosphorelay signal transduction[END_REF]. A typical twocomponent regulatory system consists of a membrane-associated, histidine kinase sensor protein, which senses a specific environmental condition, and a cytoplasmic response regulator, which enables the cell to respond via regulation of gene expression when this condition varies.

Upon stimulation by a specific ligand or signal, the histidine kinase sensor protein undergoes autophosphorylation at a conserved histidine residue. The phosphoryl group is then transferred to the cognate response regulator, which can, in turn, activate or repress the transcription of target genes. The gradual increase in the phosphorylation level of Spo0A is mediated by various two-component sensor kinases (Fujita and Losick, 2005). B. subtilis harbors five histidine kinases, the cytoplasmic KinA and the membrane-bound kinases, KinB to KinE [START_REF] Jiang | Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis[END_REF].

Numerous external signals are perceived by these kinases including polyketides (Lopez et al., 2009), combination of glycerol and manganese (Shemesh and Chai, 2013), osmotic conditions (Rubinstein et al., 2012) or potassium level (Grau et al., 2015). In response to these or other unknown signals, Spo0A~P level increases in the cells and modulates the transcription of different genes (Figure 22). KinB and KinC were shown to induce sliding motility (Grau et al., 2015), the KinC and KinD proteins seem to mainly trigger biofilm development under aerobic conditions [START_REF] Mhatre | From environmental signals to regulators: modulation of biofilm development in Gram-positive bacteria[END_REF], and KinA and KinB present the major input during entry into sporulation.

The two component system DegS/DegU also play an important role in biofilm formation. DegU is a global response regulator whose phosphorylation is carried out by its cognate sensor histidine kinase, DegS. DegU is involved in motility, chemotaxis, biofilm formation, synthesis of secreted enzymes, and production of γ-poly-dl-glutamic acid [START_REF] Verhamme | DegU and Spo0A jointly control transcription of two loci required for complex colony development by Bacillus subtilis[END_REF][START_REF] Marlow | Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm[END_REF]. The role of DegU in biofilm formation is quite complex, because DegU controls et al., 2009). However, under conditions where DegU~P levels in the cell are high, biofilm formation is inhibited due to a lack of transcription from epsA-epsO and tapA-sipW-tasA operons. In this scenario, a high percentage of the cells in the community enter the sporulation pathway. Although the biological significance of such negative regulation is less well understood, these data suggest an as yet undefined link between high DegU~P levels and high Spo0A~P levels [START_REF] Marlow | Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm[END_REF].

Recent works have indicated that the DegS/DegU two-component regulatory system is activated when the flagellum stops rotating. Data demonstrated that inhibition of flagellar rotation by deletion or mutation of the flagellar stator gene, motB, results in an increase in both degU transcription and DegU~P driven processes (exoprotease production and EPS biosynthesis). In the same way, inhibition of flagellar rotation by engaging the flagellar clutch or by tethering the flagella with antibodies also promotes an increase in degU transcription that is reflective of increased DegU~P levels in the cell [START_REF] Cairns | A mechanical signal transmitted by the flagellum controls signaling in Bacillus subtilis[END_REF]. This would provide a mechanism to allow motile cells to detect and respond to the presence of a surface during the initial stages of biofilm formation. Upon sensing a surface, DegS phosphorylates DegU to promote transcription of target genes, including bslA. In this way, the arrest of flagellar rotation acts as an additional signal to initiate matrix synthesis.

The cyclic nucleotide signaling pathways

In most organisms that choose between motile and sessile lifestyles, second messengers as cyclic nucleotides are involved in the decision-making. Bis-(3′,5′)-cyclic diguanosine monophosphate (c-di-GMP) has emerged as a nearly ubiquitous bacterial nucleotide second messenger over the past decade. c-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) characterized by the GGDEF domain and, degraded by specific phosphodiesterases (PDEs) which can feature either EAL or HD-GYP domains. Many DGCs and PDEs actually harbor GGDEF and EAL domains in the same protein, with usually one domain being enzymatically active and the other being degenerate and exerting a regulatory influence.

A majority of these enzymes harbor diverse N-terminal sensory input domains that control their activities in response to intra-or extracellular signals. c-di-GMP is sensed by a wide variety of cdi-GMP-binding effectors that control diverse targets (Figure 23).

In principle, c-di-GMP can also interact with RNAs, i.e. riboswitches, and diverse classes of proteins. Riboswitches are usually located in the untranslated 5′ regions of mRNAs (5′-UTR) and can fold into different conformations depending on c-di-GMP binding, which can promote or inhibit transcriptional termination, translation or even self-splicing of the mRNA [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF][START_REF] Hengge | Bacterial signal transduction by c-di-GMP and other nucleotide second messengers[END_REF]. c-di-GMP triggers diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. In E. coli and many other Gramnegative bacteria, high c-di-GMP levels lead to inhibited motility and enhanced biofilm formation, whereas decreased c-di-GMP levels may promote biofilm disassembly and lead to activation of virulence pathways [START_REF] Hengge | Principles of c-di-GMP signalling in bacteria[END_REF]. Recent work in Gram-positive species has revealed that c-di-GMP plays similar roles in Gram-positives, though the precise targets and mechanisms of regulation may differ. In B. subtilis, the existence of a functional c-di-GMP signaling system that directly inhibits motility and directly or indirectly influences biofilm formation has been demonstrated [START_REF] Chen | Evidence for cyclic di-GMP mediated signaling in Bacillus subtilis[END_REF]. In B. subtilis, intracellular c-di-GMP levels are modulated by a set of three diguanylate cylases, the two GGDEF domain proteins DgcK and DgcP (formerly YhcK and YtrP) and the GGDEF-EAL domain protein DgcW (YkoW), and one phosphodiesterase, named PdeH (formerly YuxH).

Overproduction of DgcK or DcgP significantly increases intracellular c-di-GMP. DgcW also appears to function primarily as a DGC, as its overproduction elevates the c-di-GMP levels in the cell [START_REF] Gao | Functional Characterization of core components of the Bacillus subtilis cyclic-di-GMP signaling pathway[END_REF]. In vitro, these three DGCs are capable of synthesizing c-di-GMP [START_REF] Gao | Functional Characterization of core components of the Bacillus subtilis cyclic-di-GMP signaling pathway[END_REF]. PdeH is EAL domain protein which is a functional PDE in vitro [START_REF] Gao | Functional Characterization of core components of the Bacillus subtilis cyclic-di-GMP signaling pathway[END_REF].

Moreover, deletion of pdeH alone increases cytoplasmic c-di-GMP levels and results in impaired flagellar swarming motility [START_REF] Chen | Evidence for cyclic di-GMP mediated signaling in Bacillus subtilis[END_REF][START_REF] Gao | Functional Characterization of core components of the Bacillus subtilis cyclic-di-GMP signaling pathway[END_REF]. The EAL domain of the YkuI protein has a conserved active site; the corresponding purified catalytic domain binds c-di-GMP, but neither the domain nor the full-length protein exhibits catalytic activity in vitro [START_REF] Minasov | Crystal structures of YkuI and its complex with second messenger cyclic Di-GMP suggest catalytic mechanism of phosphodiester bond cleavage by EAL domains[END_REF]. B. subtilis encodes two GGDEF domain containing proteins, YdaK and YybT, that have degenerate active site sequences lacking conserved residues in the cyclase active site. YdaK appears to be catalytically inactive but can bind c-di-GMP, suggesting that YdaK functions as a receptor rather than a DGC [START_REF] Gao | Functional Characterization of core components of the Bacillus subtilis cyclic-di-GMP signaling pathway[END_REF]. YybT contains a PDE domain that degrades cyclic diadenylate monophosphate (c-di-AMP) and the GGDEF domain that cannot synthesize c-di-GMP but exhibits weak ATPase activity in vitro (Rae et al. 2010).

The current knowledge on the cellular roles of c-di-GMP in B. subtilis is limited to motility control, mediated by the interaction between the cyclic-diguanylated receptor DgrA (formerly YpfA, PilZ-domain protein) and the flagellar component MotA upon elevated intracellular c-di-GMP levels. Recent works reveal that EPS production and therefore also potentially biofilm formation, are regulated by c-di-GMP via the effector protein YdaK encoded within the putative EPS-synthesis operon ydaJ-N (Nicolas et al., 2012;Bedrunka and Graumann, 2017). Under the experimental conditions used in this study, YdaK is directed in its activity via the DGC DgcK. YdaK are indispensable for the production of an unknown EPS synthesized on biofilm promoting medium (Bedrunka and Graumann, 2017).

Protein phosphorylation in bacteria

Protein phosphorylation, the process by which a phosphate (PO4) group is added to a protein molecule by a protein kinase (Figure 24), is a critically important regulatory mechanism and the most widespread type of post-translational modification occurring in both prokaryotic and eukaryotic organisms. Kinases are enzymes which covalently transfer phosphate groups from high-energy donor molecules, such as the γ-phosphate of adenosine triphosphate (ATP), to specific target molecules called substrates. The removal of phosphates, dephosphorylation, is performed by another class of enzymes termed phosphatases.

Historically, protein phosphorylation was first described in Eukarya with the demonstration of enzymatic protein phosphorylation in 1954 [START_REF] Burnett | The enzymatic phosphorylation of proteins[END_REF] and followed up with the work on phosphorylase by Nobel Laureates Edwin Krebs and Edmund Fischer [START_REF] Fischer | Conversion of phosphorylase b to phosphorylase a in muscle extracts[END_REF]. Phosphorylation of proteins in Eukarya is known to be quite an abundant phenomenon. In humans, over 500 protein kinases have been identified. They interact with their substrates (and each other) to create a large signaling network that regulates the cell cycle and myriad other housekeeping processes. Anomalies in this complex networks can lead to cancer development [START_REF] Brognard | Protein kinase signaling networks in cancer[END_REF] and other types of cellular malfunction, and this The substrate is attracted to the catalytic site of the protein kinase, with the hydroxyl (OH) group of the to-be-phosphorylated residue (Ser, in this example) bound to a negatively-charged residue of the kinase. The γ-phosphate of the ATP co-factor binds to a magnesium ion, which is stabilized by negatively-charged residues of the kinase. (C) A nucleophilic attack is performed by the oxygen of the substrate OH group on the γ-phosphorous of ATP. (D) The reaction proceeds with the breaking of the phosphoester bond between the γ-phosphate and ADP, resulting in the phosphorylated residue (P-Ser) and ADP (Modified from [START_REF] Ubersax | Mechanisms of specificity in protein phosphorylation[END_REF].

incited a very exhaustive scrutiny by the scientific community. Most protein kinases in Eukarya phosphorylate serine, threonine, or tyrosine residues and belong to the structural family described by Hanks et al. (1988). They are therefore referred to as Hanks-type kinases, sharing a common fold in their cytosolic kinase domain. The typical receptor-type kinases contain a cytosolic kinase domain attached to a transmembrane helix, connecting to an extracellular ligand binding domain, which is responsible for kinase activation upon ligand binding. The kinase domain can also be soluble, and in this case, the kinases are usually activated via phosphorylation by kinases further 'upstream' in the regulatory cascade.

The presence of this post-translational modification was neglected for a long time in bacteria [START_REF] Rubin | Protein phosphorylation[END_REF]. Indeed, as early attempts to detect its presence were unsuccessful, it was proposed that protein phosphorylation was a regulatory mechanism that emerged late in evolution to meet the specific needs of organisms composed of multiple and differentiated cells.

Protein phosphorylation in bacteria was evidenced in the late 1970s through the pioneering work of several groups that showed the presence of phosphorylated amino acids in bacterial proteins and, in some cases, characterized the phosphorylated proteins. The first evidence for the presence of phosphorylation in bacteria came in 1978 with the identification of serine and threonine phosphorylated protein in Salmonella typhimurium [START_REF] Wang | Evidence for protein kinase activities in the prokaryote Salmonella typhimurium[END_REF] and later in Escherichia coli [START_REF] Manai | Analysis of the protein-kinase activity of Escherichia coli cells[END_REF] but it was not until the 1990s with the characterization of the first eukaryotic-like serine/threonine protein kinase, Pkn1, from

Myxococcus xanthus (Muñoz-Dorado et al., 1991) that it was generally accepted that serine/threonine phosphorylation takes place in bacteria. During the second half of the 1990s, the availability of complete genome sequences revealed the widespread presence of eukaryotelike serine/threonine protein kinases (STPK) and phosphatases.

The acceptance of tyrosine phosphorylation in bacteria has been longer underway. Although phosphotyrosine was demonstrated as early as 1986 in E. coli [START_REF] Cortay | Characterization of the phosphoproteins of Escherichia coli cells by electrophoretic analysis[END_REF] and further established in Acinetobacter calcoaceticus in 1990 [START_REF] Dadssi | Evidence of protein-tyrosine kinase activity in the bacterium Acinetobacter calcoaceticus[END_REF], it was not before the purification and characterization of the first autophosphorylating protein tyrosine kinase Ptk from Acinetobacter johnsonii [START_REF] Grangeasse | Characterization of a bacterial gene encoding an autophosphorylating protein tyrosine kinase[END_REF] that protein tyrosine phosphorylation was generally accepted to take place in bacteria. Part of the reason for this is related to the analytical challenges in detection of tyrosine phosohorylated proteins due to low occupancy of phosphorylation sites [START_REF] Mijakovic | Transmembrane modulatordependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases[END_REF] and that, unlike serine/threonine phosphorylation, tyrosine phosphorylation is mediated mainly by a bacteria specific kinase showing no resemblance to eukaryotic-like tyrosine kinases [START_REF] Cozzone | Protein phosphorylation on tyrosine in bacteria[END_REF]. These kinases are grouped in a family named BY-kinases (Bacterial tYrosine kinases) (Figure 25) [START_REF] Grangeasse | Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology[END_REF]. The first endogenous substrate of a tyrosine kinase was identified in 2003: the UDP glucose deshydrogenase is activated by phosphorylation in B. subtilis [START_REF] Mijakovic | Transmembrane modulatordependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases[END_REF] and E. coli [START_REF] Grangeasse | Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase[END_REF]. The number of characterized bacterial tyrosine kinases and substrates has increased steadily and now is clear that serine/threonine and tyrosine phosphorylation largely affects bacterial physiology.

Bacteria employ at least two other phosphorylation systems that are not present in Eucarya: the two-component systems (TCS) and the phosphoenolpyruvate-dependent phoshotransferase system (PTS) (Figure 25) that are known as the major signaling systems in bacterial cells [START_REF] Klumpp | Phosphorylation and dephosphorylation of histidine residues in proteins[END_REF][START_REF] Beier | Regulation of bacterial virulence by two-component systems[END_REF]Deutscher et al., 2006). In the initial phase of research on bacterial protein phosphorylation, the kinases that came to the fore were the histidine/aspartate kinases of TCS. These systems involve a sensor kinase and a response regulator. The first TCS component, the histidine kinase is stimulated by a particular environmental or an intracellular signal, and it autophosphorylates on a key histidine residue using ATP as phosphate donor. Upon signal-induced autophosphorylation, these kinases can transfer the phosphate on an aspartate of the receiver domain of a response regulator. The response regulator is activated by phophorylation ultimately leading to activation or repression of transcription of specific genes [START_REF] Stock | Two-component signal transduction[END_REF]. To maintain a faithful flow of information through the signaling pathway, any given pair of histidine kinase and response regulator must selectively recognize each other, and must not cross-react with other TCS. In other words, the systems must be insulated via specific protein-protein interactions between the kinase and response regulator in a given TCS. Two-component systems are involved in a large number of cellular functions as chemotaxis [START_REF] Bischoff | Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis[END_REF], competence [START_REF] Weinrauch | A Bacillus subtilis regulatory gene product for genetic co,petence and sporulation resembles sensor protein members of the bacterial twocomponent signal-transduction systems[END_REF]), sporulation (Burbulus et al., 1991), virulence [START_REF] Leroux | Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant[END_REF], nitrogen assimilation [START_REF] Magasanik | Reversible phosphorylation of an enhancer binding protein regulates the transcription of bacterial nitrogen utilization genes[END_REF]. TCS are also integrated in complexes signal cascades or phosphorelay, notably involved in sporulation control and cellular cycle [START_REF] Hoch | Two-component and phosphorelay signal transduction[END_REF].

The second bacterial system is the phosphoenolpyruvate-dependent phoshotransferase system (PTS) (Figure 25) in which a phosphate group from phosphoenolpyruvate (PEP) is passed down a chain of specific proteins, all reversibly phosphorylated, and finally transferred to an incoming sugar. The PTS is thus primarily involved in translocation of sugars across the membrane, but phosphorylated forms of some or its components (HPr, EIIA) also have other regulatory roles in the cell [START_REF] Deutscher | P-Ser-HPr--a link between carbon metabolism and the virulence of some pathogenic bacteria[END_REF].

Other protein phosphorylation systems however exist; for example McsB, formerly thought to be a tyrosine kinase, was reported to be a protein arginine kinase [START_REF] Fuhrmann | McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR[END_REF] and opening up a new chapter in bacterial protein phosphorylation. Recently, 121 arginine phosphorylation sites from 87 proteins were identified in B. subtilis by phosphoproteomic analysis. It was also demonstrated that protein arginine phosphorylation is involved in the regulation of some critical cellular processes such as competence, motility, protein degradation and stress response (Elsholz et al., 2012). Moreover, some atypic enzymes catalyzing either both phosphorylation and dephosphorylation (IDHK/P and HPrK/P) or sharing no resemblance with enzymes of the systems described above, have been identified in some bacterial species. Altogether, this suggests that the number of enzymes dedicated to protein phosphorylation is likely underestimated in bacteria.

Phosphoproteomes are helpful to identify kinase-specific substrates. Several phophoproteome studies have been performed during the last 10 years and thank to technical progress especially in mass spectrometry, more and more phosphorylated proteins have been identified [START_REF] Macek | The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis[END_REF][START_REF] Soufi | Stable isotope labeling by amino acids in cell culture (SILAC) applied to quantitative proteomics of Bacillus subtilis[END_REF]Derouiche et al., 2012;Mijakovic and Macek, 2012). Additionally, interactomics identified kinase-protein interactions revealing not only potential substrates, but also anchors and modulators (Shi et al., 2014b). Therefore, phosphoproteomics and interactomics are powerfull and complementary approaches to provide an expanded view of kinases and phosphatase-centered signaling networks.

Serine/Threonine phosphorylation

Bacteria possess serine/threonine kinases, which are structurally similar to those found in Eukarya. This family of protein kinases has been initially defined by Hanks et al. (1988), who described its basic structural motifs. They are known as Hanks-type serine/threonine kinases or eukaryotic-like serine/threonine kinases (eSTKs). Their kinase domains are typically organized into 12 subdomains that fold in a characteristic two-lobed catalytic core structure, with the catalytic active site lying in a deep cleft formed between the two lobes. The structurally highly conserved kinase domain linked to a transmembrane helix, connecting to an extracellular sensing domain, whose structure can vary considerably. The extracellular domains are in general expected to bind ligands that trigger the kinase activity. The activation mechanism of bacterial Hanks-type kinases is most likely to involve kinase dimerization promoted by ligand binding [START_REF] Greenstein | Allosteric activation by dimerization of the PknD receptor Ser/Thr protein kinase from Mycobacterium tuberculosis[END_REF]. However, not all bacterial Hanks-type kinases are transmembrane proteins; they can also be cytoplasmic (Figure 26). One notable example is the kinase PknG from Mycobacterium tuberculosis, which is involved in survival inside macrophages, and mediates the sensing of nutritional stress [START_REF] Cowley | The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo[END_REF]. It is a cytosolic protein, with the core kinase domain flanked with an N-terminal Trx domain (involved in Redox sensing) and a C-terminal regulatory TPR domain. PknG can in fact be exported into the macrophages, and its autophosphorylation in the Trx domain is crucial for promoting M. tuberculosis survival [START_REF] Scherr | Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G[END_REF].

eSTKs are involved in the regulation of many aspects of the bacterial physiology including virulence, cell division, central and secondary metabolisms (Pereira et al., 2011). This versatility is notably reflected by the variety of subdomains that accompany the conserved catalytic kinase domain. Among these additional domains, a set of membrane eSTKs possesses the so-called PASTA motifs (for Penicillin binding proteins And Serine/Threonine kinase Associated) that bind β-lactam antibiotics and that are also found in the C-terminus of some class of B penicillinbinding proteins (PBPs) [START_REF] Yeats | The PASTA domain: a beta-lactam-binding domain[END_REF][START_REF] Jones | Evolution of transmembrane protein kinases implicated in coordinating remodeling of gram-positive peptidoglycan: inside versus outside[END_REF]. PASTA motifs are not found in Eukaryotes and are restricted to bacteria, notably to Firmicutes and Actinobacteria. Importantly, membrane eSTKs with PASTA motifs (PASTA-eSTKs) play a major role in the regulation of bacterial cell division and morphogenesis [START_REF] Manuse | Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis[END_REF].

Once autophosphorylated or phosphorylated by another kinase, activated kinases phosphorylate protein substrates, but their substrate specificity is very relaxed. Many bacterial eSTKs have been found to phosphorylate a large pool of substrates, and among the most promiscuous members of the family are the PrkC from B. subtilis [START_REF] Absalon | CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis[END_REF][START_REF] Pietack | In vitro phosphorylation of key metabolic enzymes from Bacillus subtilis: PrkC phosphorylates enzymes from different branches of basic metabolism[END_REF], PknB from Staphylococcus aureus (Lomas- [START_REF] Lomas-Lopez | Several enzymes of the central metabolism are phosphorylated in Staphylococcus aureus[END_REF], and several M. tuberculosis kinases: PknA, PknB, and PknD [START_REF] Villarino | Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions[END_REF][START_REF] Molle | The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation[END_REF][START_REF] Veyron-Churlet | Phosphorylation of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA regulates mycolic acid biosynthesis[END_REF].

For serine and threonine phosphorylation, bacteria mostly use eSTKs but also some atypical bi functional kinases/phosphorylases exist like the isocitrate dehydrogenase (IDH) or the HPr kinase/phosphorylase (HprK/P). Moreover, several other non-eSTKs have been described, including B. subtilis SpoIIAB, RsbT, and RsbW. SpoIIAB phosphorylates the anti-anti-sigma factor SpoIIAA on a Ser as part of a regulatory circuit that controls the activity of the sigma factor σ F in sporulation [START_REF] Najafi | Site of phosphorylation of SpoIIAA, the anti-anti-sigma factor for sporulation-specific sigma F of Bacillus subtilis[END_REF]. Although SpoIIAB is not an eSTK, its phosphatase partner SpoIIE is a eukaryote-like Ser/Thr phosphatase [START_REF] Duncan | Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division[END_REF]. RsbT and RsbW, which are members of the ATPase/kinase superfamily, phosphorylate their substrates on a Ser as part of a complex regulatory network that controls the activity of σ B , the major sigma factor involved in the stress response [START_REF] Yang | Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor[END_REF]. Eukaryotic-like serine/threonine kinases are either cytoplasmic or inserted in the membrane. Their kinase domain contains the 12 conserved signatures defined by Hanks. Both of them possess a variety of additional subdomains (illustrated by the blue and white striped ovals) that regulate their activity or that are proposed to influence their cellular localization and/or to favor their interaction with their substrates. They are able to be autophosphorylated on, but not exclusively, their activation loop (Adaptated from [START_REF] Garcia-Garcia | Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria[END_REF] (Pereira et al., 2015); 9 (Fisher et al., 1996); 10 (Min et al., 1993); 11 [START_REF] Kim | In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis[END_REF]; 12 [START_REF] Dufour | Interactions between a Bacillus subtilis anti-sigma factor (RsbW) and its antagonist (RsbV)[END_REF]; 13 [START_REF] Gaidenko | The PrpC serine-threonine phosphatase and PrkC kinase have opposing physiological roles in stationary-phase Bacillus subtilis cells[END_REF]; 14 [START_REF] Singh | Control of the phosphorylation state of the HPr protein of the phosphotransferase system in Bacillus subtilis: implication of the protein phosphatase PrpC[END_REF]; 15 [START_REF] Arigoni | SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis[END_REF]; 16 [START_REF] Yang | Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor[END_REF]; 17 [START_REF] Chen | Phosphorylation and RsbX-dependent dephosphorylation of RsbR in the RsbR-RsbS complex of Bacillus subtilis[END_REF]; 18 [START_REF] Brody | Catalytic function of an alpha/beta hydrolase is required for energy stress activation of the sigma (B) transcription factor in Bacillus subtilis[END_REF]; 19 (Martin-Vestraete et al.,1999) ; 20 (Pompeo et al., 2015) ; 21 [START_REF] Foulquier | PrkC-mediated phosphorylation of overexpressed Yvck protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells[END_REF].

Tyrosine phosphorylation

A majority of protein-tyrosine kinases identified and functionally characterized in bacteria belong to the family of bacterial protein-tyrosine kinases (BY-kinases) [START_REF] Grangeasse | Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology[END_REF].

BY kinases possess a transmembrane domain that can function as both an anchor and a sensor, and a cytosolic catalytic domain that can autophosphorylate as well as phosphorylate intracellular substrates (Figure 27). Their active site is situated in a catalytic domain capable of binding ATP and transferring its γ-phosphate to the hydroxyl group of tyrosine residues [START_REF] Lee | Structure of Escherichia coli tyrosine kinase Etk reveals a novel activation mechanism[END_REF][START_REF] Olivares-Illana | Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus[END_REF]. This domain is characterized by four structural motifs.

The The catalytic domain of BY-kinases requires an activator domain to reach its full catalytic potential. The activator domain enhances the ATP-binding affinity of the catalytic domain, and may assist in positioning the protein substrate [START_REF] Grangeasse | Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase[END_REF][START_REF] Mijakovic | Transmembrane modulatordependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases[END_REF]. MinD (Shi et al., 2014b;[START_REF] Gao | The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis[END_REF] or the transcriptional regulator SalA [START_REF] Garcia-Garcia | Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria[END_REF], which stimulate its kinase activity. The same is true for the two activators CapA1 and CapA2 that both activate the BY-kinase CapB2 of Staphylococcus aureus [START_REF] Gruszczyk | Comparative analysis of the Tyr-kinases CapB1 and CapB2 fused to their cognate modulators CapA1 and CapA2 from Staphylococcus aureus[END_REF].

Roles of protein phosphorylation in the regulation of B. subtilis cell differentiation

The Gram-positive bacterium B. subtilis is a model for studying phenotypic heterogeneity and transitional developmental programs in prokaryotes, as it can express distinct cell types associated with specific phenotypes: motility, genetic competence, biofilm formation, protease production, and sporulation. Spo0A is a key determinant for cell differentiation as mention above (Chapter 4). The Spo0A protein is activated by protein phosphorylation, which depends on the Kin family of sensor histidine kinases directly or indirectly via a phosphorelay (mediated by the phosphor transfer proteins Spo0F and Spo0B). These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of cell-fate decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine, threonine and tyrosine residues of protein substrates, has been proposed to play a role in bacterial signaling and cell differentiation [START_REF] Cousin | Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation[END_REF]. These phosphorylation reactions are generally catalyzed by Ser/Thr protein kinases (STPKs) and protein-tyrosine kinases (BY-kinases).

Role of protein phosphorylation in sporulation

In B. subtilis, four Ser/Thr kinases of the Hanks-type family have been characterized to date: PrkA, PrkC, PrkD (or YbdM), and YabT. All of them are implicated at different levels of the sporulation process: onset, dormancy, germination, and outgrowth (Figure 28). When the level of Spo0A~P is sufficient, the first compartment-specific transcription factor σ F is activated, definitely engaging the sporulating cell into a specific program of genes expression [START_REF] Tan | Spore formation in Bacillus subtilis[END_REF]. The activity of σ F is controlled by the anti-sigma factor SpoIIAB (a serine kinase) which binds and inhibits σ F (Figure 12), but is also capable to phosphorylate the antianti-sigma factor SpoIIAA. SpoIIAB binding specificity is strongly dependent on ATP/ADP ratio.

I. INTRODUCTION 60

SpoIIAB binds to σ F in the presence of ATP, but ADP favours binding to SpoIIAA and release of an active σ F . SpoIIAA can be phosphorylated by SpoIIAB in the presence of ATP, resulting in σ F inhibition, as SpoIIAA~P cannot bind to SpoIIAB [START_REF] Alper | An adenosine nucleotide switch controlling the activity of a cell typespecific transcription factor in B. subtilis[END_REF]. The serine phosphatase SpoIIE preferentially desphosphorylates SpoIIAA~P in the forespore, allowing dephosphorylated SpoIIAA to bind to SpoIIAB, which permits σ F activation [START_REF] Duncan | Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division[END_REF]. Recently, SpoIIAB was found to be efficiently phosphorylated by the Hanks-type kinases PrkD and YabT. Specifically, SpoIIAB is phosphorylated by PrkD at serine 13. This residue is located on the interface interacting with the anti-anti-sigma factor SpoIIAA suggesting alternative route to destabilize the SpoIIAA/SpoIIAB complex, and its phosphorylation is therefore likely to have a regulatory role (Shi et al., 2014a). In the schematic spore, the core is in yellow, the cortex in green and the coat in gray. The two types of spore signal receptors are represented: the GerA, B, and K in sticks, the STPK PrkC in balls and stick (transmembrane domain). The two-component cascade leading to Spo0A phosphorylation is presented in the framed rectangle and regulations by STPKs are listed in the white rectangles. Other possible cross-regulations by two-component systems, BY-kinases, or other phosphorylations are not represented here in order to not overload the drawing (Pompeo et al., 2016) Interestingly, the expression of two genes encoding the STPKs of the Hanks-type family PrkA and YabT increases strongly during sporulation under the control of the spore-specific sigma factors, σ E and σ F , respectively. These two kinases have been indeed shown to participate in the regulation of several mechanisms occurring during the initiation of sporulation (Bidnenko et al., 2013;[START_REF] Yan | Eukaryote-like Ser/Thr protein kinase PrkA modulates sporulation via regulating the transcriptional factor σ(K) in Bacillus subtilis[END_REF]Pompeo et al., 2016).

PrkA is a STPK that only possesses a catalytical domain and localizes in the coat of the forespore [START_REF] Eichenberger | The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis[END_REF]. This protein also shows a distant homology to eukaryotic cAMPdependent protein kinases and several essential residues of their active site are apparently conserved in PrkA. Using a B. subtilis crude extract, it has been proposed that PrkA phosphorylates an unidentified 60-kDa protein on Ser residue(s) [START_REF] Fischer | Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase[END_REF]. However, no PrkA autophosphorylation was detected. That is surprising since STPKs generally need to be autophosphorylated to be active. However, even if the enzymatic properties of PrkA are poorly characterized, the role of this protein in sporulation seems clearly established. Actually, deletion of the prkA gene leads to a sporulation defect corresponding to a delay in the entry into sporulation and a decrease in the number of spores. It has recently been shown that PrkA was involved in the synthesis of the σ K transcription factor [START_REF] Yan | Eukaryote-like Ser/Thr protein kinase PrkA modulates sporulation via regulating the transcriptional factor σ(K) in Bacillus subtilis[END_REF]. Indeed, PrkA increases the expression of σ K and its downstream target genes, by inhibiting the negative transcriptional regulator ScoC (Hpr). However, the complete mechanism of this regulation, potentially via the kinase activity of PrkA, is not known.

The second regulatory kinase YabT is an unusual Hanks-type STPK that has no extracellular sensing domain (Figure 29). Its transmembrane domain anchors it to the membrane and YabT concentrates at the asymetrical septum during the sporulation (Bidnenko et al., 2013). Between the transmembrane helix and the catalytic domain, there is a lysine and arginine-rich domain, which can bind DNA. YabT is the first bacterial Hanks kinase that exhibits DNA-binding capacity.

DNA binding is not sequence specific and it activates the kinase, which in turn phosphorylates the general recombinase RecA (phosphorylation on Ser 2) in vitro (Bidnenko et al., 2013).

Consistently, RecA was previously identified in a phosphoproteome study revealing the same phosphorylated residue [START_REF] Soufi | Stable isotope labeling by amino acids in cell culture (SILAC) applied to quantitative proteomics of Bacillus subtilis[END_REF]. Interestingly, single-stranded DNA is more efficient in activating the kinase, suggesting that YabT activation may be related to DNA damage.

Phosphorylation of the recombinase stimulates the formation of mobile RecA foci, which are important for spore development under DNA-damaging conditions. As for prkA, deletion of the yabT gene leads to a sporulation delay. Furthermore, resistance to DNA damages decreases in the yabT mutant spores. Both phenotypes were also observed in a recA mutant [START_REF] Shafikhani | Hpr (ScoC) and the phosphorelay couple cell cycle and sporulation in Bacillus subtilis[END_REF]Bidnenko et al., 2013). In most cells, the mobile RecA foci disassemble just before the completion of the mature spore. In cells with extensive chromosomal damage, RecA foci persist, and by a presently unknown mechanism, prevent the production of mature spores (Bidnenko et al., 2013). Thus, YabT regulates RecA activity in the forespore in order to allow DNA damage repair before nucleoid compaction in thespore [START_REF] Sciochetti | Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant[END_REF]. Similarities between the bacterial STPK YabT and the eukaryotic STPKs C-Abl and Mec1 have been found: all these kinases are activated by DNA and phosphorylate proteins involved in DNA damage repair mechanisms (Bidnenko et al., 2013).

In vitro, YabT is also able to phosphorylate RacA, another DNA-related protein involved in DNA anchoring to the cell pole (Ben-Yehuda et al., 2003;Shi et al., 2014b). RacA can be dephosphorylated in vitro by SpoIIE, a serine protein phosphatase known to modulate the phosphorylation state of the anti-anti σ F factor SpoIIAA as I mentioned before. Interestingly, in a yeast two-hybrid screen, YabT and SpoIIE have been found associated to the same protein partners suggesting that they function as a kinase/phosphatase couple during sporulation (Shi et al., 2014b). Another potential substrate of YabT is the global transcriptional regulator AbrB 2012). (B) YabT and RecA are involved in checking chromosome integrity during spore formation. YabT (in red) is synthesized 2-3 h after the onset of sporulation. It localizes to the septum, where it encounters and binds DNA. Activated YabT phosphorylates RecA (green), whichs forms a mobile focus moving along the chromosome. If nonreparable damage is encountered, the RecA focus is maintained 6 h after initiation of sporulation and this is incompatible with the formation of a mature spore. If no irreparable damage is encountered, RecA focus is dissassembled, and sporulation proceeds normally [START_REF] Cousin | Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation[END_REF] A B which is phosphorylated on Ser86 in vivo [START_REF] Soufi | Stable isotope labeling by amino acids in cell culture (SILAC) applied to quantitative proteomics of Bacillus subtilis[END_REF] as well as in vitro by YabT, PrkC and PrkD (Kobir et al., 2014). AbrB is a global gene regulator involved in transition phases (i.e., from exponential to stationary growth phase) that also antagonizes sporulation by repressing the expression of Spo0A [START_REF] Phillips | Bacillus subtilis sporulation and stationary phase gene expression[END_REF]. It has been proposed that AbrB phosphorylation serves as an additional input for a subtle control of AbrB activity. Indeed, AbrB phosphorylation inhibits its ability to bind its DNA targets (Kobir et al., 2014). In addition, a strain expressing phosphomimetic AbrB produces fewer spores and sporulates much slower. Because the YabT kinase is produced just after the onset of sporulation, it is the best candidate for AbrB phosphorylation in sporulation conditions.

During the dormancy of spores, regulation of enzymatic activities and protein synthesis by phosphorylation reactions also exist. When the mature spore is released by the mother cell, it is metabolically dormant and environmentally resistant. The spore is protected by thick layers: the cortex and the coat, and contains a high level of dipicolinic acid (DPA) and a low amount of water. However, it has been recently shown that the spore RNA profile is highly dynamic a few days following sporulation [START_REF] Segev | RNA dynamics in aging bacterial spores[END_REF]. During this short period, spores are responsive to environmental changes and can adapt their RNA content consequently. For example, the overall metabolism is down regulated, in particular protein synthesis, which is an energyintensive cellular process. This regulation is mediated by phosphorylation of the elongation factor Tu (EF-Tu). Indeed, phosphorylated EF-Tu is unable to hydrolyze GTP and remains bound to the ribosome which leads to a dominant negative effect in elongation, thus inhibiting protein synthesis (Pereira et al., 2015). It has been proposed that the kinase involved in this phosphorylation is YabT because it is present in the spore during dormancy and it is able to phosphorylate EF-Tu in vitro on Thr63. In vivo experiments confirmed that YabT is responsible of EF-Tu phosphorylation in the spore, since no phosphorylated EF-Tu was found in a yabT mutant (Pereira et al., 2015). Moreover, in vitro phosphorylation of EF-Tu by PrkC was previously reported on Thr384 but the in vivo regulatory function of this phosphorylation has not been unraveling so far [START_REF] Absalon | CpgA, EF-Tu and the stressosome protein YezB are substrates of the Ser/Thr kinase/phosphatase couple, PrkC/PrpC, in Bacillus subtilis[END_REF].

Spores of B. subtilis can remain dormant for years but return to life quickly after exposure to nutrients or muropeptides [START_REF] Setlow | Spore germination[END_REF]. PrkC is a classical Hanks-type kinase that has a cytosolic catalytic domain linked via a transmembrane domain to the extracellular PASTA sensing domains. One of the expression peaks of prkC is detectable during spore germination (Figure 30.A). At this time, extracellular domain of PrkC can bind the muropeptides (fragments derived from cell walls of dividing cells), and this triggers the kinase activity (Figure 30

.B).

Activated PrkC phosphorylates the essential ribosomal GTPase EF-G, and this is strongly stimulated by the presence of muropeptides (Shan and Dworkin, 2010). It has been proposed that PrkC phosphorylates EF-G in the spore to allow re-initiation of protein synthesis. But, it is unlikely that this phosphorylation is the only cause of germination. PrkC also promotes the expression of yocH, a muralytic enzyme encoding gene. YocH is exported and digests the peptidoglycan (PG) of growing bacteria, thus producing more muropeptides that in turn stimulate germination [START_REF] Shah | Induction and regulation of a secreted peptidoglycan hydrolase by a membrane Ser/Thr kinase that detects muropeptides[END_REF][START_REF] Libby | The eukaryotic-like Ser/Thr Kinase PrkC regulates the essential WalRK two-component system in Bacillus subtilis[END_REF]. Moreover, the phosphorylation of several proteins has been shown to be important for germination. These include the spore specific proteins SspA and SspB involved in DNA protection, the ribosomal protein RpsJ, the elongation factors EF-Tu and EF-G, and the phosphocarrier protein HPr [START_REF] Rosenberg | Phosphoproteome dynamics mediate revival of bacterial spores[END_REF]. But, for many of them, the kinase that catalyzes their phosphorylation is still unknown. Muropeptides derived from peptidoglycan of growing bacteria signal the return of favorable growth conditions. YocH generates muropeptides by cleaving the peptidoglycan and these fragments are bound by PASTA domains of PrkC. The activated kinase then phosphorylates the cellular substrates, including EF-G, which presumably stimulates protein synthesis and germination. PrkC also activates yocH transcription in a phosphorylation-dependent manner, and this reinforces the forward-feeding regulatory loop [START_REF] Cousin | Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation[END_REF].

A B

Therefore, the regulation of sporulation and spore revival by phosphorylation on Ser and Thr residues is an important mechanism that can be mediated by several types of kinases like STPKs and HprK/P or even other atypical protein kinases not yet identified. This network of regulations may also be complicated by cross-talk with other phosphorylation systems like BY-kinases [START_REF] Cousin | Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation[END_REF], two-component systems, or recently identified Arg phosphorylations.

Role of protein phosphorylation in biofilm formation

Biofilms are multicellular communities of bacteria with highly complex structures and distinct morphological features (Chapter 4). Recent studies in B. subtilis suggest that serine, threonine and mainly tyrosine phosphorylation plays an important role in the regulation of biofilm formation, in addition to the well-known mechanisms of transcriptional regulation and proteinprotein interactions (Vlamakis et al., 2013;[START_REF] Mielich-Süss | Molecular mechanisms involved in Bacillus subtilis biofilm formation[END_REF][START_REF] Mhatre | From environmental signals to regulators: modulation of biofilm development in Gram-positive bacteria[END_REF].

The two component system DegS/DegU, which influences expression of genes involved in motility and complex colony formation is also regulated via serine phosphorylation. Indeed, DegS is phosphorylated in its input domain by the Hanks-type Ser/Thr kinase PrkD. This kinase is cytosolic and it was shown in vitro to phosphorylate DegS at the residue serine 76.

Phosphorylation of DegS by PrkD increases efficiency of phosphotransfer to DegU, which in turn modulates its transcriptional activity [START_REF] Jers | Bacillus subtilis two-component system sensory kinase DegS is regulated by serine phosphorylation in its input domain[END_REF].

BY-kinases play also important roles in the biosynthesis of secreted polysaccharides in bacteria.

B. subtilis possesses two BY-kinases, originally known as YwqD and YveL, which were renamed PtkA and PtkB, respectively [START_REF] Mijakovic | Protein-tyrosine phosphorylation in Bacillus subtilis[END_REF] and their modulator TkmA and TkmB (EpsA), respectively. In B. subtilis EPS biosynthesis is controlled, at the postranslational level, by the BYkinase PtkB and its activator, TkmB, in addition to the known regulation of the epsA-epsO operon at the transcriptional level (Gerwing et al., 2014). A recent study proposed a feedback mechanism for the regulation of EPS biosynthesis in B. subtilis [START_REF] Elsholz | Self-regulation of exopolysaccharide production in Bacillus subtilis by a tyrosine kinase[END_REF]. According to the study, EPS interacts with the extracellular domain of TkmB, which is a receptor, to control PtkB kinase activity. In the absence of EPS, the kinase is inactivated by autophosphorylation. The presence of EPS inhibits autophosphorylation and promotes the phosphorylation of a glycosyltransferase EpsE, belonging to the EPS biosynthetic pathway, thereby stimulating the production of EPS. B. subtilis also possesses a second BY-kinase/kinase activator pair, PtkA and TkmA, respectively. The genes for the two proteins are located in a presumptive operon with two other genes, ptpZ and ugd (Figure 31). The ptpZ gene encodes a phosphatase that counteracts the activity of BY-kinase by dephosphorylating the kinase as well as the targets of the kinase; ugd encodes a UDP-glucose dehydrogenase (Ugd). The biological functions of TkmA, PtkA, and PtpZ have been investigated in previous studies. It was shown that the protein targets of the PtkA kinase include Ugd and TuaD (involved in teichuronic acid biosynthesis), as well as the single-strand DNA-binding proteins SsbA and SsbB [START_REF] Mijakovic | Protein-tyrosine phosphorylation in Bacillus subtilis: a 10-year retrospective[END_REF]Mijakovic et al., 2006).

Recent studies also emphasised the roles of TkmA and PtkA in biofilm formation. The kinase activator TkmA contributes significantly to biofilm formation but largely independently of its cognate kinase, PtkA [START_REF] Gao | The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis[END_REF]. Further, the biofilm defect caused by a ΔtkmA mutation can be rescued by complementation by epsA, suggesting a functional overlap between TkmA and TkmB and providing a possible explanation for the role of TkmA in biofilm formation (Figure 32). The importance of TkmA in biofilm formation depends largely on medium conditions; the tkmA deletion mutant had a very severe biofilm defect in the biofilm medium LBGM. By contrast, the defect was marginal in another commonly used biofilm medium, MSgg. The molecular basis for the medium dependence is likely due to differential expression of tkmA and epsA in the two different media and to complex regulation of these genes by both Spo0A and DegU. This study provides genetic evidence for possible cross talk between a BY-kinase activator (TkmA) and a noncognate kinase (PtkB) and an example of how environmental conditions may influence such cross-talk in regulating biofilm formation in B. subtilis [START_REF] Gao | The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis[END_REF]. Interestingly, recent studies also showed broad cross-phosphorylation among BY-kinases and other types of kinases (e.g., Ser/Thr kinases), as well as overlapping activities among different pairs of BY-kinases and kinase activators. Interactomics data also suggest the ability of TkmA/PtkA and TkmB/PtkB to switch partners, at least at the protein-protein interaction level. Strong interactions between

TkmA and PtkB and between TkmA and PtkA but not between TkmB and PtkA were observed (Figure 32.A) (Shi et al., 2014a). All these studies imply that the complex regulation and biological significance of the BY-kinases may have been underestimated.

Most bacterial protein kinases were found to be specific for one protein substrate, therefore substrate promiscuity of PtkA was initially surprising to some degree. However, taking into et al., 2014 and[START_REF] Gao | The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis[END_REF].

B A

account that eukaryal protein kinases usually phosphorylate a large number of proteins and the high number of phosphoproteins detected in bacterial phosphoproteomes, relaxed kinase specificity is not entirely unexpected. Phosphoproteomics studies mapped an increasing number of tyrosine-phosphorylated proteins. In vitro studies indicated that PtkA can indeed phosphorylate many of them (Jers et al., 2010;[START_REF] Mijakovic | Protein-tyrosine phosphorylation in Bacillus subtilis: a 10-year retrospective[END_REF] and the consequences of PtkA-dependent phosphorylation vary considerably depending on its substrate (Figure 33). Furthermore, MinD and SalA were revealed as PtkA interactants and they also exhibit the capacity to activate PtkA function (Shi et al., 2014b). Thus, it has been proposed that TkmA, MinD, and SalA represent three alternative anchoring points for PtkA, directing its localization and activity toward different substrates throughout the cell cycle.

Bacterial serine/threonine and tyrosine kinases also form complex cascades of mutual activation, in which one kinase phosphorylates another (cross-phosphorylation), and which serve as signal integration and amplification devices, like the Eukaryal kinases (Baer et al., 2014;Shi et al., 2014a). The Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases.

Role of protein phosphorylation in the regulation of cell cycle and DNA-related processes in bacteria

In this part presented as a review paper, some recent information about the role of protein phosphorylation involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria are discussed. Recent phosphoproteomics and interactomics studies reveal an increasing number of bacterial proteins phosphorylated on Ser/Thr/Tyr involved in a variety of cellular processes related to DNA metabolism. These data suggest the existence of many regulatory pathways controlled by phosphorylation during DNA replication, segregation and cell division. This review highlights the growing evidence for phosphorylation of proteins implicated in many DNA-related processes and the existence of scaffold proteins that act as signaling integrators by facilitating interaction and co-localization of kinases and their targets. Similar to eukaryotes, bacterial scaffolds could offer versatile strategies to dynamically coordinate cell processes via signaling

INTRODUCTION

In all living cells, many cellular processes are controlled through the reversible phosphorylation of proteins on serine, threonine and tyrosine (Ser/Thr/Tyr) which results from the opposing action of kinases and phosphatases. While the role of Ser/Thr and Tyr -protein kinases (STYKs) and phosphatases in cellular regulations is extensively documented in eukaryotes, the Ser/Thr/Tyr signaling network in bacteria has a more recent history [START_REF] Hunter | Signaling-2000 and beyond[END_REF]Pereira et al., 2011;[START_REF] Dworkin | Ser/Thr phosphorylation as a regulatory mechanism in bacteria[END_REF][START_REF] Kalantari | Serine/ threonine/tyrosine phosphorylation regulates DNA-binding of bacterial transcription regulators[END_REF][START_REF] Manuse | Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis[END_REF]. Analysis of microbial genomes revealed that Hanks-type Ser/Thr kinases (eSTKs) and phosphatases (eSTPs) are also widespread in bacteria [START_REF] Zhang | Genomewide survey of putative serine/threonine protein kinases in cyanobacteria[END_REF][START_REF] Zhu | Contribution of eukaryotic-type serine/threonine kinase to stress response and virulence of Streptococcus suis[END_REF]. However, prokaryotic Tyr phosphorylation is carried out by a different family of enzymes, the bacterial tyrosine kinases or BY-kinases, which do not share the structural homology with eukaryotic STYKs. Instead, BY-kinases possess structural features characteristic of P-loop ATPases. BY-kinases as well as eSTKs have been shown to regulate cell-cycle events, cell adaptation to environmental cues, and virulence in many species [START_REF] Wright | Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens[END_REF][START_REF] Zhu | Contribution of eukaryotic-type serine/threonine kinase to stress response and virulence of Streptococcus suis[END_REF][START_REF] Kalantari | Serine/ threonine/tyrosine phosphorylation regulates DNA-binding of bacterial transcription regulators[END_REF].

Phosphorylation provides a sensitive and dynamic way to regulate protein activity, stability, protein interaction and subcellular localization. Global regulators are often targeted by more than one kinase, altering the expression of many genes. The phosphorylation of the transition state transcription regulator AbrB of Bacillus subtilis by three Ser/Thr kinases, PrkC, PrkD, and YabT, leads to the deregulation of numerous target genes during the transition of vegetative to stationary growth (Kobir et al., 2014). In the human pathogen bacteria Staphylococcus aureus, the phosphorylation of the global regulator MgrA also modulates gene expression in a growth phase-dependent manner [START_REF] Truong-Bolduc | Posttranslational modification influences the effects of MgrA on norA expression in Staphylococcus aureus[END_REF]. By adding a local negative charge, phosphorylation can positively or negatively affect the DNA binding of transcriptional factors to their regulatory sequences, thus participating in eliciting the cellular response. A recent example of tyrosine phosphorylation-mediated positive regulation is illustrated by the regulator SalA, which undergoes a conformational change upon phosphorylation at tyrosine 327 that enhances its DNA binding affinity for scoC upstream regulatory sequences (Derouiche et al., 2015a). Conversely, phosphorylation of the virulence regulator SarA of S. aureus at serine 75 within the HTH-DNA binding region negatively modulates its ability to bind DNA leading to the negative regulation of gene expression [START_REF] Didier | Phosphorylation of the virulence regulator SarA modulates its ability to bind DNA in Staphylococcus aureus[END_REF].

It was also observed that modulation by phosphorylation of the DNA binding of transcriptional factors plays a role in the regulation of chromosomal replication. The two component regulators MtrA of Mycobacterium tuberculosis and CtrA of Caulobacter Crescentus are illustrative examples that phosphorylation acts to temporally restrain access of the replication initiator protein DnaA to the origin DNA sequence oriC [START_REF] Didier | Phosphorylation of the virulence regulator SarA modulates its ability to bind DNA in Staphylococcus aureus[END_REF][START_REF] Pini | Cell Cycle Control by the master regulator CtrA in Sinorhizobium meliloti[END_REF]. A similar strategy was proposed to account for the role of the master regulator Spo0A in negatively controlling DNA replication initiation by inhibiting the DnaA-dependent DNA duplex unwinding during sporulation in Bacillus subtilis (Castilla-Llorente et al., 2006;[START_REF] Xenopoulos | Regulation of growth of the mother cell and chromosome replication during sporulation of Bacillus subtilis[END_REF]Boonstra et al., 2013). Hence, taking advantage of the versatility of transcription factors combined with the limited time frame during which they contact their regulatory sequence, bacterial cells have evolved multifunctional regulatory proteins able to act in both gene expression and replication initiation control.

However, little is known about the role of phosphorylation in directly regulating the DNA-binding activity of proteins involved in other DNA-mediated processes. Large-scale phosphoproteome studies have been conducted in bacteria, covering gram-negative as well as gram positive species [START_REF] Macek | The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis[END_REF][START_REF] Macek | Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation[END_REF][START_REF] Voisin | The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases[END_REF][START_REF] Soufi | The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins[END_REF][START_REF] Ravichandran | Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species[END_REF][START_REF] Prisic | Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases[END_REF][START_REF] Schmidl | The phosphoproteome of the minimal bacterium Mycoplasma pneumoniae: analysis of the complete known Ser/Thr kinome suggests the existence of novel kinases[END_REF][START_REF] Ge | Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins[END_REF][START_REF] Manteca | Phosphoproteome analysis of Streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation[END_REF][START_REF] Misra | Analysis of the serine/threonine/tyrosine phosphoproteome of the pathogenic bacterium Listeria monocytogenes reveals phosphorylated proteins related to virulence[END_REF][START_REF] Bai | Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum[END_REF]Elsholz et al., 2012;[START_REF] Esser | Change of carbon source causes dramatic effects in the phospho-proteome of the archaeon Sulfolobus solfataricus[END_REF][START_REF] Hansen | The Escherichia coli phosphotyrosine proteome relates to core pathways and virulence[END_REF][START_REF] Soares | Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium[END_REF][START_REF] Yang | Global phosphoproteomic analysis reveals diverse functions of serine/threonine/tyrosine phosphorylation in the model cyanobacterium Synechococcus sp. strain PCC 7002[END_REF][START_REF] Kennelly | Protein Ser/Thr/Tyr phosphorylation in the Archaea[END_REF][START_REF] Ortega | Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch[END_REF][START_REF] Ravikumar | Quantitative phosphoproteome analysis of Bacillus subtilis reveals novel substrates of the kinase PrkC and phosphatase PrpC[END_REF][START_REF] Nakedi | Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG[END_REF][START_REF] Pan | dbPSP: a curated database for protein phosphorylation sites in prokaryotes[END_REF]. These databases reveal an increasing number of bacterial proteins phosphorylated on Ser/Thr/Tyr, involved in a variety of cellular processes related to DNA metabolism [START_REF] Pan | dbPSP: a curated database for protein phosphorylation sites in prokaryotes[END_REF]. Recently, a protein-protein interaction (PPI) network centered on B. subtilis S/T/Y protein kinases and phosphatases emerged as a powerful tool in bacterial signal transduction research, highlighting the existence of many regulatory pathways controlled by phosphorylation during DNA replication, chromosomal segregation and cytokinesis (Shi et al., 2014b). As with transcription factors, we can anticipate that phosphorylation could modulate protein-binding properties to DNA as well as to other proteins. Therefore, a great challenge is now to understand the role of protein phosphorylation in the coordination and integration of the different DNA processes during the bacterial cell cycle. In this review we will focus on novel aspects of regulation in bacteria that resemble those taking place in eukaryotes. We will highlight the growing evidence for phosphorylation of proteins involved in many DNA-related processes and the existence of scaffold proteins that act as signaling integrators by facilitating interaction and co-localization of kinases and their targets.

IDENTIFICATION OF PHOSPHO-PROTEINS INVOLVED IN DNA-RELATED PROCESSES IN BACTERIA

In living cells, the genome is continuously exposed to endogenous and exogenous damaging agents. If not repaired, the replication of damaged chromosomes can cause fork stalling or collapse and subsequent genome instability and possibly cell death [START_REF] De Septenville | Replication fork reversal after replication-transcription collision[END_REF][START_REF] Merrikh | Replicationtranscription conflicts in bacteria[END_REF][START_REF] Cortez | Preventing replication fork collapse to maintain genome integrity[END_REF][START_REF] Dungrawala | The Replication checkpoint prevents two types of fork collapse without regulating replisome stability[END_REF]. Sensing and repairing DNA damage is therefore necessary to ensure chromosome integrity. Upon detection of damage, eukaryotic cells relay information through signal transduction cascades to coordinate a biological response including cell cycle arrests and DNA repair pathways [START_REF] Iyer | Checkpoint regulation of replication forks: global or local?[END_REF]. These signaling cascades generally involve the successive activation of protein kinases, punctuated by the phosphorylation of protein effectors of DNA damage checkpoint and processing (Subramanian and Hochwagen, 2014). These checkpoints involve sensor proteins that identify DNA-related disorders, converting them into phosphorylation events that modulate the functions of specific target proteins. In eukaryotes, the evolutionary conserved ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad 3related) Ser/Thr kinases are considered a paradigm for such transduction cascades [START_REF] Cimprich | ATR: an essential regulator of genome integrity[END_REF]. These two master regulators act in response to double strand breaks (ATM), as well as in replication initiation control and repair of damaged forks (ATR) to coordinate cell cycle, repair, and replication (Shechter et al., 2004a,b;[START_REF] Cimprich | ATR: an essential regulator of genome integrity[END_REF][START_REF] Lee | Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response[END_REF].

In most bacteria, the replication of the circular chromosome requires the coordinated action of conserved proteins to perform the initiation, elongation and termination steps [START_REF] Langston | DNA replication: keep moving and don't mind the gap[END_REF][START_REF] Langston | Whither the replisome: emerging perspectives on the dynamic nature of the DNA replication machinery[END_REF]. Initiation is mediated by the replication initiator protein DnaA, which binds to specific origin sequences. This forms a nucleoprotein-structure leading to the unwinding of the duplex DNA for subsequent loading of the replication machinery or replisome. A bacterial replisome is usually composed of a type III DNA polymerase, a DNA helicase, and a sliding clamp with an associated clamp loading protein complex. During elongation, the replication factory encounters numerous road blocks or damage that can cause fork arrests [START_REF] Pomerantz | What happens when replication and transcription complexes collide?[END_REF][START_REF] Mettrick | Stability of blocked replication forks in vivo[END_REF]. As in eukaryotes, mechanisms are required to repair and/or restart the ongoing replication fork to preserve genomic integrity [START_REF] Merrikh | Replicationtranscription conflicts in bacteria[END_REF]. In all living organisms, chromosome replication must be coordinated with other cellular pathways [START_REF] Sclafani | Cell cycle regulation of DNA replication[END_REF].

In bacteria, however, whether phosphorylation events play a role in sensing replication stresses remains to be ascertained. Recent studies highlight kinase activation by crossphosphorylation in B. subtilis and M. tuberculosis, suggesting the existence of bacterial signaling cascades comparable to those found in eukaryotes (Baer et al., 2014;Shi et al., 2014a). Although the complement of proteins targeted by these potential regulatory cascades are not yet known, increasing numbers of large scale mass spectrometry-based proteomics studies find that proteins involved in various DNA-related processes are also phosphorylated in bacteria [START_REF] Macek | The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis[END_REF][START_REF] Macek | Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation[END_REF][START_REF] Voisin | The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases[END_REF][START_REF] Soufi | The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins[END_REF][START_REF] Ravichandran | Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species[END_REF][START_REF] Prisic | Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases[END_REF][START_REF] Schmidl | The phosphoproteome of the minimal bacterium Mycoplasma pneumoniae: analysis of the complete known Ser/Thr kinome suggests the existence of novel kinases[END_REF][START_REF] Ge | Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins[END_REF][START_REF] Manteca | Phosphoproteome analysis of Streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation[END_REF][START_REF] Misra | Analysis of the serine/threonine/tyrosine phosphoproteome of the pathogenic bacterium Listeria monocytogenes reveals phosphorylated proteins related to virulence[END_REF][START_REF] Bai | Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum[END_REF]Elsholz et al., 2012;[START_REF] Esser | Change of carbon source causes dramatic effects in the phospho-proteome of the archaeon Sulfolobus solfataricus[END_REF][START_REF] Hansen | The Escherichia coli phosphotyrosine proteome relates to core pathways and virulence[END_REF][START_REF] Soares | Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium[END_REF][START_REF] Yang | Global phosphoproteomic analysis reveals diverse functions of serine/threonine/tyrosine phosphorylation in the model cyanobacterium Synechococcus sp. strain PCC 7002[END_REF][START_REF] Kennelly | Protein Ser/Thr/Tyr phosphorylation in the Archaea[END_REF][START_REF] Ortega | Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch[END_REF][START_REF] Ravikumar | Quantitative phosphoproteome analysis of Bacillus subtilis reveals novel substrates of the kinase PrkC and phosphatase PrpC[END_REF][START_REF] Nakedi | Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG[END_REF][START_REF] Pan | dbPSP: a curated database for protein phosphorylation sites in prokaryotes[END_REF]; Table 1). Another approach, based on yeast two-hybrid detection of protein binding partners of B. subtilis protein kinase and phosphatase, identifies numerous potential DNA-binding protein substrates, some of them already characterized as phosphoproteins in other studies, or further validated in vivo and/or in vitro (Shi et al., 2014b; Table 2, Figure 1). This study lays the foundation for investigating the role of phosphorylation in modulating DNA-related processes in this bacteria.

An example of phosphorylated multifunctional protein playing a role in various DNA processes is the single-stranded DNA-binding protein SSB (Mijakovic et al., 2006;[START_REF] Vujaklija | Detecting posttranslational modifications of bacterial SSB proteins[END_REF]. In bacteria, SSB proteins assemble onto DNA as homodimers or tetramers, each unit composed of an N-terminal DNA binding domain and a C-terminal intrinsically disordered tail that mediates interaction with numerous proteins [START_REF] Kozlov | Intrinsically disordered C-terminal tails of E. coli single-stranded DNA binding protein regulate cooperative binding to single-stranded DNA[END_REF]. The bacterial SSB proteins are mainly associated with the replication machinery at the replicating forks and serve as central hubs to coordinate DNA replication and repair [START_REF] Lecointe | Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks[END_REF][START_REF] Costes | The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks[END_REF][START_REF] Antony | Multiple C-terminal tails within a single E. coli SSB homotetramer coordinate DNA replication and repair[END_REF][START_REF] Bentchikou | Are the SSB-interacting proteins RecO, RecG, PriA and the DnaBinteracting protein rep bound to progressing replication forks in Escherichia coli?[END_REF]. As in bacteria, the eukaryotic single strand binding protein RPA has multiple roles in protecting ssDNA, sensing and promoting repair of DNA damage via PPIs [START_REF] Oakley | Replication protein A: directing traffic at the intersection of replication and repair[END_REF][START_REF] Maréchal | RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response[END_REF]. In particular, phosphorylation of RPA negatively regulates its binding to DNA as well as its interaction with protein partners [START_REF] Binz | The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction[END_REF][START_REF] Oakley | RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions[END_REF]. B. subtilis possesses two single strand binding proteins SSBA and B, respectively phosphorylated at a tyrosine, identified as Tyr82 in SSBA (Mijakovic et al., 2006). Additionally, a phospho-site at Thr52 was also identified in SSBB (Table 1; Jers et al., 2010;Elsholz et al., 2012). Examination of the 3D-structure of the SSB/DNA complex shows that Tyr82 (as well as the Thr52 neighbor residue Trp54) are involved in stacking with DNA [START_REF] Yadav | Genetic recombination in Bacillus subtilis: a division of labor between two single-strand DNA-binding proteins[END_REF]. This strongly supports the observation that phosphorylation of SSBA at residue Tyr82 negatively modulates its binding onto DNA in vitro and regulates the cell response to DNA damage (Mijakovic et al., 2006). Therefore, the role of SSB protein phosphorylation in modulating the cellular DNA damage response might be evolutionarily conserved in eukaryotes and in prokaryotes.

Another strong case was made for the interaction of the DNA replicative helicase DnaC by the Ser/Thr kinase PrkD, validated by in vitro assays and high correlation of co-expressions (Shi et al., 2014b). Phosphorylation of numerous components of the DNA replication machinery, as well as of proteins involved in the different steps of the replication cycle, have been identified in one or more bacteria among Escherichia, Pseudomonas, Helicobacter, Camplylobacter, Listeria, Bacillus, Mycoplasma, Clostridium, Deinoccocus, Synechoccus, and Streptococcus genus (Tables 1,2). Orthologs of the highly conserved DNA polymerase PolA, DNA helicase DnaC and single stranded binding protein SSB were found phosphorylated, potentially reflecting the existence of generic regulatory mechanisms to control their activity across species.

The conservation of phosphorylation within protein domain families is indicative of proteins with high regulatory potential [START_REF] Maathuis | Conservation of protein phosphorylation sites within gene families and across species[END_REF]. Remarkably, several phosphoproteomic analyses have reported phosphorylation of the two largest subunits β and β' of the RNA polymerase machinery at serine, threonine or tyrosine, in seven bacterial species (Table 1). In eukaryotes, the recruitment of factors to the elongating RNAPII is regulated by the differential and dynamic phosphorylation of serine, threonine and tyrosine residues within the C-terminal domain of the Rpb1 large subunit during the transcription cycle [START_REF] Heidemann | Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription[END_REF]. Although the existence of phosphorylation of the main components of the RNA polymerase in both bacteria and eukaryotic cells does not imply similar regulatory mechanisms, it does suggest that evolutionary constraints on regulating gene expression led to targeting the transcription machinery for modulating its activity and its ability to interact with other factors.

BIOLOGICAL ROLE OF PHOSPHO-PROTEINS IN COORDINATING DNA-METABOLISM WITH TRANSITION STATE AND CELLULAR DEVELOPMENT IN B. SUBTILIS

A large panel of proteins involved in various DNA replication and repair pathways are identified with phospho-sites, suggesting that phosphorylation could play a role in coordinating the different cellular responses to DNA damage and integrity of the replicating chromosome (Table 1). Regulation by phosphorylation of DNArelated processes has been documented in B. subtilis. One process involves the DNA recombinase RecA, a multifunctional protein

B1XQW1_SYNP2

Escherichia coli K12 and O157:H7 species (ECOLI and ECO57:H7) [START_REF] Macek | Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation[END_REF][START_REF] Hansen | The Escherichia coli phosphotyrosine proteome relates to core pathways and virulence[END_REF][START_REF] Soares | Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium[END_REF], Helicobacter pylori (HELPY) [START_REF] Ge | Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins[END_REF], Campylocacter jejuni (CAMJE) [START_REF] Voisin | The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases[END_REF], Bacillus subtilis (BACSU) [START_REF] Macek | The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis[END_REF]Jers et al., 2010;Elsholz et al., 2012;Shi et al., 2014b), Clostridium acetobutilicum (CLOAB) [START_REF] Bai | Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum[END_REF], Listeria Monocytogenes (LISMO) [START_REF] Misra | Analysis of the serine/threonine/tyrosine phosphoproteome of the pathogenic bacterium Listeria monocytogenes reveals phosphorylated proteins related to virulence[END_REF], Pseudomonas fluorescens Pf0-1 (PSEPF) [START_REF] Ravichandran | Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species[END_REF], Streptococcus agalactiae serotype 1a (STRA1) [START_REF] Burnside | Serine/threonine phosphatase Stp1 mediates post-transcriptional regulation of hemolysin, autolysis, and virulence of group B Streptococcus[END_REF], Streptococcus pneumoniae serotype 2 (STREP2) [START_REF] Sun | Phosphoproteomic analysis reveals the multiple roles of phosphorylation in pathogenic bacterium Streptococcus pneumoniae[END_REF], Streptococcus coelicolor (STREPCO) [START_REF] Manteca | Phosphoproteome analysis of Streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation[END_REF], Thermus thermophilus HB8 (THET8) [START_REF] Takahata | Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilus HB8[END_REF], Thermoanaerobacter tengcongensis (THETN) [START_REF] Lin | Optimization of titanium dioxide enrichment of phosphopeptides and application in the Thermoanaerobacter tengcongensis phosphoproteome analysis[END_REF], Mycobacterium tuberculosis (MYCTO and MYCTU) [START_REF] Prisic | Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases[END_REF], Mycobacterioum bovis (MYCBO) [START_REF] Nakedi | Comparative Ser/Thr/Tyr phosphoproteomics between two mycobacterial species: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG[END_REF], Mycoplasma pneumoniae (MYCPN) [START_REF] Schmidl | The phosphoproteome of the minimal bacterium Mycoplasma pneumoniae: analysis of the complete known Ser/Thr kinome suggests the existence of novel kinases[END_REF], Synechococcus sp. (SYNP2) [START_REF] Yang | Global phosphoproteomic analysis reveals diverse functions of serine/threonine/tyrosine phosphorylation in the model cyanobacterium Synechococcus sp. strain PCC 7002[END_REF]. http://dbpsp.biocuckoo.org/browse.php [START_REF] Pan | dbPSP: a curated database for protein phosphorylation sites in prokaryotes[END_REF]. Protein target names are given according to HAMAP standard (http://hamap.expasy.org) and UniProtKB (http://www.uniprot.org/uniprot). Phosphoproteins provided with links to the dbPSP database (http://dbpsp.biocuckoo.org/) are labeled in blue. Phosphoproteins identified by other approaches are indicated in black. KO identifiers are provided for each family of proteins with links to the Kegg database for functional information [START_REF] Kanehisa | KEGG for integration and interpretation of large-scale molecular data sets[END_REF]. Validation (Val) is provided by (a) ability to interact with both a protein kinase (PK) and a cognate protein phosphatase (PP), (b) phosphorylation confirmed in vivo (*) and/or in vitro ( # ), (c) identified phosphosite(s), and (d) existence of phosphorylation at tyrosine (Y) serine (S) or threonine (T) in other bacterial orthologs (orth). 1 (Shi et al., 2014b); 2 [START_REF] Soufi | The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins[END_REF]; 3 (Bidnenko et al., 2013); 4 (Elsholz et al., 2012); 5 (Mijakovic et al., 2006); 6 [START_REF] Macek | The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis[END_REF]; 7 (Jers et al., 2010).

playing important roles in homologous recombination and repair of DNA double-strand breaks, as well as initiating the cellular SOS response to DNA damage in bacteria [START_REF] Kuzminov | DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination[END_REF][START_REF] Chen | Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures[END_REF][START_REF] Butala | The bacterial LexA transcriptional repressor[END_REF]. Recently, RecA was described as playing a role in monitoring and maintaining chromosome integrity at the onset of spore development (Bidnenko et al., 2013). This novel activity is regulated by phosphorylation by the ssDNA-dependent Ser/Thr kinase YabT, expressed during sporulation. Although the molecular mechanism underlying this activity is not yet understood, RecA phosphorylation at the N-terminal S2 plays a role in temporarily restraining sporulation furtherance upon encountering DNA damages. From the recent finding that RecA is also subjected to tyrosine phosphorylation by PtkA, we can anticipate a more complex picture for the regulation of its activities in B. subtilis (Shi et al., 2014b).

The DNA-binding developmental protein RacA illustrates a prospective role of phosphorylation in coordinating chromosome segregation with the asymmetric division taking place during sporulation in B. subtilis. During sporulation, RacA anchors the segregating chromosome at the cell pole of the forespore in a DivIVA-dependent manner (Ben-Yehuda et al., 2003;[START_REF] Wu | RacA and the Soj-Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis[END_REF]Ben-Yehuda et al., 2005;[START_REF] Perry | The Bacillus subtilis DivIVA protein has a sporulation-specific proximity to Spo0[END_REF]. In the B. subtilis PPI network, RacA is connected by both the Ser/Thr kinase YabT and cognate phosphatase SpoIIE, suggesting it may be a substrate of both (Shi et al., 2014b). This assumption, further confirmed in vitro, was also supported by the existence of a high positive correlation of expression of racA and spoIIE genes across numerous physiological conditions, indicating they are part of the same biological processes.

Nucleoid associated proteins (NAPs) are histone-like proteins involved in modifying the structural organization of chromosomes by changing its topology at a global or local level [START_REF] Dillon | Bacterial nucleoid-associated proteins, nucleoid structure and gene expression[END_REF]. As a consequence of their DNAbinding properties, NAP proteins are found to modulate different DNA-processes and act as global regulators of gene expression [START_REF] Dorman | Genome architecture and global gene regulation in bacteria: making progress towards a unified model?[END_REF]. NAP proteins are diverse and present in all living cells [START_REF] Dillon | Bacterial nucleoid-associated proteins, nucleoid structure and gene expression[END_REF][START_REF] Dorman | Genome architecture and global gene regulation in bacteria: making progress towards a unified model?[END_REF]. The examination of phosphoproteomics data in the database of Phosphorylation Sites in Prokaryotes dbPSP reveals that many representatives of this class of proteins are phosphorylated, including HupA, HupB, H-NS (and H-NS like StpA), Dps and structural maintenance chromosome (SMC) proteins (Table 1). A particular feature of this class of proteins is the occurrence of phospho-sites across a broad spectrum of bacterial orthologs (Table 1), suggesting that they could be functionally regulated by phosphorylation. The recent discovery that phosphorylation of the histone-like protein HupB by STKPs kinases negatively modulates its ability to bind DNA in M. tuberculosis supports this assumption [START_REF] Gupta | HupB, a nucleoid-associated protein of Mycobacterium tuberculosis, is modified by serine/threonine protein kinases in vivo[END_REF]. The phosphorylation state of the HupB protein correlates with the expression of pknE and pknF kinases. Their expression is high during the exponential growth phase and low when reaching the stationary phase. This differential phosphorylation strongly points to a potential mode of regulation of HupB which favors interaction with DNA during the stationary phase to promote compaction of the chromosome.

MinD, A SIGNALING SCAFFOLD-LIKE PROTEIN

Kinases are regulated by forming specific associations with arrays of different proteins, including kinase inhibitors and activators, as well as scaffold and anchoring proteins that target the kinasecomplexes to specific subcellular sites. Scaffold proteins are versatile hubs that spatially and temporally tether signaling components in eukaryotic cells. Interactome-based research in yeast provides a definition of classical signaling scaffold proteins as (i) having an active role in signaling while being itself devoid of catalytic activity relevant for signaling, and (ii) being able to promote the interaction of multiple components of signaling cascades at a particular cellular location to regulate their activity [START_REF] Zeke | Scaffolds: interaction platforms for cellular signalling circuits[END_REF]. A eukaryotic scaffold protein paradigm is the Ste5 protein of the mitogen-activated protein kinase (MAPK) pathway in yeast, which insulates three protein kinases MAPKKK Ste11, MAPKK Ste7, and MAPK Fus3 to promote a signaling cascade [START_REF] Good | The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation[END_REF].

Recent report of a yeast two-hybrid based PPI network centered on tyrosine-kinase in B. subtilis provide a 2D map of potential BY-kinase interacting partners (Shi et al., 2014b). Numerous protein partners are contacted by cognate kinase/phosphatase pairs, supporting the hypothesis that they are substrates. However, there are exception to this rule. The bacterial cell division regulator MinD is contacted by both the tyrosine kinase PtkA and tyrosine phosphatase PtpZ in the PPI network, but not targeted for phosphorylation by PtkA (Shi et al., 2014b). Strikingly, PtkA has been found to phosphorylate the septum site selection protein DivIVA, which recruits MinD at the cell poles via MinJ. Subcellular localization studies reveal that PtkA localizes at the cell pole in a MinD-dependent fashion. Septum defect phenotypes are observed in absence of PtkA and aggravated in a zapA deficient background, strongly suggesting that PtkA could play a role during cell division. All these observations support the hypothesis that B. subtilis MinD, in addition to its characterized role in recruiting the FtsZ-negative regulator MinC to prevent the formation of a Z-ring at the cell poles [START_REF] Gregory | Bacillus subtilis MinC destabilizes FtsZ-rings at new cell poles and contributes to the timing of cell division[END_REF], also acts as an intracellular organizer of signaling components involved in the regulation of cytokinesis (Shi et al., 2014b).

MinD belongs to the class of ParA-like AAA+ ATPases and bears structural homology to PtkA (Derouiche et al., 2015b). However, it lacks the tyrosine cluster determinant for autophosphorylation and is thus not a catalytically active kinase. Importantly, similar to TkmA, the transmembrane and cognate modulator of PtkA, necessary for the general activation of the kinase in the cell, MinD binds to and activates PtkA. In the PPI network, MinD is also found to interact with the second B. subtilis BY-kinase PktB, the Ser/Thr Hanks kinase YabT and a phosphatase (PtpZ), suggesting a potential ability to bring various components of tyrosine, but also serine/threonine phosphorylation pathways in close proximity (Figure 2). In many ways, MinD fulfills the definition of a signaling protein scaffold that allows several actors belonging to different pathways to co-localize at the cell poles. Remarkably, in the B. subtilis PPI phosphotyrosine network, MinD exhibits an interacting landscape similar to that of TkmA (Shi et al., 2014b; Figure 2). As a transmembrane protein, TkmA is classified as a receptor protein, activating PtkA upon sensing external inputs, while MinD perfectly matches with the definition of a classical scaffold, enforcing the proximity of kinase/phosphatase and substrate at a specific time and location during the bacteria cell cycle. However, the potential ability of TkmA to contact more than one kinase, a phosphatase and various PtkA substrates suggests that this receptor could also act as a docking platform to activate tyrosine kinase signaling events (Jers et al., 2010).

MinD acting as a scaffold is therefore proposed to promote PtkA-mediated phosphorylation of DivIVA in B. subtilis (Shi et al., 2014b). Phosphorylation of DivIVA and other homologs by Ser/Thr kinases is described as playing a role in regulating apical growth and cell polarity in Actinobateria, as well as well as orchestrating peptidoglycan synthesis during cell elongation in streptococci [START_REF] Fleurie | Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae[END_REF][START_REF] Hempel | The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces[END_REF][START_REF] Fleurie | Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division[END_REF]Pompeo et al., 2015). A real question about the role of phosphorylation of DivIVA at tyrosine in regulating its dynamic from the cell poles to the future septum is now addressed in B. subtilis. In this bacterium, PtkA could modulate DivIVA at the cell pole through landing/activation by MinD. The potential presence of other signaling components, such as the phosphatase PtpZ, would also add to the fine-tuning of such regulations. The interaction of the sporulation YabT Ser/Thr kinase with MinD might also provide a clue toward understanding how DivVIA switches from a MinCD(J)-mediated control of FtsZ assembly during vegetative growth, to a RacA mediated chromosome anchorage to the pole of the fore spore compartment at the onset of sporulation (Ben-Yehuda et al., 2003;[START_REF] Wu | RacA and the Soj-Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis[END_REF]Ben-Yehuda et al., 2005;[START_REF] Perry | The Bacillus subtilis DivIVA protein has a sporulation-specific proximity to Spo0[END_REF][START_REF] Bramkamp | A novel component of the division-site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD[END_REF]. This novel function of MinD as a signaling scaffold is thus expected to participate in coordinating cytokinesis, the terminal step of the cell cycle, with other pathways during the different Bacillus life-styles.

MinD/ParA-like proteins are widely found distributed among bacteria. They are not only involved in different aspects of regulation of cell division and chromosome segregation, but also in polar localization of cellular machineries, such as those directing conjugative transfer or chemotaxis, as well as those organizing the polar positioning of Type IV pili [START_REF] Kirkpatrick | Cell polarity: ParA-logs gather around the Hub[END_REF][START_REF] Lutkenhaus | The ParA/MinD family puts things in their place[END_REF][START_REF] Treuner-Lange | Regulation of cell polarity in bacteria[END_REF]. ParA/MinD-like proteins are often described as protein-hubs responsible for the architectural and spatial organization of necessary components driving these processes [START_REF] Lutkenhaus | The ParA/MinD family puts things in their place[END_REF]. In the asymmetrically dividing bacterium C. crescentus, the mechanisms underlying the segregation of the cell fate determinants to the two structurally distinct cell poles also requires MinD/ParA-like proteins to achieve polar localization of chromosomes and cell division [START_REF] Thanbichler | MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter[END_REF]. The MinD-like ATPase MipZ forms a dynamic complex with the DNA partitioning protein ParB at the chromosomal origin of replication, and also negatively regulates the polymerization of FtsZ [START_REF] Du | MipZ: one for the pole, two for the DNA[END_REF][START_REF] Kiekebusch | Localized dimerization and nucleoid binding drive gradient formation by the bacterial cell division inhibitor MipZ[END_REF]. Through ATP hydrolysis, MipZ acts as a molecular switch to couple chromosome segregation with the control of mid-cell positioning of the FtsZ ring in C. crescentus [START_REF] Thanbichler | MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter[END_REF]. Although its role in signaling scaffold is not proven, recent studies highlight the importance of ParB phosphorylation in the localization and function of proteins involved in chromosome segregation in mycobacteria [START_REF] Baronian | Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system[END_REF]. Lastly, the discovery that the ParA-like BY-kinase CspD, encoded by the cps capsular operon in S. pneumoniae, which plays a role in coordinating capsular polysaccharide with chromosome segregation through interaction with ParB, leads to the hypothesis that this kinase could act as molecular scaffold [START_REF] Nourikyan | Autophosphorylation of the bacterial tyrosine-kinase CpsD connects capsule synthesis with the cell cycle in Streptococcus pneumoniae[END_REF]. Although no proteins have so far been found to be phosphorylated by CpsD, ATP hydrolysis leading to CpsD autophosphorylation plays a major role in regulating the dynamic of localization of the ParB proteins bound to the chromosomal replication origin, and provides a molecular switch to couple the cell cycle with cell elongation [START_REF] Nourikyan | Autophosphorylation of the bacterial tyrosine-kinase CpsD connects capsule synthesis with the cell cycle in Streptococcus pneumoniae[END_REF].

CONCLUSIONS

Large-scale profiling of phosphoproteins and phosphosites by mass spectrometry-based proteomics, differential phosphoproteomics across physiological conditions and kinase-related phosphoproteomes are helpful to identify kinasespecific substrates and understand the functional dynamics of phosphorylation networks. Additionally, interactomics identifies kinase-protein interactions revealing not only potential substrates, but also anchors and modulators. Hence, phosphoproteomics and interactomics are powerfull and complementary approaches to provide a blueprint of kinase and phosphatase-centered signaling networks. The ever growing data from large scale phosphoproteome studies in bacteria, coupled with identification of kinase, phosphatase, substrates, and modulators provides important insights into cellular regulations. It also provides a unique opportunity to understand how phosphorylation participates in the regulation of protein activities, their interactions with other proteins, and their localizations in the bacterial cell.

In many bacteria, phosphorylation of proteins involved in various DNA-related processes, together with biological evidence of their role during cell cycle, suggests the existence of checkpoint regulations akin to eukaryotes. In all living organisms, cell cycle events involve the temporal and spatial coordination of chromosome replication and segregation with cytokinesis, the entire process being also coordinated with cell growth. In eukaryotes, the existence of highly intricate signaling networks is ensured by quality control and checkpoint proteins that coordinate these sequential events and trigger cell cycle arrests when things go wrong. In bacteria, the different processes are more interwoven. Consequently, the accurate execution of each step relative to others in time and space is crucial and involves strict regulatory mechanisms. The finding, in B. subtilis, that cell division MinD may act as a signaling hub to reinforce the proximity of various components of the Tyr and Ser/Thr phosphorylation pathway, along with the septum site protein DivIA, again hints at the existence of scaffold-mediated signal transduction in bacteria. In addition, the recent discovery that bacterial kinase are themselves able to tune their activities through phosphorylation add to the complexity of the picture (Shi et al., 2014a). However, it also suggests that, just as in eukaryotes, such bacterial scaffolds could offer versatile strategies to dynamically coordinate cell processes via signaling.

II. RESULTS

II. RESULTS -CHAPTER 1

Phosphorylation of YabA by the serine/threonine kinase YabT regulates Bacillus subtilis differentiation processes.

1. Phosphorylation of YabA by the serine/threonine kinase YabT regulates Bacillus subtilis differentiation processes.

Phosphorylation of proteins plays a key role in regulating many aspects of the cell cycle by integrating environmental and cellular signals. In eukaryotes, the interplay between the cell cycle and the other cellular processes mostly involves post-translational modifications (PTM) of proteins, including phosphorylation by Ser/Thr/Tyr Hanks-kinases (Hanks et al., 1988). In Phosphorylation of SSBA increases its binding to single-stranded DNA and may therefore modulate the DNA damage response (Mijakovic et al., 2006). Moreover, the Hanks-type kinase PrkD has been shown to interact in vitro with DnaC, the replicative helicase (Shi et al., 2014).

RecA, the general DNA recombinase, is required for survival and replication re-initiation. During sporulation, phosphorylation of RecA by the Hanks kinase YabT is involved in chromosome integrity during spore development (Bidnenko et al., 2014).

In this study, we report that the main initiator controller YabA is also specifically phosphorylated on its Threonine 71 residue by YabT. YabA plays a major role in down regulating initiation replication through interaction with the initiator protein DnaA and the clamp polymerase DnaN (Noirot-Gross et al., 2002;Noirot-Gros et al., 2006). However, YabA is a structural hub protein able to interact with other protein partners, indicating it might be multifunctional. Through its unique overall tri-dimentional structure composed of N-terminal four helix-bundle tetramer connected to four monomeric C-terminal domains by a highly flexible linker, YabA is capable to physically interact with more than one protein at a time, thus providing a suitable platform to integrate intracellular signals to replication initiation [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]; II.RESULT-Chapter

3.1).

We show in vivo that the YabT-dependent phosphorylation of YabA is not involved in initiation control, but rather influences differentiation processes such as sporulation and biofilm formation. Our results therefore highlight the multifunctional role of YabA as well as its role in integrating physiological signals to connect chromosomal replication initiation control with cell development. subtilis couples the developmental decision-making with the replication status is not fully understood. In B. subtilis, multiple regulatory processes contribute to regulating DNA replication at the onset of sporulation (Veening et al., 2009). Essentially, these mechanisms target the master initiator protein DnaA and modulate its activity (Katayama et al., 2010;Skarstad and Katayama, 2013;[START_REF] Zakrzewska-Czerwinska | Regulation of the initiation of chromosomal replication in bacteria[END_REF] ). The initiation controller YabA also exerts its regulatory control as part of a multimeric complex with DnaA and DnaN, associated with the replication fork during most of the replication cycle (Cho et al., 2008;[START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]Hayashi et al., 2005;Noirot-Gros et al., 2002;Noirot-Gros et al., 2006;Soufo et al., 2008). YabA is a small Zn-binding protein with an overall tri-dimensional structure composed of four N-terminal helix-bundle in a tetramer connected to four monomeric C-terminal domains by flexible linkers [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. Its unique structure defines YabA as a hub-like protein, able to interact with several partners to control and coordinate replication initiation with other cellular processes [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]Noirot-Gros et al., 2006). In cells engaged in sporulation, the SirA protein acts, similarly to Soj, by inhibiting DnaA loading at replication origin to prevent new rounds of replication initiation (Duan et al., 2016;Jameson et al., 2014;Rahn-Lee et al., 2011).

INTRODUCTION

In B. subtilis, the decision to enter the differential states of sporulation or biofilm involves the phosphorylation of a key regulator, Spo0A. The intra-cellular concentration of Spo0A-P is the determining factor that drives the expression of several gene clusters which are necessary for sporulation or production of the biofilm matrix. A well-characterized mechanism for coupling the initiation of sporulation with the DNA replication involves the protein Sda that promotes the reduction of Spo0A-P through inhibition of the activity of the phosphorelay kinase KinA (Burkholder et al., 2001;[START_REF] Hoover | Changes in DnaA-dependent gene expression contribute to the transcriptional and developmental response of Bacillus subtilis to manganese limitation in Luria-Bertani medium[END_REF]Veening et al., 2009). During vegetative growth, R II. RESULTS

91

the expression of the sda gene is positively regulated by DnaA which also acts as a transcriptional regulator of many genes involved in replication stress and sporulation (Breier and Grossman, 2009;Burkholder et al., 2001). Recent studies established that most of the DnaA transcriptional network is indirectly shaped by Sda, through the DnaA-mediated activation of the sda gene expression (Seid et al., 2017;Smith and Grossman, 2015;Washington et al., 2017).

In eukaryotes, the interplay between the cell cycle and the other cellular processes mostly involves the post-translational modifications (PTM) of proteins by Ser/Thr/Tyr Hanks-type kinases (Hanks et al., 1988;[START_REF] Stancik | Serine/Threonine Protein Kinases from Bacteria, Archaea and Eukarya Share a Common Evolutionary Origin Deeply Rooted in the Tree of Life[END_REF]. The phosphorylation of proteins plays a key role in regulating many aspects of the cell cycle by integrating environmental and cellular signals [START_REF] Endicott | The structural basis for control of eukaryotic protein kinases[END_REF]. In eukaryotic cells, DNA replication stress triggers coordinated responses involving signal transduction cascades, leading to the phosphorylation of specific proteins involved in DNA damage checkpoint and processing (Subramanian and Hochwagen, 2014).

The phosphorylation of proteins involved in DNA replication and repair has been identified in various bacteria [START_REF] Garcia-Garcia | Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria[END_REF]Shi et al., 2014). In B. subtilis, the phosphorylation of the single-strand DNA binding proteins SsbA on a tyrosine residue was found to modulate its binding to DNA (Mijakovic et al., 2006) while the replicative helicase DnaC was identified as a substrate of the Hanks-type kinase PrkD (Shi et al., 2014). In B. subtilis, phosphorylation at serine or threonine residues is catalyzed by the three Hanks-type Ser/Thr kinases PrkC, PrkD and YabT.

YabT is a transmembrane kinase devoid of the classical extracellular signal receptor domain but containing a DNA-binding motif essential to its activation (Bidnenko et al., 2013). This particular Ser/Thr kinase is mainly expressed at early stage of sporulation as well as in biofilms (Nicolas et al., 2012). Notably, YabT was found to phosphorylate the general DNA recombinase RecA to enforce chromosome integrity during spore development (Bidnenko et al., 2013) YabT also play a role in down regulation of protein synthesis by phosphorylation of the elongation factor EF-Tu (Pereira et al., 2015). Growing evidence in B. subtilis thus points at the involvement of different classes of kinases, kinase activators and substrates, and phosphatases in the regulation of processes related to cell cycle and development.

In this study, we report that the replication initiation controller YabA is also specifically phosphorylated by YabT. The phosphorylated residue was identified by biochemical and genetic approaches and functional analysis indicates a regulatory role of YabA phosphorylation during sporulation and biofilm formation that is mediated by Spo0A. These results highlight the multifunctional nature of YabA and its potential role in integrating physiological signals to connect and coordinate chromosomal replication initiation control with cell development.

RESULTS AND DISCUSSION

YabA is phosphorylated by YabT at Threonine 71 within the flexible inter-domain region

A purified His6-tagged recombinant form of YabT was incubated with the native YabA protein at a 1:25 ratio in the presence of 32 P-γ-ATP (Figure 1). YabT was found to self-activate by autophosphorylation and to phosphorylate YabA in vitro (Figure 1A). The YabA phosphorylation site was identified by mass spectrometry as the threonine residue 71 (Figure 1B) and validated by MaxQuant (Figure S2). To confirm the functional relevance of this phosphorylation site, this residue was replaced with the non-phosphorylatable residue alanine and the phospho-mimetic residue aspartate by site directed mutagenegis. The YabA-T71A and T71D mutant proteins were purified and tested in vitro for YabT-mediated phosphorylation. In both cases, the substitution of T71 prevented the transfer of a phosphoryl group by YabT, further indicating that T71 is the targeted phosphosite on YabA. YabA is composed of a core of four N-terminal α-helices, individually connected to a globular C-terminal region by a 14 residues long linking region [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. The mapping of T71 onto the YabA tetramer 3D-structure revealed it is located within the flexible hinge region (Figure 1C). This extended linker was postulated to confer the high degree of flexibility observed in the YabA structure [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]). These observations suggest that the phosphorylation at T71 could have an effect on the overall intrinsic flexibility of YabA.

Phosphorylation at T71 does not affect YabA interaction pattern

The flexibility of proteins was described to be important for the formation of protein complexes [START_REF] Marsh | Protein flexibility facilitates quaternary structure assembly and evolution[END_REF]. Previous work established that YabA interacts with multiple partners [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]Noirot-Gros et al., 2002;Noirot-Gros et al., 2006). The mapping of the binding surfaces on YabA highlighted two interacting domains. The free C-terminal domains of the YabA tetramer were found to be engaged in binding, in an exclusive manner, the initiator DnaA and the β-subunit of the replicative DNA polymerase DnaN, through a partially overlapping interacting surface. Similarly, an intersecting binding interface with the signaling and metabolic proteins TlpA and AcuB, respectively, was delineated on the tetrameric helical bundle formed by the N-terminal domain [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]Noirot-Gros et al., 2006). We first investigated whether the phosphorylation of the residue T71 in the inter-domain hinge region of YabA could affect its protein-interaction landscape. The T71A and T71D substitutions were transferred on the yabA gene fused to the activating domain (AD) and binding domain (BD) of the yeast transcriptional factor GAL4 and the YabA-fusions were tested for their ability to interact with the different protein partners in a yeast two-hybrid assay (Figure S3). The interaction patterns of the non-phosphorylatable and phosphomimetic forms of YabA were found to be similar to that of the wild type, indicating that the residue T71 was not likely to participate in modulating protein interactions.

Phosphorylation at T71 does not affect replication initiation control

We then investigated the role of YabA phosphorylation in replication initiation control. B. subtilis mutant strains carrying mutations at the yabA genomic locus leading to T71A and T71D substitutions were constructed, and tested for potential initiation-defect phenotypes. Using origin-proximal (ori) and terminus-proximal (ter) primer pairs (Table S2), we monitored the ratio of origin-to-terminus (ori-to-ter) genomic sequences as a means of detecting over-initiation events. In the ∆yabA null strain, the ori-to-ter ratio was twice as high as compared to the wildtype (Figure 2A), as already shown in previous studies (Murray and Koh, 2014;Soufo et al., 2008). However, the deletion of yabT as well as the expression of the non-phosphorylatable or the phosphomimetic versions of yabA gave rise to ori-to ter ratio similar to the wild type (Figure 2A). This indicated that the T71 residue was not involved in down regulation of replication initiation and that YabT-dependent phosphorylation of YabA was not required for DnaAmediated replication initiation control.

Previous studies established that YabA also exerts its function as part of a heterocomplex with DnaA and DnaN that localizes at the replication machinery. GFP-YabA fusion was found to form a discrete foci co-localizing with DnaN, and centrally localized at the nucleoid during most of the bacterial cell cycle (Hayashi et al., 2005;Noirot-Gros et al., 2002;Noirot-Gros et al., 2006;Soufo et al., 2008). We constructed B. subtilis strains conditionally expressing a gfp-yabA, a gfp-yabA-T71A or a gfp-yabA-T71D gene fusion from the ectopic chromosomal site amyE in B. subtilis.

These constructs were placed in their cognate wt-yabA, yabA-T71A and yabA-T71D genetic background (Table S1). Examination of the foci localization patterns revealed that the YabA subcellular localization remained unaffected by the substitutions at T71 (Figure 2B). Altogether, these experiments showed that YabA-T71A and YabA-T71D have retained all the functional properties of a wild type YabA, suggesting that phosphorylation of T71 is not involved in initiation control. Together with the retention of ability to interact with all there protein partners, these results provided evidence that the substitutions at T71 did not affect the structural and functional integrity of YabA.

YabA phosphorylation enhanced sporulation

Analysis of the transcriptional profiles of yabT from transcriptomes of B. subtilis cells exposed to many environmental and nutritional conditions revealed that yabT was expressed at an early stage of sporulation (data available at www.basysbio.eu/bsubtranscriptome/seb). We examined the physiological conditions under which the yabT and yabA genes exhibited correlated expression profiles (Figure S4). While yabA is highly expressed during all conditions, the expression of yabT is triggered only i) during sporulation, with a maximum expression peak three hours after the onset of sporulation, ii) during biofilm formation and iii) after glucose exhaustion (Figure S4). Interestingly, YabT has already been shown to be involved in the chromosome integrity control during sporulation. We therefore investigated whether YabA phosphorylation could play a role during sporulation. Our wild type, non-phosphorylatable and phosphomimetic

YabA strains were induced to sporulate by the exhaustion method in DSM media. The point when cells stop to divide is considered as the onset of sporulation (T0). We determined the survival efficiency of mature spores, expressed as the ratio of heat-resistant spores obtained 18 hours after T0 (Figure 3A). The yabA deleted strain was found to be significantly affected in sporulation, reaching 30% of efficiency compared to 70% of resistant spores for the wild type (P=0.0038), indicating that YabA could play a role during sporogenesis. We observed that the yabA-T71A strain exhibited the same level of sporulation as the wild-type strain ( ̴ 70%) while the yabA-T71D mutant reached the highest sporulation efficiency slightly above 100% (P=0.0043), indicating that YabA phosphorylation at T71 could enhance sporulation. Note that the sporulation efficiencies greater than 100% found in the yabA-T71D strain derivatives might result from the existence of short chains or doublets leading to an artificial excess of spore counts after heating treatment. The yabT mutant strain produced a final yield of mature spores similar to the wild-type, as previously observed. Examination of the ∆yabT yabA-T71D double mutant revealed that the phosphomimetic mutant derivative of yabA exhibited the same propensity to stimulate sporulation in a yabT-deficient genetic background (Bidnenko et al., 2013). This indicates that YabT and YabA are part of the same pathway and suggests that YabTdependent phosphorylation of YabA may play a role in enhancing sporulation.

The effect of YabA phosphorylation in spore yields was visible in the early stage of the sporulation process by microscopy analysis (Figure S5A). At T2, about twice as many cells were committed to sporulation (i.e exhibited an asymmetrical septum) in the yabA-T71D (27%, P<0.0001) as compared to the wild-type (14.5%). In contrast, in the yabA-T71A strain the number of stage T2-sporulating cells was reduced by 1.6 fold (9%, P=0.006) compared to the wild type (Figure 3B, Figure S5A). The ∆yabA strain showed the most severe defect in spore R II. RESULTS

97

production with a 4.25 fold reduction (3.4%) compared to the wild type (P<0.0001). The sporulation increase phenotype observed in yabA-T71D was maintained over 6 hours up to the production of mature spores (Figure S5C). Altogether, the results obtained at early (T2) and late (T8) sporulation stages suggest that the phosphorylation of YabA at T71 enhances the commitment of Bacillus cells to sporulation, leading to an increase in spore production, while the absence of phosphorylation only delays the entry into sporulation without affecting the final spore yield.

Our next question was to determine whether this phosphorylation-mediated stimulation of sporulation is related to YabT activity. The yabT gene was inserted in a plasmid under the transcriptional control of the IPTG-inducible Pspac promoter. The resulting construct pDG148flag-yabT, which allows the expression of the Flag-tagged YabT was then introduced in the wild-type yabA strain. Increased intracellular levels of YabT at T2 in crude extracts of cells harboring the pDG148flag-yabT construct was confirmed by a Western blotting assay (Figure S5D). Samples induced by the addition of IPTG 0.5 or 1 mM yielded signals of comparable intensities, indicating that YabT was efficiently produced in vivo and was not degraded by host proteases. Similar to yabA-T71D, we observed that the number of stage T2-sporulating cells was enhanced about 60% upon addition of IPTG (P=0.0075), indicating a correlation between the level YabT expression and the efficiency of entry into the sporulation process (Figure 3B, Figure S5B). A comparable increase in sporulating cells at T2 was observed with statistical significance (P=0.04) upon the expression of the untagged YabT protein in YabA wild type cells, that was abolished in YabA-phosphorylation mutant cells, suggesting that the increase requires the integrity of the YabA phosphosite and did not result from unrelated YabT function (Figure S5B).

Taken together, our findings highlighted a correlation between increased intracellular level of YabT, YabA phosphorylation and efficiency of entry into the sporulation process that support the hypothesis that the YabT-mediated phosphorylation of YabA at T71 plays a regulatory role during sporulation.

Effect of YabA phosphorylation at T71 on sporulation early developmental program

Sporulation is an adaptive developmental program engaged when the bacteria encounter conditions of starvation, leading to the liberation of highly resistant spores (Piggot and Hilbert 2004). Sporulation in B. subtilis is a strictly regulated process linked to the phosphorylation of the master regulator Spo0A, modulated by a multicomponent phosphorelay system [START_REF] Sonenshein | Control of sporulation initiation in Bacillus subtilis[END_REF][START_REF] Stephenson | Molecular insights into the initiation of sporulation in Grampositive bacteria: new technologies for an old phenomenon[END_REF][START_REF] Vishnoi | Triggering sporulation in Bacillus subtilis with artificial two-component systems reveals the importance of proper Spo0A activation dynamics[END_REF]. Only cells expressing a high level of Spo0A~P can initiate sporulation [START_REF] Chastanet | Just-in-time control of Spo0A synthesis in Bacillus subtilis by multiple regulatory mechanisms[END_REF]Fujita and Losick, 2005;[START_REF] Worner | Phosphorylation and functional analysis of the sporulation initiation factor Spo0A from Clostridium botulinum[END_REF]. Then, compartment-specific transcriptional programs that drive sporulation are activated by successive sporulation-specific sigma factors σ F , σ E , σ G , and σ K (Steil, Serrano et al.

2005

). We evidenced that the phosphorylation state of YabA differentially affects sporulation at an early stage. To better understand the role of YabA phosphorylation at the onset of sporulation, we constructed a Pspo0A-gfp transcriptional fusion and compared the expression profiles in our yabA-T71 mutant strains after initiation of sporulation (Figure 4). When cells enter the sporulation developmental program, phosphorylated Spo0A accumulates and activates its own expression (Fujita and Losick, 2005;Molle et al., 2003) (Figure 4A). Indeed, the fluorescence profile of the gfp expressed from Pspo0A in the wild-type yabA background increased with time (Figure 4B). Expression from Pspo0A in the yabA-T71A strain was found to be similar to that R II. RESULTS

99

obtained in the wild-type background, while it is reduced two-fold in the absence of yabA, hinting at a regulatory role of yabA in spo0A transcription. Interestingly, expression of the spo0A

promoter was most efficient in the presence of the YabA-T71D protein, suggesting that indeed, accumulation of Spo0A~P was enhanced in this strain.

To further investigate the role of YabA phosphorylation on the sporulation transcription program we examined the expression of promoters driven by the early sporulation specific sigma factors σ F and σ E . At the onset of sporulation, elevated levels of Spo0A~P activates the transcription factor σ H that will trigger the expression of numerous genes including the spoIIA operon, encoding for σ F as well as its regulators, and σ E (Piggot and Hilbert 2004). Both sporulation σ factors are first held in an inactive form in the mother cell and their activation is linked to the formation of the asymmetric division septum. Activation of the σ F factor occurs in the forespore and is followed by the activation of σ E through cleavage of the pro-σ E form (Figure 5A) [START_REF] Hilbert | Compartmentalization of gene expression during Bacillus subtilis spore formation[END_REF]). To investigate the impact of YabA phosphorylation on the early steps of sporulation, we constructed luminescent reporters for the promoter PspoIIA, controlled by the Spo0A~P-regulated σ H factor, as well as for the promoter PspoIIID, controlled in a σ Edependent manner. Both genes were transcriptionally fused with the luc gene encoding the firefly luciferase. Luciferase expression profiles were examined for 20 hours after sporulation (Figure 5B). In the wild type and yabA-T71A and -T71D genetic backgrounds the expression peak of the PspoIIA-luc and PspoIIID-luc fusions occurred at a similar time but not with a similar intensity. Compared to the wild-type, the expression of PspoIIA-luc was diminished in the yabA-T71A strain but was significantly higher in a yabA-T71D background, consistent with sporulation kinetics and spore yields obtained earlier (Figure 3). Indeed, the lower expression in yabA-T71A

could account for the sporulation delay observed in this strain (Figure 3B) while the elevated expression in yabA-T71D is in accordance with the increased sporulation efficiency triggered by the phosphomimetic mutation. PspoIIID expression is the highest in the yabA-T71D background.

These observations are in agreement with the hyper-sporulation phenotype exhibited in the yabAT71D strain. We also noticed that the expression of these early promoters was strongly delayed in the absence of YabA, which is consistent with a low sporulation efficiency of the

YabA phosphorylation inhibits biofilm formation

Sporulation is an energy consuming process which becomes irreversible after engulfment of the prespore by the mother cell. Early after starvation, cells might also adopt other solutions to survive, such as cannibalism, competence or biofilm formation (Chai et al., 2008;Vlamakis et al., 2013). The decision process is linked to the Spo0A~P to Spo0A ratio, which gradually increases after starvation. Biofilm formation and sporulation are repressed when Spo0A~P is low (Chai et al., 2008;Hamon and Lazazzera, 2001). At intermediate levels of Spo0A~P, the biofilm developmental program is induced, whereas only cells expressing high levels of Spo0A~P can enter into sporulation (Chai et al., 2008). We hypothesized that the phosphorylation of YabA we earlier found to correlate with enhancement of sporulation and Spo0A-mediated gene expression would, as a corollary, affect biofilm formation. B. subtilis is described to develop different biofilms from surface-associated to air-liquid pellicle (Vlamakis et al., 2013). We looked at the capacity of our strains to form air-liquid biofilm. We first verified that all strains were able to grow in MSgg medium: only the growth of the ∆yabA strain was slowed down (Figure 6A).

When cultured in glass tubes, the yabA-T71A and ∆yabT mutant derivatives strains exhibited similar re-suspended biofilm cell densities than the wild type YabA while biofilms formed by the ∆yabA and the yabA-T71D strains were 44% and 77% less, respectively, suggestion a negative role of YabA phosphorylation (Figure 6B). Biofilm cell density was also investigated upon overexpression of a Flag-tagged YabT. We found that overexpression YabT correlated with a decrease of biofilm cell density (Figure 6C), supporting a role of YabT-mediated phosphorylation of YabA in preventing biofilm formation. The tridimensional structures of air-liquid biofilms pellicles forms in 12 well culture plates were visualized by confocal microscopy and their biovolume quantified using the software Imaris (Figure 7A). Again, the wild type YabA, the yabA-T71A and ∆yabT displayed similar biofilm structures with statistically comparable biovolumes.

Contrastingly, both the yabA-T71D and the ∆yabA strains totally failed to form a pellicle (Figure 7A).

R II. RESULTS

103

We set out to confirm our findings from the angle of colony architecture formed on MSgg medium (Figure 7B). B. subtilis macrocolonies exhibit sophisticated three-dimensional structural designs on agar media, reflecting their ability to produce exopolysaccharides (EPS) polymers that compose the biofilm matrix, as well as other proteins important for the biofilm structure integrity (Branda et al., 2006;[START_REF] Romero | Amyloid fibers provide structural integrity to Bacillus subtilis biofilms[END_REF]. The enhanced matrix production in biofilms has been attributed to the derepression of the epsA-O and tapA-sigW-tasA operons which are normally inhibited by the regulator SinR (Kearns et al., 2005;[START_REF] Leiman | SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis[END_REF]. This is achieved via the protein SinI, an antagonist of SinR, which alleviates the repression of the genes encoding the components of the biofilm matrix and which expression is activated at a low-to-intermediate level of Spo0A~P (Figure S6) (Chai et al., 2008;Chu et al., 2008;Kearns et al., 2005). We observed a clear difference in colony morphology in the absence of YabA compared to the wild-type strain (Figure 7B). The ∆yabA strain displayed a hyper-wrinkled morphotype, indicative of a high production of extracellular matrix (ECM) associated with localized zones of cell death that trigger mechanical forces to form wrinkled structures (Figure 7B) [START_REF] Ghosh | Modeling cell-death patterning during biofilm formation[END_REF][START_REF] Asally | Localized cell death focuses mechanical forces during 3D patterning in a biofilm[END_REF][START_REF] Leiman | SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis[END_REF][START_REF] Spiers | Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose[END_REF][START_REF] Spiers | The Pseudomonas fluorescens SBW25 wrinkly spreader biofilm requires attachment factor, cellulose fibre and LPS interactions to maintain strength and integrity[END_REF]. Our observations suggested that the absence of YabA could lead to the de-regulation of the EPS synthesis pathway. However, we also observed that the overall production of biofilm cell mass is diminished in the ∆yabA (Figure 6B). This reduction of cell mass in the ∆yabA biofilms could be attributed to the decrease of cell growth observed in this strain (Figure 6A), associated with cell death during static growth in liquid as well as on semi-solid agar surfaces. Observation of the macrocolony morphology of the yabA-T71A revealed only a subtle difference compared to that of the wild-type strain, the later displaying a slight wrinkle phenotype at the center of the colony, not found in yabA-T71A

(Figure 7B). Interestingly, the yabA-T71D strain exhibited a smooth phenotype indicative of a defect in EPS production (Figure 7B). The deletion of the yabT gene induced a similar absence of wrinkles as in yabA-T71A. This phenotype is not affected when combined with the nonphosphorylatable mutation of YabA (∆yabT yabA-T71A strain), while the ∆yabT yabA-T71D strain exhibited the same smooth colony structure as the yabA-T71D strain. This indicated that YabT and YabA act in the same pathway regarding biofilm formation. Altogether, these results suggest that the YabA phosphorylation at T71 mediated by YabT prevents biofilm formation. Our results further support the role of YabT-mediated phosphorylation of YabA in negatively regulating biofilm formation through a Spo0A-directed repression of transcription of biofilm operons. et al., 2008;[START_REF] Deloughery | Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR[END_REF]. This could provide a foundation for the YabAphosphorylation mediated inhibition of biofilm formation. The mechanism underlying this novel role of YabA remains to be further characterized. However, the discovery that YabA can carry out another function that seems to be unrelated to its primary function in replication initiation control contributes to the complexity of its biological role in B.

CONCLUSION

subtilis. Many proteins are found to be multifunctional in eukaryotic but also bacterial cells.

These multitasking proteins, also called moonlighting proteins, exert a variety of activities in different biological pathways, thus contributing to cellular network complexity [START_REF] Jeffery | Protein species and moonlighting proteins: Very small changes in a protein's covalent structure can change its biochemical function[END_REF].

Post-translational protein modifications are important mechanisms that regulate protein activities in a reversible way. The role of protein phosphorylation in triggering a switch from a given function to another unrelated function is documented, most particularly in eukaryotes [START_REF] Gancedo | The Expanding Landscape of Moonlighting Proteins in Yeasts[END_REF][START_REF] Jeffery | Protein species and moonlighting proteins: Very small changes in a protein's covalent structure can change its biochemical function[END_REF]. However, bacterial proteins were also described as performing more than one function spanning processes such as metabolism, virulence, stress or even DNA maintenance [START_REF] Huberts | Moonlighting proteins: an intriguing mode of multitasking[END_REF][START_REF] Mani | MoonProt: a database for proteins that are known to moonlight[END_REF].

YabA belongs to a family of replication controllers, largely conserved in low GC sporulating or non sporulating Gram-positive bacteria. The YabT-targeted residue T71 of YabA is located in a structurally flexible domain linking the well conserved N-terminal and C-terminal parts. This flexible hinge is divergent among the YabA family of proteins and the residue T71 is not conserved in many other related bacteria. Thus, this novel function of YabA might provide B.

subtilis cells with a useful cross talk between the different cellular processes for fine tuning cell fate decision and to better respond to environmental changes.

MATERIALS AND METHODS

Bacterial strains and growth conditions

B. subtilis strains used in this work are listed in Table S1, PCR primers are listed in Table S2 and the plasmids constructs in Table S3. All B. subtilis strains constructed in this study are derived from the 168 (trp -) derivative CCBS185, containing a neomycin resistance gene under the control of the Lambda Pr promoter (λPr-neo) inserted into upp gene. The yabA-T71A and yabA-T71D mutants were constructed at locus by an improved mutation delivery approach as follows.

The primers pair yabA fwd + YabAT71AR and YabAT71AF + yabA rev were used to amplify two Cells were routinely grown in LB medium containing, when needed, spectinomycin 60 μg ml -1 , phleomycin 2 μg ml -1 , neomycin 5 μg ml -1 , chloramphenicol 7 μg ml -1 , or ampicillin 100 μg ml -1 .

Protein purifications

The N-terminal 6xHis-tagged YabT protein (cytosolic part containing the kinase domain) was synthesized in the chaperone overproducing strain E. coli M15 carrying a pQE-30 derived vector [START_REF] Jers | Bacillus subtilis two-component system sensory kinase DegS is regulated by serine phosphorylation in its input domain[END_REF]. Culture was grown under shaking at 37 °C to OD600 0.5, induced with 0.1 mM IPTG and grown for an additional 2 hours. Cells were then disrupted by sonication and 6xHistagged protein were purified on Ni-NTA columns (Qiagen) according to manufacturer's instructions, desalted on PD-10 columns (GE-Healthcare) and stored in a buffer containing 50 mM Tris-Cl pH 7.5, 100 mM NaCl and 10% glycerol. Protein concentrations were estimated using the Bradford assay (Bio-Rad) with BSA as standard.

For YabA expression and purification, the yabA gene and derivatives genes carrying mutations T71A and T71D were amplified from pGBDU-yabA, pGBDU-yabA-T71A, pGBDU-yabA-T71D bait plasmids with yabA-NdeI and yabA-XhoI primers sets and cloned into NdeI and XhoI sites of pSMG201, which allow cloning of ORF under the control of the T7 promoter. To purify the proteins, E. coli strain ER2566 was transformed with the resultant plasmids. Cells were cultured in LB medium supplemented with 30 μg ml -1 kanamycin. When the OD600 reached 0.

Data processing and phosphopeptide validation

A database search was performed with XTandem (version 2011.12.01. Cells were rinsed in MM and mounted on 1.2 % pads. When required, cells were stained with FM4-64 (Molecular Probes) to visualize the cell membrane or with DAPI to visualize the nucleoid. Fluorescence microscopy was performed using Leica DMRA2. System control and image processing were performed using MetaMorph software.

Microscopy analysis of sporulation

Cultures growing in a CH medium were induced to sporulate by transfer to resuspension medium (SM), essentially as described by Sterlini & Mandelstam (1969). Samples were taken at time T2 and mounted on 1.2% agarose pads. The cells were stained with DAPI to visualize the nucleoid, and with FM4-64 (Molecular Probes) to visualize the cell membrane. Fluorescence microscopy was performed on a Leica DMR2A using a 100 UplanAPO objective with an aperture of 1.35 and equipped with CoolSnap HQ camera (Roper Scientific). System control and image processing were performed using MetaMorph software. The indicated counts of cells were obtained from three to four independent experiments performed on over 400 cells per strain.

Sporulation efficiency

To quantify spore formation, cells were induced to sporulate by nutrient exhaustion in Difco sporulation medium (DSM) (Schaeffer et al., 1965). Overnight precultures were used to inoculate 5 ml of DSM and grown at 37°C for 18 h. Serial dilutions were plated on LB before and after a 20 min heat shock at 80 °C. Colonies were counted after 24 hours of incubation at 37°C, and the percentage of spores was calculated as the ratio of colonies forming units in heated and unheated samples.

Luciferase and GFP transcriptional fusions

To analyze genes expression, spoIIA and spoIIID transcriptional fusions with the butterfly luciferase gene luc [START_REF] Bidnenko | Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF] were used to transform B. subtilis strains where they integrated by single crossover. This event reconstructs natural regulatory region of gene upstream the fusion and an intact copy of gene downstream. For the detection of luciferase activity, strains were grown in LB medium until exponential phase (OD600 0,4-0,5). The cells were centrifuged and re-suspended in fresh DSM media to obtain OD600 1,0. The pre-cultures were next diluted in respective media to OD600 0.025. Then 200 μl was distributed into each well in a 96-well black plate (Corning) and 10 μl of luciferin was added to reach a final concentration of R II. RESULTS 112 1.5 mg/ml (4.7 mM). The cultures were incubated at 37°C with agitation in a Perkin-Elmer Envision 2104 Multilabel Reader equipped with an enhanced-sensitivity photomultiplier for luminometry. The temperature of the clear plastic lid was maintained at 37°C to avoid condensation. Relative Luminescence Units (RLU) and OD600 were measured at 5-min intervals.

For spo0A expression, pBSBII-spo0A plasmid containing a GFP transcriptional fusion was used to transform B. subtilis strains. The plasmid was integrated by single crossover at chromosomal loci of the targeted gene. GFP expression was measured after induction of sporulation (T0) by the resuspension method using a TECAN instrument.

Biofilm formation

Strains were grown in LB to OD600 of 1,0. For biofilm formation, 10 µL of culture were added to The tubes were incubated without agitation at 30 °C for 44 hours. For quantification, the MSgg medium was eliminated and the biofilm was re-suspended in 1 ml of H2O to measure the optical density by spectrometry at 600 nm.

Analysis of air-liquid biofilm pellicles

Strains were grown in LB to OD600 of 1.0 and inoculated in 12 wells culture plates containing Biovolumes were calculated based on total volume (μm3) per area (μm2) from n≥3 samples.

Complex colony formation

Cells were grown in LB shaking at 37°C to OD600 of 1,0. 10 µL cultures were then spotted on a dried MSgg plate (1.5% agar) and incubated for 96 hours at 30 °C. Colonies were measured and photographed using a camera. For each sample, a representative image from 3 examined colonies is presented.

YabT overexpression

For YabT overproduction, yabT gene was amplified using oligonucleotides YabT-F-SalI and YabT-R-SphI (Table S2); digested by SalI and SphI enzymes and cloned at pDG148 and pDG148F plasmids (Table S3). Expression of the cloned yabT gen in B. subtilis was induced by 0.5 and 1 mM IPTG during sporulation and biofilm conditions. The expression of YabT was determined by inmuno-detection. The FLAG-tagged YabT were visualized using the primary mouse ANTI-FLAG M2 monoclonal antibodies (Sigma-Aldrich; dilution 1:10.000) and the secondary goat peroxidase-coupled anti-mouse IgG antibodies (Sigma-Aldrich; dilution 1:20.000). The immunedetection was developed using the kit Clarity Western ECL substrate (Bio-Rad) as per the manufacturer on a Chemidoc (Biorad) imaging system. The images were analyzed using Image Lab.

Figure S2

P-T71

LP 0.9 (MaxQuanT)

Transcriptomic analyses of T71 phosphosite mutants

As we previously showed, YabA is phosphorylated by the Hanks-type serine/threonine kinase YabT at the threonine 71 residue located in the flexible inter-domain region. We also demonstrated that the phosphorylation of YabA is not involved in replication initiation control, The aim of this study is to identify genes that are differentially expressed during exponential phase and at the onset of sporulation, in the non-phosphorylatable yabAT71A, the phosphomimetic yabAT71D and yabA null strains as compared to the wild-type strain. We then extracted RNA in triplicates from corresponding cells grown in sporulation medium, at midexponential growth, at stage T1 and stage T3. Cultures were performed as described in supplementary materials and methods (1.2.1). RNAs were purified and checked for their quality and integrity in a Bionalyzer system (Agilent). An example is given in the Figure 1, which shows a good quality of total RNA profiles, with traces of multimeric forms.

129

For all analysis, a significant p-value (adjusted) of < 0.05 was chosen. In all conditions tested, a high number of genes, whose expression was significantly altered in the phosphomimetic yabAT71D strain and in the yabA null strain, were identified (Figure 2, Table 1). Red dots represent significantly differentially expressed genes in each condition, as compared to the wildtype strain. We previously demonstrated that the yabAT71A and WT strains showed a similar phenotype in sporulation and biofilm formation (See previous article). As expected from these studies, yabAT71A transcriptomic profile was roughly identical to that of the wild-type strain in all three conditions tested. However, we demonstrated that the behavior of the yabAT71D strain was different from that of the WT and yabAT71A strains, both in sporulation and biofilm conditions. Not surprisingly, gene expression was highly altered in this strain. Finally, we demonstrated that the ∆yabA strain showed replication initiation defect and a delayed entry in sporulation. As expected from these data, a high number of up-and down-regulated genes were identified in this strain. In both the yabAT71D and the ∆yabA strains, gene expression is altered as soon as the exponential phase. We already identified significant effects of YabA phosphorylation in cell differentiation (see previous article). We therefore focused our searches on known genes involved in these pathways within the set of genes differentially expressed in each strain, as compared to the wildtype strain. A list of the most important genes that regulate sporulation and biofilm formation was then established. This list includes genes encoding the histidine kinases kinA-KinE, the phosphorelay genes, the sigma factors, the spo genes, the rap genes (see Figure 12-INTRODUCTION) and also genes that play an important role during the development of biofilms, such the anti-repressor sinI and the repressor sinR, the eps operon required for biosynthesis of the extracellular matrix, the tapA-sipW-tasA operon, required for the production of amyloid fibers, autolytic genes, motility genes, etc… (see Figure 20-INTRODUCTION). We also included in this first analysis genes involved in replication and DNA repair, kinases and phosphatases (data shown in the annexes) and other highly impacted genes. The lists of genes differentially expressed are provided in the annexes. We thereafter established Heatmaps, showing differentially expressed genes involved in the pathways of interest (Figure 3). We observed a clear difference in the expression pattern of genes involved in sporulation (Figure 3A) and biofilm (Figure 3B) in the phophomimetic yabAT71D strain at stage T3. At this stage, a high number of spo genes were up-regulated in the strain yabAT71D (dark orange, increased levels of correlation), weather most of them were down-regulated in the yabA null strain. These transcriptomic data are in agreement with the enhanced (yabAT71D strain) and delayed (yabA null strain) sporulation phenotypes we previously observed. In contrast, a high number of genes involved in biofilm were down-regulated in the yabAT71D strain (violet, decreased levels of correlation). These data also correlate to the inhibition of biofilm formation previously observed in this strain.

-

-fli -hag -lyt -motA-B -flg -sigD -epsG-H-I-J-K-M -yqcG-yqcF -sfr -epsA-B-C-D-E-F -sipW-tasA -bslA -sinI -aprE -ydak -comQ

B

The expression patterns of sporulation and biofilm genes were similar in the yabAT71A and wildtype strains, both at T1 and T3 (Figure 3). These transcriptomic data fit with the fact that both strains exhibit equivalent sporulation (at T18) and biofilm phenotypes, except a slight delay in the entry of sporulation evidenced for the yabAT71A strain. Interestingly, we observed at T1 in the yabAT71A strain, a slight but significant down-regulation of spo0B, sigH and spoIIAA-spoIIAB-sigF operon. All these genes are involved in early sporulation. These data may support the delayed entry in sporulation observed for the yabAT71A strain in our previous studies. Furthermore, we also observed at T1 that the histidine kinases kinA to kinE genes and the spoIIAA-spoIIAB-sigF operon are up-regulated in the yabAT71D strain. These data suggest a premature entry of the yabAT71D strain into the sporulation pathway. By contrast, the spoIIAA-spoIIAB-sigF operon is strongy repressed at T1 in the ∆yabA strain, which could explain the delayed entry in sporulation of this strain.

Interestingly, the rapA-phrA and rapE-phrE operons were strongly down-regulated in the yabAT71D strain at T1 (Log2FC: -9,6 and -9.4, P< 0.00005) and T3 (Log2FC: -12.6 and -14.3, P< 0.00005). Raps are an interesting group of modulator proteins which activity is "switchable" through the action of signaling molecules (quorum-sensing). Most rap genes are associated in an operon with their cognate phr (phosphatase repressor). phrs code for small peptides that are exported from the cell, undergo peptide processing and are then re-imported as mature signaling peptides into the cell, where they interact with and inhibit their cognate Raps towards the target response regulator [START_REF] Pottathil | The extracellular Phr peptide-Rap phosphatase signaling circuit of Bacillus subtilis[END_REF]. RapA, B, and E act by dephosphorylating the Spo0F~P response regulator of the phosphorelay. These Raps proteins therefore inhibit the accumulation of Spo0A~P and play a major role in negatively regulating the sporulation initiation. Transcription of the genes encoding the Rap is under the control of physiological conditions such as competence (RapA and RapE) and vegetative growth (RapB), which are alternatives to sporulation [START_REF] Perego | Multiple protein aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in Bacillus subtilis[END_REF][START_REF] Jiang | Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis[END_REF]. Deletion of rapA increases the sporulation efficiency (35% more spores than in the wild type), whereas deletion of rapE only slightly increase sporulation (only 12%). The rapA rapE double mutant does not exhibit an additive phenotype and shows a sporulation efficiency similar to that of the ∆rapA strain [START_REF] Jiang | Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis[END_REF]. Therefore, the strongly down-regulation of rapA and rapE observed in the yabAT71D strain, leading to a fast accumulation of Spo0A~P intracellular levels, could explain the hypersporulation phenotype of this strain.

A consequence of elevated levels of Spo0A~P is the prevention of the expression of the antirepressor sinI leading to full repression of the EPS operon by the master regulator SinR. This R II. RESULTS 134 could explain the inhibition of genes involved in biofilm formation observed in strain yabAT71D.

The master regulator for biofilm formation, SinR, directly represses the EPS operon and yqxM-sipW-tasA. Under appropriate conditions for initiating biofilm formation, SinR repression is relieved by expression of the anti-repressor SinI that sequesters SinR, thereby resulting in the production of exopolysaccharides and amyloid fibers [START_REF] Kearns | A master regulator for biofilm formation by Bacillus subtilis[END_REF], Chai, Chu et al. 2008, Chu, Kearns et al. 2008). Unfortunately, we did not observe any down-regulation of the anti-repressor sinI in strain yabAT71D. This gene was instead slightly up-regulated (Log2FC: 0.652, P< 0.05) leading the subsequent activation of the yqxM-sipW-tasA operon (Log2FC: 2.97, P< 0.00005) and the EPS operon. Interestingly, our transcriptomic data showed that only a part of the EPS operon (epsA to epsF) is up-regulated (Log2FC < 1, P< 0.005), whereas genes epsG to epsK were strongly down-regulated (Log2FC < -9, P< 0.005). A new processive antitermination mechanism has been recently evidenced in B. subtilis. This mechanism is required for eps expression to ensure the full synthesis of the long eps transcript (16 kb) and is mediated by a novel RNA element, called 'EAR' (eps-associated RNA). EAR is located between the second (epsB) and third (epsC) gene of the eps operon [START_REF] Irnov | A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales[END_REF]. The presence of the EAR element within the eps operon is required for readthrough of distally located termination signals. In the absence of this antitermination mechanism, the eps transcription is truncated at intermediary points of the operon, resulting in an exopolysaccharide-deficient phenotype. Analysis of the effect of EAR on eps operon expression revealed that deletion of the EAR did not reduce epsC, epsD and epsE mRNA levels but markedly decreased expression of the distal part of the operon (epsF, epsH and epsM). A dramatic drop was observed in epsF-O gene transcription by microarray and qRT-PCR analyses, supporting a positive regulatory relationship between the EAR element and expression of the second half of the eps operon [START_REF] Irnov | A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales[END_REF]. The EAR element promotes eps synthesis via two possible models, which are not mutually exclusive. Constitutive antitermination could result from cellular cofactors that assemble with EAR and the transcription elongation complex to promote processive antitermination. Alternatively, processive antitermination may require an unknown signal in addition to the EAR element. EAR antitermination could be coordinated with the functional roles of genes located upstream and downstream of epsF. The most proximal upstream gene, epsE, is a glycosyltransferase that acts as a 'molecular clutch' by arresting flagellar movement. Downstream of epsF are mostly genes required for polymerization and extracellular assembly of the exopolysaccharides. Therefore, although the de-repression of SinR under biofilm conditions would permit transcription initiation of eps, transcript elongation could prematurely terminate at the epsF locus. This could result in a population of non-motile cells that await accumulation of an unknown physiological or quorum-based signal before allowing full EPS production [START_REF] Irnov | A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales[END_REF]. The EAR cofactors and the molecular mechanism of its action remain unknown. We observed the same transcription profile for eps genes in the yabAT71D strain than in an EAR deleted strain. These data strongly suggest that YabAphosphorylation could play a role in the regulation that is mediated by the EAR element.

Examining the highly impacted genes, we observed that the immR gene (Log2FC: -11.05, P< 0.0005), the rapI-phrI genes (Log2FC< -9, P< 0.0005) and the subsequent ImmR regulon (Figure 4) were strongly down-regulated in the yabAT71D strain. These genes are involved in the transfer of the mobile genetic element ICEBs1 (Figure 4). The mobile genetic element ICEBs1 is an integrative and conjugative element present in the genome of the B. subtilis. During normal exponential growth, ICEBs1 gene expression is repressed by the element's repressor ImmR and there is almost no excision of ICEBs1. Upon production of active RapI, a cell sensory response regulator aspartate phosphatase dedicated to the control of transfer of ICEBs1, or during the RecA-dependent SOS response, ImmR is inactivated and ICEBs1 gene expression is de-repressed [START_REF] Auchtung | Identification and characterization of the immunity repressor (ImmR) that controls the mobile genetic element ICEBs1 of Bacillus subtilis[END_REF][START_REF] Bose | Regulation of horizontal gene transfer in Bacillus subtilis by activation of a conserved site-specific protease[END_REF]. The conditions that are optimal for ICEBs1 conjugation, that is, stationary growth and high cell density, coincide with conditions that induce sporulation. As sporulation and conjugative transfer of ICEBs1 are likely not to be compatible, it is conceivable that sporulation will be blocked in cells having activated ICEBs1 conjugation. Our transcriptomic data showed that in the wild-type strain, the ImmR regulon is repressed at exponential phase and thereafter induced at stationary phase (T1), as expected. The expression pattern of ICEBs1 genes is the same in the yabAT71A background. By contrast, weather its expression is normally repressed during the exponential phase in the yabA strain, the ICEBs1 genes are highly derepressed at T3 in this strain. Higher mobility of ICEBs1 could therefore be the source of the deadlock of the sporulation process observed in this strain. Interestingly, the ICEBs1 genes are strongly repressed in the phosphomimetic yabAT71D in all conditions, which may account for the higher sporulation efficiency observed in this strain. These results suggest that YabA-phosphorylation is involved in the repression of the ICEBs1 conjugation genes at stationary phase. Furthermore, it has been demonstrated in vitro and in vivo that RapI can also dephosphorylate Spo0F~P and that ectopic expression of rapI inhibits sporulation [START_REF] Parashar | Conformational change-induced repeat domain expansion regulates rap phosphatase quorum-sensing signal receptors[END_REF][START_REF] Singh | Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling[END_REF]. Higher expression of rapI at T3 in strain yabA and most efficient dephosphorylation of Spo0A~P may therefore also account for sporulation inhibition in this strain. Thus, RapI probably has a dual function: activation of ICEBs1 conjugation genes and inhibition of sporulation.

R II. RESULTS

136

In addition, the nucleoid-associated protein Rok, wich regulates the expression of several genes including comK [START_REF] Hoa | Rok (YkuW) regulates genetic competence in Bacillus subtilis by directly repressing comK[END_REF]Albano et al., 2005;Kovacs and Kuipers, 2011;[START_REF] Marciniak | Regulation of ykrL (htpX) by Rok and YkrK, a novel type of regulator in Bacillus subtilis[END_REF] can bind to several region in the mobile genetic element ICEBs1 (Seid et al., 2017) and repress ICEBs1 (Smits and Grossman, 2010). Rok therefore contributes to keep ICEBs1 quiescent in the genome and has a similar effect than that observed in the phosphomimetic strain.

Furthermore, the small hydrophobine bslA gene is significantly down-regulated in a ∆rok strain, which in addition shows reduced complex colony structure formation (Kovacs and Kuipers, 2011). Transcriptomic analysis showed that bslA is up-regulated in the yabAT71D strain in all conditions, at exponential phase (LogFC: 1.02, P< 0.005) and stationary phase T1 and T3 (LogFC < 1, P< 0.005). Recently, interactions between the replication initiation protein DnaA and Rok have been identified by ChIP-seq assays. DnaA is associated with eight previously identified regions containing clusters of DnaA boxes (Smith and Grossman, 2015) and with 36 additional regions that were also bound by Rok. Interestingly, association of DnaA to these 36 additional regions was dependent on the presence of Rok. Mutant analyses support a model in which DnaA represses these genes through its interaction with Rok (Seid et al., 2017). YabA is associated with at least some of the chromosomal regions specifically bound by DnaA, including oriC (Merrikh and Grossman, 2011). Nevertheless, YabA alone appears incapable of binding DNA and its association with chromosomal regions bound by DnaA in vivo depends on DnaA (Merrikh and Grossman, 2011). YabA associates with four regions containing DnaA-boxes (rpmH/dnaA, yydA/yycS, ywlC/ywlB, and ywcI/vpr) in a DnaA-dependent manner (Merrikh and Grossman, 2011). Focusing on these 4 transcription units, we considered them as candidates for a direct regulation by both DnaA and YabA. Out of these four candidates, we identified 3 transcription units significantly impacted in the yabAT71D mutant at T3 (Table 2). ywIC and ywcI are repressed by DnaA. Our transcriptomic data show that both ywIC and ywcI are highly expressed in the yabAT71D strain, weather dnaA is significantly down-regulated. Expression of dnaA is indeed repressed by the master regulator Spo0A, which has been found to influence, directly or indirectly, the expression of over 500 genes during the early stages of development (Molle et al., 2003). We know however that YabA phosphorylation positively influence the accumulation of Spo0A~P. YabA may therefore contribute to the repression of ywIC and ywcI either directly through a protein/protein interaction with DnaA, either indirectly through the transcriptional regulation of dnaA by Spo0A~P. In both cases, phosphorylation of YabA may counteract its regulatory role. Therefore it will be difficult to find a direct role of YabA-phosphorylation during stationary phase.

OTHERS GENES

Region Gene yabAT71D

rpmH/dnaA dnaA -1,543 yydA/yycS yydA (0.273) ywIC/ywlB ywIC 0,942 ywcI/vpr ywcI 1,041 Table 2. Expression of genes where DnaA and YabA were found associated and in which its expression is affected by yabAT71D mutant. Each value indicates the log-fold change in expression of the indicated gene in the yabAT71D mutant at T3 relative to the wild-type at T3. Data are averages of three independent experiments, and except for the numbers in parentheses, all are statistically significant (adjusted p < 0.05).

We also examined the expression of 36 additional regions that were also bound by Rok and DnaA (Table 3) (Seid et al., 2017). Each value indicates the log-fold change in expression of the indicated gene in the yabAT71D mutant at T3 relative to the wild-type at T3. Data are averages of three independent experiments, all are statistically significant (adjusted p < 0.05).

Region

Interestingly, we observed that expression of 25 of these regions was significantly altered in the yabAT71D mutant. These results suggest that YabA may have a role in gene regulation through association with both Rok and DnaA, and that this gene regulation may be regulated by YabA phosphorylation (Table 3). However, both Rok and DnaA are transcriptional regulators which transcription is repressed by Spo0A (Molle et al., 2003). High levels of activated Spo0A observed in the yabAT71D strain could then repress dnaA and rok, leading to de-regulation of the genes controlled by Rok and DnaA. Therefore, our transcriptomic data support the phenotypes found in our previous study but also reveal a high degree of complexity in the regulatory mechanism that could be trigger by YabA and modulated by YabA phosphorylation. Further work is needed to deciphering the regulatory role of YabA phosphorylation during sporulation and biofilm formation.

Supplementary materials and methods

Cell culture for RNA extraction

Cells were inoculated in 2 mL of LB media from a fresh colony and incubated at 37ºC overnight.

This pre-culture was used to inoculate 150 ml of CH media at OD600nm =0.015. Incubation at 37ºC was carried out until cells reached the exponential phase (OD600nm=0.4-0.5). At this time 30 ml of cells were collected and mixed with 15 ml ice-cold killing buffer (20 mM TrisHCl pH = 7.5; 5 mM MgCl2, 20 mM NaN3) before centrifugation for 10 min at 4700 rpm (4ºC). Pellet was frozen in liquid nitrogen and stored at -80ºC. The remaining 120 ml CH culture were allowed to grow to reach an OD600nm=1 in which cells were induced to sporulate by the resuspension method as described in Garcia-Garcia et al., 2017. At T1 and T3, cells were harvested and frozen as described above. All strains and conditions were performed in triplicate.

RNA extraction

Bacterial pellets were resuspended in 500 µL lysis buffer (4 M guanidinethiocyanate, 25 mM sodium acetate pH = 5.2, 0.5 % N-lauroylsarcosinate) and were transferred into a tube containing small glass beads (volume equivalent to 500 µL). 500 µL of Phenol was incorporated before proceeding to cellular disruption with a Fastprep, for 40 sec at power 6.5 (two cycles).

Samples were cooled in ice and centrifuged at maximum speed for 5 min at 4 °C. Supernatants were carefully recovered and mixed with 1 mL of chlorophorm/isoamyl-alcohol 24:1. Then, samples were centrifuged and the aqueous phase was washed twice with equivalent volumes of chlorophorm/isoamyl-alcohol. 1/10 volume of 3 M sodium acetate (pH 4.2) and 1 ml isopropanol were then added. Samples were kept ON at -20 °C to lead RNA precipitation.

Precipitated RNA was centrifuged for 15 min at 15000 rpm at 4 °C and washed with 1 mL 70 % ethanol. Finally, RNA pellets were dried and resuspended in 75 µL ddH2O. To eliminate DNA, samples were treated with QIAGen RNase-Free DNase set as per manufacturer's instructions and cleaned with Norgen Concentration Micro Kit as per manufacturer's instructions. RNA was checked on the Bioanalyser system (Agilent) for its quality and integrity. The RNA Integrity Number (RIN) must be above 8 and the ribosomal ratio (23S/16S or 28S/18S) should be as high as possible, the ideal value being 2.

RNAseq

II. RESULTS -CHAPTER 2

YabA protein exhibits an unusual ATP/GTPase activity

YabA protein exhibits an unusual ATP/GTPase activity

Previous in vitro phosphorylation studies revealed a slightly YabA auto-phosphorylation when the protein was incubated with γ-ATP 32 and without the presence of YabT. This autophosphorylation was also detected in the YabA mutant proteins. Interestingly, we observed a huge band at the bottom of the gel in some of YabA mutant proteins and not in YabA wild-type (Figure 1). We supposed that this band corresponded to free γ-ATP 32 and YabA could hydrolyzed this ATP, therefore the band disappeared.

From this observation, in vitro ATPase assays were initiated to detect a potential YabA ATPase activity. However, later we confirmed that auto-phosphorylation and the band at the bottom of the gel disappeared when we used a new purified protein. We constructed and purified untagged YabA and phospho-mutants (YabAT71A and YabAT71D) proteins in order to confirm that T71 was indeed the YabT-dependent phosphorylation site as described in García-García et al. 2017. The first proteins were purified by applying a 20 % ammonium sulfate precipitation, a step that surprisingly leads to a >95% purity of YabA. Initially, this purification procedure was not followed by a Gel Filtration step. We thus suppose that the initial auto-phopshorylation observed in our proteins and the bands at the bottom of the gel were due to contaminants that co-precipitated with our proteins.

The YabA and YabA phopho-mutant proteins, purified by 20 % ammonium sulfate precipitation followed by gel filtration, were then used to determine if YabA hydrolyzes ATP. First, we performed an in-gel ATPase assay (Figure 2). YabA and phospho-mutant proteins were first electrophoresed in a clear native gel (CN-PAGE 4-16%). The gel was then incubated with ATP and lead nitrate. ATP hydrolysis is correlated with the development of lead white precipitates.

For the in-gel ATPase activity assay, CN-PAGE is highly preferred compared with blue native gels (BN-PAGE) because the dye Coomassie Blue applied on BN-PAGE can dissociate detergent-labile subunits when proteins form complexes. Furthermore, lead precipitate detection is very difficult in the presence of the Coomassie Blue dye. The CN-PAGE gels are colorless; therefore, the in-gel assays can be directly performed without any interference with the Coomassie Blue dye [START_REF] Wittig | High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes[END_REF].

Under conditions of native gel electrophoresis, YabA and YabA phospho-mutant proteins migrated as a major band with an apparent molecular mass of 242 kDa, except for YabAT71D, which migrated faster, indicative of a different charge to mass ratio. Different upper bands were also detected, that might correspond to different conformations and/or oligomerisation states of the YabA protein. Indeed, we previously demonstrated that in solution, YabA forms a tetramer composed of N-terminal four helix-bundle tetramer connected to four monomeric Cterminal domains by a highly flexible linker [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. Although tetramers of YabA was shown to be the most predominant form in solution, higher molecular weight forms could be also identified by Sec-Mall analysis (Fericoli et al., 2016). Furthermore, the inter-domain linker of YabA confers a high degree of flexibility, leading to a multiplicity of conformation of the C-terminal domains relative to the tetrameric core. Replacement of the threonine 71 residue located in the flexible region by an aspartate residue (YabAT71D) introduces a supplementary negative charge which could modify this conformation and affect the oligomerisation state of the protein, therefore explaining the different migration pattern observed for YabAT71D (Figure

2.A).

Interestingly, we found that a clear ATPase activity was associated to all the different YabA bands, strongly suggesting that this activity was displayed by YabA. This ATPase activity was also found associated with non-phosphorylatable (T71A) and phosphomimetic (T71D) mutants

(Figure 2.B).

R II. RESULTS

147

To further corroborate these surprising results, we also used conventional radioactive TLC assays with both [α-32 P] ATP and [α-32 P] GTP. YabA, YabAT71A and YabAT71D proteins were incubated with radiolabelled ATP or GTP; reactions were stopped on ice after 15, 30, 60, 90 and 120 min of incubation (Figure 3). The results show that YabA derivatives possess an atypical "ATP /

GTPase" activity, leading to the accumulation of yet uncharacterized products. As shown in Figure 3.A, the hydrolysis activity of the parental YabA protein was almost null, whatever the substrate was. The YabAT71A derivative exhibited a slight hydrolysis activity. By contrast, the phosphomimetic YabAT71D mutant protein exhibited a highest hydrolysis activity.

We also observed that several intermediates were produced when ATP was hydrolyzed, suggesting a gradual degradation, whereas only one final product was evidenced when GTP was used as the substrate. These results suggest that this unusual hydrolytic activity is modulated by

C

R II. RESULTS

149

phosphorylation of the YabA T71 residue and that GTP may be the true substrate. Also the ingel ATPase assay suggested that ATPase activity strictly correlates with YabA migration bands, we wanted to validate that the ATPase activity was not due to undesirable contaminants in the protein purification process. For that purpose, we purified the YabA protein with another methodology, based on the addition of a histidine tag (6xHis), and later removed by a TEV protease (see II.RESULT-Chapter 3.1; [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. After affinity purification, we repeated the TLC using this new protein batch (YabA-3). Before performing the TLC assay, the purity and sizes of YabA proteins was checked by loading 15 µg of protein on a SDS-PAGE (Figure

3.C).

Both YabA proteins purified by the two different approaches migrated to the expected size, corresponding to a monomer of YabA (~13 kDa). For each purification batch, a satisfactory purity was obtained. Interestingly, the purity of YabA-3 was higher, and TLC evidenced that GTP hydrolysis by YabA-3 occurred, with an activity even slightly higher (Figure 3.B).

Our work therefore evidenced an unusual nucleotide hydrolysis activity linked to the YabTdependent phosphorylation of YabA onto the T71 residue. If YabA can hydrolyze GTP and ATP, the protein must bind these nucleotides for further hydrolysis. We therefore used a sophisticated biophysical technique to detect ligands interaction named Differential Scanning Fluorimetry (DSF). DSF uses a real-time PCR instrument to monitor thermally induced protein denaturation by measuring changes in fluorescence of a dye that binds preferentially to unfolded protein (such as Sypro Orange, which binds to hydrophobic regions of proteins exposed after unfolding). This experiment is also known as Protein Thermal Shift Assay, because shifts in the apparent melting temperature can be measured upon the addition of stabilizing or destabilizing binding partners or buffer components (Figure 4) [START_REF] Niesen | The use of differential scanning fluorimetry to detect ligand interaction that promote protein stability[END_REF]. Basically, the purified protein is combined with a hydrophobic dye in solution. The dye exhibits a basal intrinsic fluorescence, and its fluorescence greatly increases when it binds to hydrophobic patches on a protein. When properly folded, most hydrophobic parts of proteins are hidden and fluorescence remains low. These hydrophobic surfaces are therefore exposed only when the protein melts, meaning, when it unfolds, loses its native structure. Thus, when the protein is Unfortunately, similar DSF experiments with YabA were not as easy to interpret (Figure 6). Even in the absence of ligand (green curves), a high initial fluorescence background was observed.

However, multiple thermal unfolding transitions occurred when temperature was shift from 25°C to 95°C, suggesting that YabA is probably a highly flexible protein in solution, as further proposed in complementary results. In the presence of non-hydrolysable ATP or GTP, the transition states slightly changed, but it was rather difficult to determinate increases in the corresponding Tm. Nevertheless, in the absence of any ligand, two distinct YabA states were observed, with a first Tm transition detected around 66°C and a second one at 78°C.

Interestingly, addition of non-hydrolysable ATP and GTP seemed to displace these transition states. This technique is incompatible with hydrophobic proteins/ligands and requires compactly folded (globular) proteins; otherwise a high initial fluorescence signal could appear and multiple transitions could be detected. Since YabA forms a tetramer with a high degree of flexibility [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF], DSF is probably not the best technique to detect YabA ligand binding.

Alternatively, Mass spectrometry (MS) appears an analytical tool of choice for the analysis of metabolites resulting from the YabA-dependent hydrolysis of GTP. MS analysis will be performed in collaboration with the « Plateforme Métabolisme-Métabolome » (Université Paris-Sud, le Moulon).

Cyclic-di-GMP

As described in INTRODUCTION-Chapter 4.2.3, cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cellcell signaling, and host colonization. Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation, whereas low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. It has been also demonstrated in B. subtilis that a functional c-di-GMP signaling system directly inhibits motility and directly or indirectly influences biofilm formation [START_REF] Chen | Evidence for cyclic di-GMP mediated signaling in Bacillus subtilis[END_REF]. B. subtilis contains three active DGCs (DgcK, DgcP, and DgcW), one active PDE (PdeH), and a single c-di-GMP receptor (DgrA) [START_REF] Gao | Functional Characterization of core components of the Bacillus subtilis cyclic-di-GMP signaling pathway[END_REF].

In order to study if YabA could regulate biofilm through the cyclic-di-GMP signaling pathway, a collaboration with MF Noirot-Gros (Argonne National Laboratory, Chicago, USA) was established. We used engineered B. subtilis strains that serve as reporters to screen for active DGCs and PDEs. This assay is based on a system that responds to changes in c-di-GMP levels via alterations in the fluorescence of a riboswitch reporter [START_REF] Gao | Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cylases and phosphdiesterases[END_REF]. Riboswitches are cisacting RNA elements generally located at the 5′ untranslated region (5′-UTR) of mRNAs that can alter gene expression by sensing metals, metabolites, or secondary messenger molecules. In response to ligand binding to a riboswitch aptamer, changes occur in the expression platform that result in regulation of downstream open reading frames. In this system, the riboswitch reporter responds to elevation of c-di-GMP levels by increasing the frequency of transcriptional termination upstream of the GFP coding sequence, thereby decreasing the steady-state levels of GFP (Figure 7). This riboswitch was integrated into the B. subtilis amyE gene in both the c-di-GMP null, highly fluorescent strain (ΔydaK ΔdgcK ΔdgcW dgcP::tet pdeH::kn amyE::Pmbox-bc1_GEMM-GFP spec) or in a non-fluorescent strain, highly synthetizing intracellular c-di-GMP, leading to the extinction of GFP expression (pdeH::kn amyE::Pmbox-bc1_GEMM-GFP spec) [START_REF] Gao | Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cylases and phosphdiesterases[END_REF]. This set of strains, called NP401 and NP400, respectively, were successfully used to characterize the DCG and PDE activities of the Clostridium c-di-GMP binding family of proteins [START_REF] Gao | Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cylases and phosphdiesterases[END_REF]. The yabA gene, placed under the control of the IPTG-inducible Pspac promoter was introduced at the lacA ectopic locus. The dgcP and pdeH genes encoding for the main B. subtilis diguanylate cyclase DgcP and the phosphodiesterase PdhE, respectively, were similarly introduced in strains NP401 and NP400 under the control of Pspac at the lacA locus in order to serve as reference set for maximal DCG and PDE activities. The GFP production was monitored in each strains (Figure 8).

The yabA gene was then introduced into both the NP401 and NP400 strains and GFP fluorescence was measured in each strain. In absence of any phosphodiesterase, the c-di-GMP is not degraded in strain NP400, leading to the accumulation of c-di-GMP and therefore to the extinction of GFP expression (Figure 8.A). Ectopic expression of pdhE restored the expression of GFP, weather neither dgcP nor yabA expression allowed the accumulation of GFP in the corresponding strains. From these data we concluded that yabA expression did not restored any phosphodiesterase activity in the corresponding strains. On the other hand, strain NP401 (Figure 8.B) was used to monitor potential diguanylate cyclase activity, which would lead to the accumulation of c-di-GMP and to the inhibition of GFP synthesis. As expected, GFP expression was high in the NP401 recipient strain, due to the absence of c-di-GMP and was not countered when PdeH was expressed. By contrast, expression of the diguanylate cyclase DgcP, as well as expression of YabA partially lowered the production of GFP, suggesting that in these contexts, intracellular level of c-di-GMP raised. From these results we concluded that YabA could mediate the production of c-di-GMP, directly or not (also all genes encoding known diguanylate cyclases have been deleted in the recipient strain). To further study more in details the DCG activity observed in the presence of YabA, NP401 derivative cells were grown in LB for 5 h and subjected to flow cytometry analysis to assess GFP fluorescence. The c-di-GMP null strain (NP401) and the strain expressing the diguanylate cyclase DgcP (NP401-dgcP) were compared with the YabA strain (NP401-yabA). As predicted for the cdi-GMP-responsive reporter, the average cell GFP fluorescence was highest in the c-di-GMP null strain and decreased in the strain expressing DgcP (Figure 9). In cells expressing YabA, two populations with distinct GFP intensities were observed, together with a population of more elongated cells. The first population exhibited a low level of GFP, corresponding to a high level of c-di-GMP, similar to that observed in cells expressing DgcP. This population is also of similar size of cells. The second population exhibited a high level of GFP, comparable to that observed in c-di-GMP null cells. However, this population also correspond to more elongated cells, indicative of filaments. In order to ensure the over expression of yabA did not affect their viability, a cells were stained with Propidium iodide (PI). Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability. It is a membrane impermeant dye generally excluded from viable cells. A few quantity of dead cells were detected in the c-di-GMP null strain and in cells expressing YabA. However, the two populations observed in the strain expressing YabA corresponded to living cells, indicating a change in morphology and not in viability. Cells were also observed by microscopy, revealing two types of cells, either small or elongated sized. The "elongation effect" may expected to results from a defect in replication control, linked to the overexpression of YabA. Therefore, as it is shown in Figure 9, the small sized cells, which exhibit a low GFP level, could reflect a role of YabA in controlling the intracellular c-di-GMP levels.

A B

These results provide a support to our hypothesis that YabA could be involved in biofilm formation via the c-di-GMP signaling pathway (directly or indirectly). Since no c-di-GMP binding signature is present on YabA, it is probably that YabA could trigger indirectly a yet unknown DGC activity. Nevertheless, all DGC encoding genes are supposed to be deleted in strain NP401.

Therefore YabA could control an unknown c-di-GMP enzyme. The second hypothesis is that YabA acts directly on the intracellular GTP pool. We previously observed in vitro that YabA exhibits an unusual GTP hydrolysis activity. If this hydrolysis activity induces a significant drop in intracellular levels of GTP, then the synthesis of c-di-GMP, which results from the cyclization of two GTP molecules, must also be down. This is not in agreement with our experimentations. Indeed, we observed that overexpression of YabA induces an increase in intracellular levels of c-di-GMP.

However, YabAT71D is associated in vitro with a high GTP hydrolysis activity and its expression in vivo is associated with a defect in biofilm formation. Since YabA only slightly hydrolyzes GTP, this activity may be not enough to affect the intracellular c-di-GMP level via availability of GTP.

Supplementary materials and methods

In-gel ATPase assay

The in-gel assay followed the principles described by [START_REF] Zerbetto | Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels[END_REF]

Thin Layer Chromatography (TLC)

The

DGC and PDE assay

Genes encoding YabA, DgcP and PdhE were introduced in the NP401 (ΔydaK ΔdgcK ΔdgcW dgcP::tet pdeH::kn amyE::Pmbox-bc1_GEMM-GFP spec) and NP400 (pdeH::kn amyE::Pmbox-bc1_GEMM-GFP spec) strains backgrounds. B. subtilis strains were streaked on LB plates with 100 μg/ml spectinomycin and allowed to grow overnight at 37°C. A single colony was used to inoculate LB culture containing 1 mM IPTG. After 5 h expression, GFP fluorescence was monitored.

Fluorescence-activated cell sorter (FACS) analysis

B. subtilis strains were streaked out on LB plates containing 100 μg/ml spectinomycin and allowed to grow overnight at 37°C. A single colony was then picked for inoculation in LB containing 250 µM IPTG. After 3 and 5 h at 37°C, 20 μl culture was diluted into 1 ml PBS, and samples were analyzed using flow cytometer with excitation at 488 nm. Only after 5h of expression cells were stained with Propidium iodide prior to monitor fluorescence to detected dead cells. Results were analyzed using FloJo software.

II. RESULTS -CHAPTER 3

Complementary results: YabA structure and architectural organization of its complex with DnaA and DnaN To address this question, a combination of physical, computational, biochemical and biological approaches were used. These approaches involved X-ray crystallography, small angle X-ray scattering (SAXS), in silico 3D-modeling, biophysical studies, mutational yeast two-and three

INTRODUCTION

In all living organisms, chromosome replication is highly regulated to ensure only one initiation event per chromosome per cell cycle (1). Bacteria have developed various strategies to prevent inappropriate re-initiation, principally by regulating the activity and/or the availability of the master initiator protein DnaA. DnaA assembles at specific DNA sequences within oriC to promote the opening of the DNA duplex, and directs the assembly of the replisome machinery by first recruiting DNA helicase (1,2). DnaA is a member of the AAA+ (ATPases associated with diverse cellular activities) superfamily that binds to and hydrolyses adenosine triphosphate (ATP). Although both ATP and adenosine diphosphate (ADP) bound forms of DnaA are proficient in oriC binding, only the ATP-bound form is active in replication initiation (3).

In bacteria, multiple homeostatic mechanisms contribute to coordinate DNA replication with the cellular cycle (1,4). In Gram negative bacteria, proteins have been identified which regulate positively or negatively the initiation of replication by forming a complex with DnaA. Positive regulators such as DiaA in Escherichia coli, or the structural homolog HobA in Helicobacter pylori promote initiation by stimulating the assembly of ATP-DnaA at oriC [START_REF] Keyamura | DiaA dynamics are coupled with changes in initial origin complexes leading to helicase loading[END_REF][START_REF] Keyamura | The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP DnaA-specific initiation complexes[END_REF][START_REF] Natrajan | The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria[END_REF]. In E. coli, the major mechanism of initiation control is the regulatory inactivation of DnaA (RIDA), mediated by the ADP-activated protein Hda (homolog of DnaA) and the ␤-clamp subunit of the replicative DNA polymerase DnaN. The interaction between Hda, DnaN and ATPbound DnaA, promotes the hydrolysis of ATP and the accumulation of inactive ADP-DnaA (8). RIDA has also been reported in Caulobacter crescentus, a Gram-negative proteobacteria phylogenetically distant from E. coli [START_REF] Collier | Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter[END_REF]. In this bacterium, the Hda homolog HdaA prevents overinitiation and co-localizes with the replication machinery in the stalked cell. However, the presence of Hda seems to be restricted to proteobacteria suggesting that other bacterial species have developed different mechanisms for the negative regulation of DnaA.

In the Gram positive and spore forming bacterium Bacillus subtilis, replication initiation control is fulfilled by various proteins, which bind to DnaA and modulate its activity during the different life-styles of the bacilli. In cells committed to sporulation, the protein SirA prevents replication reinitiation by antagonizing DnaA binding to oriC [START_REF] Jameson | Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA[END_REF][START_REF] Rahn-Lee | The sporulation protein SirA inhibits the binding of DnaA to the origin of replication by contacting a patch of clustered amino acids[END_REF][START_REF] Wagner | SirA enforces diploidy by inhibiting the replication initiator DnaA during spore formation in Bacillus subtilis[END_REF]. In vegetative cells, the primosomal protein DnaD and the ParA-like protein Soj were recently found to down regulate replication initiation by preventing the formation of a DnaA nucleofilament at oriC [START_REF] Merrikh | Control of the replication initiator DnaA by an anti-cooperativity factor[END_REF][START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF]. The main regulatory protein YabA was also found to prevent over-initiation by inhibiting the cooperative binding of DnaA to oriC [START_REF] Merrikh | Control of the replication initiator DnaA by an anti-cooperativity factor[END_REF][START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF]. Furthermore, YabA downregulates replication initiation as part of a multimeric complex with DnaA and DnaN that is associated with the replication factory [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF][START_REF] Soufo | Cell-cycle-dependent spatial sequestration of the DnaA replication initiator protein in Bacillus subtilis[END_REF][START_REF] Hayashi | Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation[END_REF][START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF]. Thus, YabA is likely to act by trapping DnaA in a manner that can be both dependent and independent of DnaN during the cell replication cycle. However, although YabA, like Hda, regulates initiation through coupling to the elongation of replication, several pieces of evidence point to a distinct mode of action. In contrast to Hda, YabA has no sequence similarity to DnaA, and YabA does not belong to the AAA+ superfamily. YabA is a small protein of 119 residues, with an unusual predicted organization composed of a leucine zipper at its N-terminus and a putative zinc-binding domain at its C-terminus [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Curiously, it lacks the classical bacterial DnaN-binding pentapeptide consensus motif [START_REF] Dalrymple | Identification of putative DnaN-binding motifs in plasmid replication initiation proteins[END_REF]. Given that YabA is conserved in low-GC Gram-positive bacteria, the protein represents the prototype of a distinct family of replication controller proteins.

To gain insight into the mechanism by which YabA interacts with multiple partners to control DNA replication, we have combined the X-ray structure of the N-terminal domain (NTD) of YabA, in silico modeling of the C-terminal domain (CTD) and small angle X-ray scattering (SAXS) of the full-length protein to derive a model of the YabA structure. The crystal structure of the NTD of YabA revealed an extended ␣-helix, four of which assemble into a helical bundle. We found that the CTD of YabA binds to a single Zn 2+ ion with a zinc binding motif resembling known short Zn binding domains. Solution studies demonstrated that YabA is a tetramer in which the monomeric CTDs are separated from the tetrameric NTDs by a flexible linker resulting in an extended conformation. We then showed that the CTD is sufficient for interaction with either DnaA or DnaN. The interacting surfaces with DnaA and DnaN have been mapped within the CTD and unveil an atypical motif for binding to the ␤-clamp. Our study provides a structural explanation for the capacity of YabA to bind multiple partners, hereby defining a novel protein hub central to DNA replication in low GC Gram-positive bacteria.

MATERIALS AND METHODS

Proteins expression and purification

The yabA gene sequence was amplified from B. subtilis (strain 168) genomic DNA by polymerase chain reaction (PCR) and inserted into pET151/D-TOPO (Invitrogen) to generate the plasmid pET151-yabA. E. coli BL21 (DE3) cells (Invitrogen) carrying pET151-yabA were grown in LB medium (with ampicillin at 100 g/l) at 37 • C until OD 600 = 0.6. Protein expression was induced with 1 mM isopropyl ␤-D-thiogalactopyranoside (IPTG) for 16 hours at 20 • C. Cells were centrifuged and resuspended in buffer L (30 mM Tris pH 8, 150 mM NaCl, 1% Triton, 1 mM ZnCl 2 and 5% Glycerol (V/V)) with protease inhibitor tablet (complete EDTA-free, Roche), lysozyme (Roche) and Dnase (Sigma-Aldrich). The cells were lysed by sonication and centrifuged at 16 000 g for 20 min. The soluble fraction was loaded on a HisTrap TM 5 ml column equilibrated with buffer A (30 mM Tris pH 8, 150 mM NaCl, 5% glycerol) and the protein eluted using a 0-100% gradient of buffer B (buffer A + 500 mM imidazole). Fractions containing YabA were incubated with TEV protease in the presence of 0.5 mM dithiothreitol (DTT) and 0.5 mM ethylenediaminetetraacetic acid (EDTA) and dialyzed for 16 h against buffer A at 4 • C. The cleaved histidine tag was subsequently removed by passage of the dialysate through a HisTrap TM column. The flowthrough fraction was concentrated and injected onto a Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated with buffer A. Fractions containing YabA were pooled and concentrated to 5 mg.ml -1 .

To produce the YabA/DnaN complex, a vector (pET Lic duet yabA-dnaN) was used which directs the coexpression of N-terminally histidine tagged YabA and DnaN. E. coli BL21 (DE3) cells (Invitrogen) harboring (pET Lic duet yabA-dnaN) were grown in LB medium (with kanamycin at 40 g/l) at 37 • C until OD 600 = 0.6. Protein expression was induced by addition of 1 mM IPTG and cells were incubated 3 h at 37 • C. Cells were harvested by centrifugation and resuspended in buffer L with a protease inhibitor tablet (complete EDTA-free, Roche), lysozyme (Roche) and Dnase (Sigma-Aldrich). The cells were lysed by sonication and centrifuged at 16 000 g for 20 min. The soluble fraction was diluted four times with buffer A and applied on a HisTrap TM 5 ml column equilibrated with buffer A. The protein complex was eluted using a 0 to 100% gradient of buffer B. Fractions containing the YabA/DnaN complex were pooled and applied to a Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated with buffer A. The pure YabA/DnaN complex was concentrated to 5 mg ml -1 .

For the preparation of YabA 1-58 and YabA 70-119 , the coding region of yabA was amplified from B. subtilis genomic DNA (strain 168) by PCR using gene-specific primers, with appended sequences to facilitate ligation independent cloning. The PCR product was inserted into the vector pET-YSBLIC3C by ligation-independent cloning methods [START_REF] Fogg | Higher-throughput approaches to crystallization and crystal structure determination[END_REF]. Plasmids encoding N-and C-terminal fragments of YabA, residues 1-58 (YabA 1-58 ) and residues 70-119 (YabA 70-119 ) respectively, were created using a deletion mutagenesis method. A whole vector amplification of pET-YSBLIC3C-YabA was performed by PCR using the primers listed in Supplementary Table S5. The recombinant plasmids pET-YSBLIC3C-YabA 1-58 and pET-YSBLIC3C-YabA 70-119 direct the expression of YabA 1-58 or YabA 70-119 , respectively, each fused to a 3C cleavable N-terminal His-tag. The recombinant YabA fragments were overproduced in E. coli BL21 (DE3) and following cell lysis, purified by steps of immobilized nickel affinity and gel filtration chromatography similar to those described above with the affinity tag subsequently being removed by HRV 3C protease.

Crystallization, structure determination and refinement

Two crystals forms of YabA were obtained using vapor diffusion methods in hanging drops consisting of 1.0 l of protein or protein complex and 1.0 l of reservoir solution containing 20% PEG 3350 (W/V), 100 mM Bis-Tris propane pH 6.5 and 200 mM potassium thiocyanate. Crystal form I was obtained using the purified YabA/DnaN complex (5 mg.ml -1 ). A second crystal form (II) was obtained using YabA (5 mg.ml -1 ) and by including 0.025% (V/V) dichloromethane in the drop. For structure determination, form II crystals were soaked in reservoir solution supplemented with 10 mM platinum potassium thiocyanate for 16 h. Crystals were then flash-cooled in liquid nitrogen (100K) using mother liquor containing step-wise additions of glycerol (15% final concentration) as cryoprotectant. Xray diffraction data were collected at the ID14EH4 (form I) and ID23EH1 (form II) beamlines of the European Synchrotron Radiation Facility (ESRF, Grenoble). Form I crystals belonged to the space group P3 2 21 with unit cell dimensions of a = b = 83.4 Å and c = 64.7 Å and diffracted to a resolution of 2.7 Å. Crystal form II belonged to the space group P6 1 22 with unit cell dimensions of a = b = 83 Å and c = 110 Å and diffracted to a resolution of 4 Å according to CC 1/2 [START_REF] Karplus | Linking crystallographic model and data quality[END_REF]. The diffraction data were indexed and integrated using XDS [START_REF] Kabsch | Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants[END_REF] and scaled with SCALA from the CCP4 program suite [START_REF]The CCP4 suite: programs for protein crystallography[END_REF]. Data collection statistics are given in Table 1.

A single high redundancy dataset was collected at the Pt edge on a form II crystal soaked into platinum thiocyanate. The structure was solved using Autosol from the PHENIX package [START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF]. The experimental map obtained at 4 Å and the two Pt sites identified were used in Autobuild which resulted in a non crystallographic symmetry (NCS) averaged, solvent flattened and improved map [START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF]. The initial model consisted of two polyalanine helices of 30 residues and was subsequently extended manually in the improved low-resolution experimental map. The model was then used for molecular replacement using the data obtained from crystal form I. The model was built manually using COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and refined using PHENIX [START_REF] Adams | PHENIX: building new software for automated crystallographic structure determination[END_REF]. Phasing and refinement statistics are indicated in Table 1. Figures depicting the structure were generated with PyMOL (26). The coordinates of YabA 1-62 have been deposited in the protein databank (pdb code 5DOL).

SEC-MALS

Samples (100 l) of YabA, YabA 1-58 and YabA 70-119 were loaded onto gel-filtration columns equilibrated with 50 mM Tris pH 8.0, 150 mM NaCl. YabA was loaded at a concentration of 2.5 mg.ml -1 onto a Superdex 200 HR 10/30 column and YabA 1-58 and YabA 70-119 were loaded at concentrations of 5 mg.ml -1 onto a Superdex 200/75 HR 10/30 column. In each case, the column eluate was successively analyzed by a SPD20A UV/Vis detector, a Wyatt Dawn HELEOS-II 18-angle light scattering detector and a Wyatt Optilab rEX refractive index monitor. Data were analyzed with Astra software (Wyatt).

Circular dichroism

Circular dichroism spectra were recorded at 20 • C on a Jasco J-810 CD spectrophotometer using a quartz cell with a 0.1 cm path length. Experiments were carried out at protein concentrations of 0.2 mg.ml -1 in a buffer of 20 mM potassium phosphate, pH 8.0. Spectra were recorded across the wavelength range of 260-185 nm. A buffer scan was also recorded and subtracted from the protein spectra to remove any contribution from the buffer. Analysis of YabA CD spectra was performed using Dicroweb (http://dichroweb. cryst.bbk.ac.uk) [START_REF] Whitmore | Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases[END_REF].

Atomic absorption spectroscopy

For AAS we used a Phillips PU9200 double beam spectrometer equipped with a flame volatilization system. Samples of YabA dissolved in deionized water at 0.5 mg.ml -1 were analyzed in triplicate using a zinc-specific lamp of wavelength 213.9 nm and the absorbance values were compared to those for a standard curve derived from samples of zinc powder dissolved in HCl.

In silico molecular modeling of CTD

Template Search and Secondary Structure Prediction: the amino acid sequence analysis was performed using the Position Specific Iterated BLAST (PSI-BLAST) and Pattern Hit Initiated BLAST (PHI-BLAST) methods at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) and using the All Non-Redundant (NR) amino acid sequence database from April 2000, which includes SwissProt, CDS translation of GenBank (gb), EMBL (emb), the DNA database of Japan (dbj) and the Protein Structure Database (pdb) [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. Default amino acid replacement matrices and gap penalties were used in all database searches. Secondary structure predictions were made using JPRED (http://www.compbio. dundee.ac.uk/jpred) through the Jalview version 2 software [START_REF] Lu | Multiple sequence alignment based on profile alignment of intermediate sequences[END_REF].

Full length YabA and two fragments encompassing residues 1-70 and 71-119 were scanned against the protein databank (PDB) by PSI-BLAST over five iterations. PHI-BLAST was also used to search templates for the C-terminal part of YabA. The templates selected by the BLAST approach were aligned with YabA sequence using ISPALIGN (Intermediate Sequence Profile Alignment) [START_REF] Lu | Multiple sequence alignment based on profile alignment of intermediate sequences[END_REF]. A manual alignment adjustment was performed with the help of Geneious Pro 4.8.4 software (Biomatters Ltd).

The identity and similarity between YabA-CTD and its templates (aminoacids 76-119) were computed using SIAS tool (http://imed.med.ucm.es/Tools/sias.html) with a BLO-SUM62 matrix. Based on alignment results, a 3D model of YabA was obtained by homology modeling using the software package Modeller 9v8 [START_REF] Eswar | Comparative protein structure modeling using Modeller[END_REF]. Image rendering and H95 position refinement were performed using Py-MOL (DeLano Scientific LLC) sculpting properties. The Qmean package was used to evaluate model quality (http: //swissmodel.expasy.org/qmean) [START_REF] Benkert | QMEAN server for protein model quality estimation[END_REF][START_REF] Benkert | Toward the estimation of the absolute quality of individual protein structure models[END_REF].

SAXS data collection

Protein samples were centrifuged for 10 min at 10 000 rpm prior to X-ray analysis in order to eliminate all aggregates. Sample concentration was measured by UV absorption at = 280 nm on a Thermo Scientific NanoDrop 1000 Spectrophotometer. For each sample, a stock solution was prepared at a final concentration of 8 mg/ml and stored at 4 • C and then directly used for the experiments. SAXS experiments were conducted on the SWING beamline at the SOLEIL synchrotron ( = 1.033 Å). The Aviex chargecoupled device detector was positioned to collect data in the q-range 0.006-0.5 Å-1 (Q = 4sin. -1 , where 2 is the scattering angle). The solution was injected in a fixedtemperature (15 • C) quartz capillary with a diameter of 1.5 mm and a wall thickness of 10 m, positioned within a vacuum chamber. A total of 80 l of monodisperse protein sample were loaded onto a size-exclusion column (SEC-3 300 Agilent), using an Agilent HPLC system and eluted directly into the SAXS flow-through capillary cell at a flow rate of 0.2 ml min -1 [START_REF] David | Combined sampler robot and high-performance liquid chromatography: a fully automated system for biological small-angle X-ray scattering experiments at the Synchrotron SOLEIL SWING beamline[END_REF]. The elution buffer consisted of 50 mM Tris-HCl pH 8, 150 mM NaCl, 5% glycerol filtered and degassed. SAXS data were collected continuously, with a frame duration of 1.5 s and a dead time between frames of 0.5 s. Data reduction to absolute units, frame averaging and subtraction were done using FOXTROT, a dedicated homemade application. All subsequent data processing, analysis and modeling steps were carried out with PRIMUS and other programs of the ATSAS suite [START_REF] Konarev | TSAS 2.1, a program package for small-angle scattering data analysis[END_REF].

SAXS data processing, analysis and molecular modeling

The experimental SAXS data for all samples were linear in a Guinier plot of the low q region, indicating that the proteins had not undergone aggregation. The radius of gyration R g was derived by the Guinier approximation I(q) = I(0) exp(-q 2 R g 2 /3) for qR g < 1.0 using PRIMUS [START_REF] Konarev | PRIMUS: a Windows PC-based system for small-angle scattering data analysis[END_REF]. The program GNOM [START_REF] Svergun | Determination of the regularization parameter in indirect-transform methods using perceptual criteria[END_REF] was used to compute the pairdistance distribution functions, p(r). This approach also features the maximum dimension of the macromolecule, D max . The overall shapes of the entire assemblies were de-rived from the experimental data using the program GAS-BOR [START_REF] Svergun | Determination of domain structure of proteins from X-ray solution scattering[END_REF]. These models were averaged to determine common structural features and to select the most typical shapes using the programs DAMAVER [START_REF] Volkov | Uniqueness of ab initio shape determination in small angle scattering[END_REF] and SUPCOMB [START_REF] Kozin | Automated matching of highand low-resolution structural models[END_REF]. An initial model of the YabA tetramer was built by assembling a tetramer of the NTD (residues 1-61) as seen in the crystal structure with four homology models of the CTD (residues 76-119). A linker of 14 residues between each NTD and CTD generated a complete full-length tetramer. To generate a model of full-length YabA using the crystal structure, and the homology model, which was compatible with the SAXS data, we performed atomic modeling using Dadimodo [START_REF] Evrard | DADIMODO: a program for refining the structure of multidomain proteins and complexes against small-angle scattering data and NMR-derived restraints[END_REF], a genetic algorithm based rigid-body refinement analysis program. The NTD and CTD structures were not modeled further but the linker region of YabA and the positions of the four CTDs were allowed to move. Continuity of the structure was assured by subsequent energy minimization using Dadimodo [START_REF] Evrard | DADIMODO: a program for refining the structure of multidomain proteins and complexes against small-angle scattering data and NMR-derived restraints[END_REF]. A SAXS 2 value was then computed for each eligible structure, using CRYSOL. The averaged scattering curve of YabA and the fitting curve calculated from the model produced by Dadimodo were superimposed with CRYSOL [START_REF] Svergun | CRYSOL -a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates[END_REF].

Yeast two-hybrid and three-hybrid assays

Full length YabA and the C-terminal region , full length DnaA and an N-terminally truncated DnaA fragment (71-440), and DnaN proteins were expressed as fusions to the GAL4 binding domain BD or activating domain AD from the vectors pGBDU-C1 and PGAD-C1, respectively. pGBDU and PGAD derivative constructs were introduced by transformation into PJ694-(␣) and (a) haploid strains, respectively. Binary interactions were tested by combinational mating of the strains expressing the BD and AD fusions as previously described [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Interacting phenotypes are tested by the ability of the diploid cells to grow on selective media SD-LUH and SD-LUA. For 3HB experiments, DNA fragments encoding full length YabA or the C-terminal domain were inserted into the p3HB vector [START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF]. The p3HB-YabA derivatives were co-expressed with either DnaA or DnaN in haploid (␣) strains and the strains expressing the different combination pairs are then mated again with haploid (a) strains expressing the appropriate AD-fusion.The ability of a given BD-protein fusion to interact with a given AD-protein fusion conditional upon the presence of a third protein is attested by the appearance of growth on SD-LUWH and SD-LUWA selective media as previously described [START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF].

Site targeted mutagenesis and analysis of Loss-of-Interaction (LOI) phenotypes by yeast-two hybrid

Site-directed saturation mutagenesis of yabA was performed by PCR amplification and fragment joining using degenerate oligonucleotides containing a randomized codon at the targeted position. The mutated PCR amplified yabA coding sequences were cloned into the pGBDU vector, in frame with the BD domain of GAL4 as previously described [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Mutations were identified by sequencing and pGBDU-yabA derivatives carrying mutations at the targeted codon leading to different amino-acid sub-stitutions were introduced into the pJ69-4 (a) yeast haploid strain by transformation. Yeast haploid cells expressing the BD-yabA mutant derivatives were mated with haploid strains of the opposite mating type (pJ69-4 (␣)), harboring the pGAD-partner constructs encoding interacting proteins DnaA, DnaN or YabA fused to the AD domain of GAL4, as previously described [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Interacting phenotypes were assessed according to the ability of the diploid forms to grow on (-LUH and -LUA) selective media. Mutants were tested for their ability to express interacting phenotypes with each of the three partners. The screening for loss-of-interaction (LOI) mutations against a single partner while maintaining the ability to bind to the other partners precludes the isolation of mutations that destabilize the tertiary structure of YabA.

Construction of GFP fusions

Wild-type and mutant yabA genes were PCR amplified from the corresponding pGBDU-yabA constructs for subsequent cloning into the vector pGS1190 to generate cfp-yabA fusions under the control of the xylose-inducible promoter P xyl [START_REF] Feucht | Improved plasmid vectors for the production of multiple fluorescent protein fusions in Bacillus subtilis[END_REF]. The cfp-yabA constructs were then integrated into the amyE locus of the B. subtilis yabA strain, JJS142 (Supplementary Table S4). For the fluorescence complementation assay, genomic DNA from a strain expressing the yfp-yabA-N85D fusion under the control of its native promoter at the locus position, ( 17) was used to transform the recipient strains carrying the xylose inducible cfp-yabA constructs at amyE locus (see Supplementary Table S4), using chloramphenicol (5g/ml) for selection.

Fluorescence microscopy

Cells expressing the CFP-tagged YabA mutant derivatives and cells co-expressing CFP-YabA and YFP-YabA were grown and treated as previously described [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. For microscopic observations, the cells were mounted on agarose slides [START_REF] Glaser | Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning[END_REF]. Images were acquired by using an HQ snap digital charge-coupled device camera and analyzed using Metamorph v3 software. Appropriate filter sets to visualize the CFP and YFP fluorescence signals were obtained from Leica/WWR. DNA was stained with DAPI, and membranes were stained with FM5-95 (Molecular Probes).

RESULTS

YabA is a tetrameric, multidomain Zn-binding protein

Homology-based sequence analysis suggested that YabA consists of N-terminal (residues 1-56) and C-terminal (residues 73-119) structural domains separated by a region of low complexity and poorly conserved sequence, which we will refer to as a 'linker' or 'hinge' (Figure 1A, S1). The N-terminal region includes a conserved leucine heptad repeat (residues 6-48) and a regular distribution of charged residues, pointing to a potential leucine zipper-like domain. The C-terminal region exhibits higher sequence conservation, with three cysteine residues (C97, C109, C112) and one histidine (H95) being invariant in YabA sequences, suggesting they participate in the coordination of a zinc ion (Figure 1A and B and Supplementary Figure S1). A PSI-BLAST search of non-redundant sequence databases using the C-terminal region of YabA as seed allowed us to assign a YabA-like function to 46 additional proteins harboring the HCCC motif in firmicutes (Supplementary Table S1).

The presence of zinc atoms in purified B. subtilis YabA was thus studied by spectroscopic methods. Atomic absorption spectra of the purified protein dissolved in deionized water revealed strong absorption at 231.9 nm diagnostic of the presence of zinc (Figure 1C). Quantitative measurements calibrated against a zinc standard curve gave a zinc:YabA molar ratio of 0.94 indicating that one zinc ion is bound per YabA chain. These data suggest that YabA defines a novel class of bacterial Zn binding proteins in which the HCCC motif could be used to coordinate one Zn per protein chain.

To assess the overall secondary structure composition, circular dichroism spectra were recorded for purified YabA, as well as NTD and CTD recombinant fragments, YabA 1-58 and YabA 70-119 (Figure 1D). The spectra for all three species exhibit shallow minima in their molar ellipticity over the wavelength range 205-225 nm and increasing molar elliptic-ity with decreasing wavelength below 205 nm. These strong secondary structure characteristics indicate that the proteins are folded. Analysis of these spectra using Dicroweb confirmed this conclusion and indicated that the secondary structures of full length YabA and its domain fragments feature a high percentage of ␣-helices. To determine the quaternary structures of YabA, YabA 1-58 and YabA 70-119 , the proteins were next analyzed by size exclusion chromatography and multi-angle laser light scattering (SEC-MALS). In these experiments, the protein samples are analyzed on a gel-filtration column and the absorbance at 280 nm and the refractive index of the eluting species are monitored together with the multi-angle laser light scattering of the sample. YabA eluted as a single peak from an S200 column with a retention time of ∼26.5 min (Figure 1E and Supplementary Figure S2) and a molecular mass of 58.7 kDa. As the theoretical molecular mass of YabA is 14.3 kDa, we concluded that recombinant YabA is a tetramer (57.2 kDa) in solution as previously reported [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. The N-terminal fragment YabA 1-58 had a retention time of ∼21.5 min on an S75 column and this peak was associated with an Mw of 27.7 kDa (Figure 1E and Supplementary Figure S2). This value is in agreement with the mass calculated for a YabA 1-58 tetramer (28.8 kDa). Interestingly, YabA 70-119 eluted from the same column as a single peak with a retention time of ∼28 min and an associated Mw of 6.2 kDa (Figure 1E, Supplementary Figure S2), indicating a monomeric state for this C-terminal domain fragment (theoretical molecular mass of 6.0 kDa). We conclude that YabA tetramer formation relies predominantly on the N-terminal portion of the molecule.

Crystal structure of YabA NTD

We attempted to crystallize YabA and obtained two crystal forms (form I and II, see 'Materials and Methods' section for details). In both cases, analysis of the unit cell contents and lattice interactions indicated that proteolysis occurred during crystallization. The final model allowed us to delineate the NTD boundary of the crystallized fragment as residing around residue F62 in the form I crystals, fragment that will be thus described as YabA 1-62 . The Form I and II crystals each contain two molecules of YabA 1-62 per asymmetric unit arranged almost identically. The structure obtained with form I crystals was refined to a final resolution of 2.7 Å with good geometry (Table 1). The YabA 1-62 protomer consists of a single 90 Å long ␣-helix comprising 15 turns with a pronounced curvature at its center (Figure 2A). YabA 1-62 assembles as a dimer of dimers in the crystal consisting of chains A and B and their symmetry equivalents A' and B' (Figure 2B). The A'B dimer resembles a pair of tweezers (Figure 2C) assembled via tight interactions of the C-terminal elements of the two helices whose arrangement can be described as a parallel coiled-coil. Two pairs of these tweezers (AB' and A'B) embrace each other to form the head-to-head tetramer (Figure 2D). The tetramer thus comprises a central four-helix bundle flanked by pairs of helical extensions arranged as coiled coils (Figure 2B andD). The packing of the molecules appears to be tight with ∼40% of the surface area of each chain buried by intermolecular interactions, with each subunit forming extensive contacts with its three neighbors. As a result, there is a total buried surface area of 9880 Å2 in the tetramer. The four-helix bundle comprises residues 1-35 of the four chains with chains A and B' running antiparallel to chains A' and B. The interior of the bundle is formed by the close packing of the aliphatic side chains of L6, V10, L13, I17, L20, L24, L27, I31 and M34 from all four chains creating a substantial hydrophobic core (Figure 2E). The heptadic arrangement of L6, L13, L20 and L27 gives rise to a leucine zipper-type interdigitation of side chains projecting from pairs of adjacent chains (A-A' and B-B'). Sets of reciprocal charge-charge interactions (four in total) between the E14 and K28 side chains on the A chains and hydrogen bonds between Q16 and Q23 side chains on the B chains presumably lend further stability to the tetramer (Figure 2E).

The flanking coiled-coil regions are formed by the Cterminal halves of chains A and B' on one side of the four helical region and chains A' and B on the other (residues . These pairs of helices, which are splayed in the four helix bundle region, converge at their C-termini (Figure 2C) where hydrophobic interactions of the side chains of M34, L41, L48, L52 and I59 with the equivalent residues in the partner chain are augmented by hydrogen bonding interactions of the side chains of N38, N45, T55 and most strikingly by reciprocal salt-bridging interactions between the side chains of E44 and R49 (Figure 2F). The crystal structure of YabA 1-62 is consistent with the circular dichroism data which suggested a high proportion of ␣-helix in the YabA 1-58 fragment and the SEC-MALS data which clearly point to a tetramer. The structure also shows that residues 59-61 previously assigned to the linker region can adopt a ␣-helical conformation.

YabA CTD in silico structure model

In the absence of a crystal structure for the CTD, we searched for relevant structural templates to build a threedimensional model of YabA CTD. Using a PSI-BLAST search we first identified Saccharomyces cerevisiae profilin (pdb code: 1YPR) which exhibited partial similarity to YabA CTD although it does not contain a zinc finger motif [START_REF] Eads | Structure determination and characterization of Saccharomyces cerevisiae profilin[END_REF]. In order to refine the template identification for the zinc finger motif, a hypothesis-driven pattern search was performed. Protein of known structures containing variations of the zinc-binding motif of YabA H-x-C-x(11)-Cx(2)-C were identified using PHI-BLAST [START_REF] Zhang | Protein sequence similarity searches using patterns as seeds[END_REF]. These include a series of zinc finger proteins that belong to the PHD, RING, CHY and FYVE domain families (Supplementary Table S2). The structure of the endosome targeting protein EEA1 (pdb code: 1JOC) ( 46) was of particular interest since the sequence similarities extend beyond the FYVE zinc-binding domain to the linker region and the NTD of YabA (Figure 3A andB). Based on a multiple alignment of the combination of homologous regions of interest from the structural templates, a 3D-model of YabA CTD was built using constraint-based homology modeling procedures implemented in Modeller 9v8 (24) (Figure 3B). To evaluate the quality of its fit to experimental structures the model was next validated by QMEAN [START_REF] Benkert | Toward the estimation of the absolute quality of individual protein structure models[END_REF]. The determined Qmean Zscores were -1.59 for CTD spanning amino acid residues 76-119 and -3 for the region corresponding to model structure from residues 63 to 119. This indicated that the structural features of the YabA CTD model were of a quality comparable to high resolution structures [START_REF] Benkert | Toward the estimation of the absolute quality of individual protein structure models[END_REF]. In addition, the YabA CTD (residues 76-119) sequence has 44% similarity and 35% identity with profilin, and accordingly the YabA model exhibited two short alpha-helices (Figure 3A, ␣2 and ␣3). Structural alignment with EEA1 revealed 12% identity and 19% similarity with YabA CTD. Similar to EEA1, the YabA zinc finger residues H95 and C110 (Figure 3A, upper case residues) and C97 and C112 (Figure 3A, lower case residues) were, respectively, inaccessible and accessible to solvent (Figure 3A, orange dots). These results stress the structural relevance of the two templates used here to build the YabA CTD model. The 3D model consists of two short helices (␣2 and ␣3) followed by zinc binding loops (Figure 3B). Interestingly, the model positions the residues of the HCCC in a fold similar to the short zinc-binding loops found in other proteins [START_REF] Krishna | Structural classification of zinc fingers: survey and summary[END_REF].

Next, the model was assessed using knowledge of surface exposed residues. A previous functional dissection of YabA identified single residue changes that selectively disrupted interactions with one partner protein, while maintaining interactions with its other protein partners [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. In view of the selective effects of these LOI mutations, the substituted residues are expected to be exposed on the surface where their mutation is unlikely to affect the overall structure and stability of the protein. The sites of the LOI residue substitutions were mapped onto the YabA model (Figure 3C). The residues important for interaction with DnaA and DnaN were found to be surface exposed, defining non-overlapping interacting surfaces on the C-terminal zinc-binding domain. Residues Y83, N85 and L86, important for interaction with DnaA, are clustered on the helical side of the CTD surface when viewed as in Figure 3C while residues V99, H100 and L110 involved in interaction with DnaN are grouped predominantly on a surface formed by protein loops. This mapping of residues important for interaction with DnaA and DnaN to the surface of the YabA CTD provides an important validation of our in silico structural model.

Low-resolution structure of the YabA tetramer

To gain insight into the overall architecture of the fulllength tetrameric YabA, we used SAXS. YabA eluted from the online HPLC as a single peak and SAXS data indicated a radius of gyration (R g ) of 63.2 Å and a maximum dimension (D max ) of 250 Å (Figure 4A). These values are very high for a low molecular weight protein, even for a tetramer. The presence of a flexible region is suggested by the shape of the Kratky plot which displays a flattened bell shape at low values of s 2 corresponding to the folded part of the protein, followed by a continuous rise at the higher angle sections [START_REF] Bernado | Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering[END_REF] (Supplementary Figure S3A). Such dual behavior is indicative of flexibility in multidomain proteins [START_REF] Bernado | Effect of interdomain dynamics on the structure determination of modular proteins by small-angle scattering[END_REF]. An extended YabA structure is also strongly suggested by the autocorrelation function with a shape characteristic of an elongated object (Figure 4A). In the crystal structure of the NTD, the C-termini of chains A and B' (residues 62) are separated by ∼130 Å from the C-termini of chains A' and B. The CTD model encompassing residues 76-119 is of around 25 Å at its maximum radius. Coupled with the observation from the SEC-MALS experiments that YabA 70-119 is monomeric, these data suggest that residues 63-75 may act as an extended flexible linker between the NTD and the CTD. This is also supported by the fact that residues 60-80 are poorly conserved in YabA sequences (Supplementary Figure S1).

Using the scattering curve, a low-resolution structure of YabA was calculated by ab initio modeling with the program GASBOR without imposing symmetry constraints. In this case, we obtained a set of very different shapes (data not shown). To limit the range of possibilities, internal P 22 symmetry was imposed, giving a shape composed of a cylindrical core with pairs of elongated arms attached to each end (Figure 4B). To gain better insights into the structure of the tetramer of YabA, we combined the crystal structure of the YabA NTD tetramer, with the CTD structure obtained by molecular modeling. Each N-terminal helix was connected to its cognate CTD domain by a linker composed of 14 residues [START_REF] Lee | Structural basis for inhibition of the replication licensing factor Cdt1 by geminin[END_REF][START_REF] Mizuguchi | JOY: protein sequence-structure representation and analysis[END_REF](64)(65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76). Modeling was performed with DADI-MODO which allows a flexible linker region to be defined in the model while maintaining structural constraints imposed by molecular modeling processes and the SAXS data (see Materials and Methods, ( 40)). The model produced by DADIMODO fits the experimental scattering pattern (Figure 4C) and exhibits the same global organization as that obtained by ab initio modeling (Figure 4B andD). Individual runs revealed different solutions with variable positions of the CTDs, suggesting an intrinsic flexibility of the linker (Supplementary Figure S3B). Nevertheless, the ab initio and structure-guided modeling approaches converge to a common architecture for YabA: a core of four NTD ␣-helices each of which is connected to a globular CTD by a linker region in an extended conformation. The SAXS data and modeling experiments suggest that the linker confers a high degree of flexibility, leading to a multiplicity of conformations of the individual CTDs relative to the tetrameric core bundle Supplementary Figure S3B). [START_REF] Dumas | Multivalent endosome targeting by homodimeric EEA1[END_REF] and profilin (1YPR) using the Joy algorithm [START_REF] Mizuguchi | JOY: protein sequence-structure representation and analysis[END_REF], highlighting the structural similarities between YabA model and it's templates. Alpha helix (red); 3 10 helix (maroon); beta strand (blue); solvent accessible residues (lower case); solvent inaccessible residues (upper case); hydrogen to bond main-chain amide(bold), hydrogen bond to main-chain carbonyl (underline). Secondary structures of YabA are indicated on top and correspond to the crystal structure (residues 1-62) and the homology model (76-119). The residues involved in Zn coordination are indicated by orange dots. (B) Cartoon representation of YabA CTD model colored as in (A). Side chains of the residues participating in Zn binding are indicated as ball and sticks. (C) Surface representation of YabA-CTD revealing surface-exposed residues important for interaction with DnaN (left) and DnaA (right) identified in a previous study [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF].

Mutually exclusive interactions of DnaA and DnaN to the YabA C-terminal moiety

Previous studies revealed that YabA forms heterocomplexes with DnaA and DnaN in vivo [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF][START_REF] Soufo | Cell-cycle-dependent spatial sequestration of the DnaA replication initiator protein in Bacillus subtilis[END_REF]. YabA was also shown to bridge DnaA and DnaN in a yeast three-hybrid assay [START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF]. It was further shown that YabA LOI-mutations disrupting interactions with DnaA or with DnaN mapped to the CTD [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Our structural and biophysical data established that the isolated CTD is monomeric, suggesting that these domains may function as independent binding sites in the YabA tetramer. We tested this assumption by determining whether YabA 62-119 , comprising the CTD globular domain together with the linker, was sufficient for interaction with DnaA or with DnaN in a yeast two-hybrid assay (Supplementary Figure S4A). We found that indeed, YabA 62-119 was able to support interaction with either partner, implying that the NTD's quaternary structure is dispensable. This scheme raised the important question of whether a single CTD monomer could form simultaneous interactions with both DnaA and DnaN or whether the interactions with the two proteins are mutually exclusive. To answer this question, we performed a yeast 3HB assay in which a constitutively expressed YabA 62-119 was tested for its ability to trigger interacting phenotypes by bridging DnaA and DnaN. We found that in contrast to the full length YabA, YabA 62-119 was not able to bind simultaneously to DnaA and to DnaN whether they are fused with to the BD or the AD domains of Gal4. This observation sheds new light on the mode of binding of YabA to its partners. We conclude that DnaA and DnaN are able to bind concurrently to a YabA tetramer but not to a single CTD.

Structural and functional determinants underlying the binding of YabA to DnaA and DnaN

Next, the model of the YabA CTD was used to predict additional residues involved in interactions with the partner proteins DnaA and DnaN. The predictions were based on residue surface accessibility calculations, sequence conservation analysis, comparison with templates and previous work [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Candidate residues were targeted for scanning mutagenesis and the interaction profiles of the corresponding YabA mutants were determined experimentally in a yeast two-hybrid assay, similar to that previously used to identify LOI mutations [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Previous work showed the importance of residues C97, C109 and C112 of the HCCC motif for interactions with both DnaA and DnaN [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. To experimentally investigate the role of the H95 of this motif, we tested the effects of targeted substitutions on the capacity of YabA to bind to its different partners. Replacement of H95 by I or L resulted in loss of interaction with both DnaA and DnaN while YabA-YabA self-interactions were preserved (Figure 5, Supplementary Table S3 and Figure S5). Similar effects were observed previously upon mutation of the conserved cysteine residues [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. This suggests that substitution of the polar H95 by hydrophobic residues results in structural defects that are confined to the CTD. Curiously, a H95G substitution did not exhibit an interaction defect in our yeast two hybrid assay (Supplementary Table S3). However this mutant, like the previously examined C97A mutant, exhibited a propensity to aggregate during expression in E. coli cells (data not shown). This is consistent with reduced stability caused by loss of Zn binding, although the direct participation of H95 in Zn coordination is not yet proved. Substitutions of the neighboring residues, F94 and I96 also impeded interaction with DnaA but had only moderate effects on interaction with DnaN (Supplementary Table S3). The hydrophobic residue F94, is highly conserved as a F or Y among YabA homologs and is buried in our model (Supplementary Figure S1). The F94Y mutation in YabA did not affect protein interactions. In contrast, substitution of F94 by a variety of polar and aliphatic residues led to specific defects in DnaA interactions (Figure 5, Supplementary Table S3 and Figure S5). These results suggest that the H95 flanking residues, F94 and I96, are also important for the folding of the CTD. Together these results support the importance of a structured domain in which Zn-coordination might play a role.

To further delineate the DnaA binding surface, we targeted residues R105 and K106, as these neighbor the surface-exposed residues N85 and L86 (previously shown to be important for DnaA binding). YabA mutants R105E, R105D and R105G lost the ability to bind to DnaA. However substitution of K106 by a range of amino acid residues had no effect on DnaA binding (Figure 5, Supplementary Table S3 and Figure S5). This suggests that R105 but not K106 is part of the DnaA binding site (Figure 5 and Supplementary Table S3). YabA substitutions of the neighboring residues E107and D108 did not exhibit LOI phenotypes for DnaA, indicating these residues are not part of the DnaA binding surface. Instead these substitutions produced a DnaN LOI phenotype. This suggests that the two interacting surfaces may be overlapping.

To further delineate the DnaN interaction surface, mutagenesis of F111, N117 and the terminal K118 and K119 residues was performed. Whilst, the substitution of K118 by various amino-acid residues had no effect on protein interactions, mutations targeting residues F111, N117 and K119 all resulted in DnaN LOI mutants (Figure 5, Supplementary Table S3 and Figure S5). The location of residues F111, N117 and K119 in the CTD model are consistent with their role in DnaN binding as they are predominantly surface exposed. Moreover, these residues are adjacent to V99 and L110 previously found to be important for DnaN binding. Taken together, these data clearly delineate the interaction determinants of DnaA and DnaN on two partially overlapping surfaces on the YabA CTD.

Effect of loss-of-interaction mutations on sub-cellular localization in B. subtilis cells

YabA forms a heterocomplex with DnaA and DnaN which co-localizes with the replication machinery for most of the cell cycle [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF][START_REF] Hayashi | Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation[END_REF]. To validate our structure predictions, we investigated the subcellular localization of several YabA DnaA and YabA DnaN -LOI mutants in living cells by transferring the corresponding point mutations into a yabA gene fused in-frame to cfp or yfp. Examination of the cellular protein levels revealed that the YabA mutant derivatives were present at similar or higher levels compare to the wild-type (Supplementary Figure S6). Wild-type YabA localizes as one or two foci per cell, with single foci displaying a midcell localization (Figure 6, [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF][START_REF] Hayashi | Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation[END_REF]). LOI with either DnaA or DnaN is associated with both loss of YabA localization and loss of initiation control [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Here, we observed that F94S and R105E substitutions in a CFP-YabA fusion give a fluorescence signal dispersed throughout the cell, similar to that of the YFP-YabA-N85D mutant identified in a previous functional study [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF] (Figure 6A and Supplementary Figure S7). Since F94S and R105E substitutions specifically impaired the binding of YabA to DnaA (but not DnaN) in the yeast two-hybrid assays, we conclude that these two residues contribute to the integrity of the YabA/DnaA interface together with residues N85 and L86 identified formerly. The R105E and F94S substitutions reduce but do not completely abolish foci formation since patches of increased fluorescence intensity can still be discerned in parts of the cells, in agreement with the observation of Goranov et al. that YabA foci can be detected in cells deficient in DnaA-mediated initiation of chromosomal replication [START_REF] Goranov | YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress[END_REF]. Thus, while these two substitutions cause significant dispersal of YabA throughout the cell, residual YabA co-localization with the replication machinery could still be observed. Examination of CFP-YabA fusions carrying the DnaN-LOI substitutions E107T, E107R, D108S or F111Y, revealed that foci formation was completely abolished as observed previously for DnaN-LOI substitutions of V99 and L110 (Figure 6A, Supplementary Figure S7). These observations support the assignment of these residues to the DnaN interacting surface. Overall, the data are consistent with the predictions of the structural model of YabA.

When co-expressed in the cell, DnaA-and DnaNinteraction deficient mutants of YabA can crosscomplement and restore YabA cellular localization and function [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Here we tested the capacity of the DnaA-LOI YabA mutant N85D of YabA, to restore the localization of the DnaN-LOI mutants (F111Y and E107T). The CFP-YabA-F111Y (or E107T) and the YFP-YabA-N85D fusion proteins are primarily dispersed in the cell when expressed alone (Figure 6A), but re-localized as sharp mid-cell foci containing both CFP and YFP fluorescence when co-expressed in the same cell (Figure 6B). This functional complementation of DnaA and DnaN LOI mutations indicates that the mutant YabA proteins are properly folded in the cell, and that the interaction deficiency of each mutant is complemented by the other in a functional heterocomplex that localizes correctly at midcell (Figure 6C). These results fully support the assignment of residues E107 and F111 to the DnaN-binding surface thus lending further experimental support to the structural model. These observations suggest that the CTD harbors the determinants for association with the replication machinery while the N-terminal domains account for the quaternary structure of YabA perhaps allowing additional levels of regulation of its activity.

DISCUSSION

In B. subtilis, YabA is proposed to exert its negative control of replication initiation by concurrent mechanisms at work at the replication origin and at the replication factory. YabA inhibits the formation of a DnaA-nucleoprotein structure at oriC by binding to free DnaA thus reducing its availability in the cell [START_REF] Merrikh | Control of the replication initiator DnaA by an anti-cooperativity factor[END_REF][START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF]. YabA also tethers DnaA with the ␤-clamp DnaN in a heterocomplex, which co-localizes with the replication factory during most of the replication cycle [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF][START_REF] Soufo | Cell-cycle-dependent spatial sequestration of the DnaA replication initiator protein in Bacillus subtilis[END_REF][START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF]. Thus, YabA acts to prevent premature re-initiation by trapping DnaA in a DnaN-independent or a DnaN-dependent manner, respectively. The question of how YabA coordinates its actions to allow the transition from initiation to elongation of replication remains obscure and a better understanding of the structural organization and dynamic character of the complexes it forms with DnaA and DnaN is needed.

Our study establishes that YabA assembles as a headto-head dimer of dimers in solution, through coiled-coil interactions of its helical NTDs. The C-terminal portions of YabA fold as independent domains linked to their cognate NTDs by a short flexible region. YabA CTD is a zinc-associated domain that mediates binding to DnaA and to DnaN. Analysis of the surfaces involved in binding to DnaA and DnaN revealed they are partly overlapping, thus ruling out the possibility that the two proteins bind concomitantly to the same CTD. As a result, ternary interactions of YabA, DnaA and DnaN are observed only if the full length YabA, able to self-assemble through its NTD, is expressed in the yeast 3HB assay. Importantly, the capacity of YabA DnaA and YabA DnaN LOI mutants to complement each other's localization defects and restore foci at the replication factory when co-expressed in B. subtilis cells provides strong evidence to substantiate the hypothesis that DnaA, YabA and DnaN can be part of the same hetero complex. Altogether our results suggest strongly that DnaA and DnaN associate with separate CTDs of the YabA tetramer.

Examination of residues important for interaction with DnaA and DnaN showed that they clustered in partially overlapping patches. Protein-protein interfaces are generally composed of different types of residues that contribute unevenly to the binding free energy. Scanning mutagenesis experiments allow three classes of residues to be defined according to their contribution to protein-protein complex formation [START_REF] Janin | Protein-protein interaction and quaternary structure[END_REF]. Interfaces are composed of (i) core residues, which become extensively buried upon complex formation and whose mutation leads to drastic lowering of complex stability. These residues have been defined as 'hot spots' and they are typically highly evolutionary conserved ( 52); (ii) rim residues which surround the core, sheltering the latter from the solvent, and whose mutation generally tends to have modest effects on complex stability [START_REF] Bogan | Anatomy of hot spots in protein interfaces[END_REF][START_REF] Moreira | Hot spots-a review of the protein-protein interface determinant amino-acid residues[END_REF] and (iii) residues which do not contribute directly to the interface but which play an important role in defining its structural integrity. Our refinement of the DnaA interacting surface revealed novel conserved residues that fall in two of these categories. Firstly, a three amino-acid residue cluster FHI at positions 94-96, encompassing the conserved H95 of the HCCC motif, was found to be important for interaction with DnaA. We propose that this cluster is most likely involved in the stabilization of the zinc-containing fold and contributes to the structural integrity of the binding surface. Secondly, we found that the surface-exposed and positively charged residue R105, is critical for DnaA binding to YabA. R105 is strictly conserved among the YabA-family of protein. We propose that R105 constitutes a binding hotspot, in agreement with the observation that arginines are frequently represented in hot spots [START_REF] Bogan | Anatomy of hot spots in protein interfaces[END_REF]. Together with N85 and L86, R105 is part of an interacting patch highly conserved in the YabA family of proteins.

YabA exerts part of its regulatory mechanism by preventing DnaA oligomerization at oriC [START_REF] Merrikh | Control of the replication initiator DnaA by an anti-cooperativity factor[END_REF][START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF]. DnaA mutant derivatives involving residues H162 and A163 were found to be insensitive to YabA control [START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF]. These two residues are located in domain III of DnaA, which is important variously for binding to ssDNA, for ATP hydrolysis and for DnaA oligomerization. In the three dimensional structure of DnaA, H162 and A163 are in close proximity to residue F120 which is known to be important for interactions with YabA and DnaD (1,[START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF][START_REF] Cho | The functional analysis of YabA, which interacts with DnaA and regulates initiation of chromosome replication in Bacillus subtils[END_REF]. Whether these residues play a role in binding to YabA directly or indirectly remains to be established. The available structural data on YabA and DnaA from B. subtilis is insufficiently detailed to allow precise inferences on the YabA:DnaA interface to be drawn. However, analysis of the of a crosslinked YabA:DnaA complexes prepared by Schoefield et al., revealed the dominance of a 1:1 complex stochiometry with a low proportion of 2:1 stochiometry [START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF]. We can anticipate that YabA could bind to one DnaA unit per YabA-CTD domain.

Proteins interacting with the bacterial sliding clamp usually possess a short linear binding consensus signature QLxLF [START_REF] Dalrymple | Identification of putative DnaN-binding motifs in plasmid replication initiation proteins[END_REF]. Remarkably, no sequence related to this consensus is present in YabA [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. Although hydrophobic residues were identified, the composition and spatial arrangement of the surface-exposed residues involved in binding to DnaN suggest a different mode of interaction for YabA. Among the YabA DnaN surface residues only one, F111, is highly conserved among the YabA family of proteins. Residues at this position are strictly hydrophobic, represented by F (82%) or L (18%) (Supplementary Table S1) suggesting this might contribute substantially to the free energy of binding. In YabA, further amino-acid residues including two hydrophobic residues (V99 and L110), two acidic residues (E107 and D108) and a positively charged residue at the C-terminus (K119) have been found to contribute to binding to DnaN. These less conserved residues might be important for tuning the affinity and/or the speci-ficity of the interaction. In summary, our results point to a YabA ␤-binding mode that differs from other ␤-binding proteins such as the replicative DNA polymerase and presumably requires a different interacting surface. This result is in agreement with the DnaN-dependent localization of YabA at the replication machinery for most of the bacterial cell cycle [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF][START_REF] Hayashi | Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation[END_REF].

The structural characterization of YabA reveals a hub architecture. The structure of YabA is reminiscent of that of the FtsZ associated protein ZapA which is recruited to the divisome machinery to stabilize the Z-ring during bacterial cytokinesis [START_REF] Mohammadi | The GTPase activity of Escherichia coli FtsZ determines the magnitude of the FtsZ polymer bundling by ZapA in vitro[END_REF][START_REF] Roach | Crystal structure and site-directed mutational analysis reveals key residues involved in Escherichia coli ZapA function[END_REF][START_REF] Small | FtsZ polymer-bundling by the Escherichia coli ZapA orthologue, YgfE, involves a conformational change in bound GTP[END_REF]. ZapA assembles into an elongated anti-parallel tetramer, in which two subunits associate via their coiled coil CTDs and two dimers then associate into a tetramer. At both ends of the tetrameric helical bundle, globular NTDs comprise the determinants for binding to FtsZ [START_REF] Roach | Crystal structure and site-directed mutational analysis reveals key residues involved in Escherichia coli ZapA function[END_REF]. An important aspect of such homo-oligomeric proteins is their ability to contact and connect different protein partners. In E. coli, ZapA recruits ZapB to the Z-ring [START_REF] Galli | FtsZ-ZapA-ZapB interactome of Escherichia coli[END_REF][START_REF] Galli | Spatial resolution of two bacterial cell division proteins: ZapA recruits ZapB to the inner face of the Z-ring[END_REF]. In B. subtilis, ZapA interacts with different components of the division machinery including SepF and EzrA [START_REF] Ishikawa | A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis[END_REF]. The architecture of YabA is also similar to the eukaryotic protein Geminin, which downregulates DNA replication through the timely inhibition of the licensing factor Cdt1 [START_REF] De Marco | Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing[END_REF][START_REF] Lee | Structural basis for inhibition of the replication licensing factor Cdt1 by geminin[END_REF]. Geminin forms a parallel coiled-coil homodimer that binds to Cdt1. It has been proposed that regulation of initiation is exerted though a switch in quaternary structure from a permissive Geminin 2 /Cdt1 heterotrimer to an inhibitory Geminin 4 /Cdt1 2 heterohexamer [START_REF] De Marco | Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing[END_REF]. Auxiliary basic and acidic residues in Cdt1 are involved in binding to PCNA, a parallel which may be extended to the recruitment of DnaN by YabA. Although these structural resemblances between proteins regulating replication in bacterial and eukaryotic cells do not imply similar regulatory mechanisms, they suggest that the evolutionary constraints on preventing re-replication have led to targeted interactions with key factors involved in both initiation and elongation.

Thus YabA, Geminin and ZapA represent examples of the exploitation of a coiled-coil helical core as a protein hub allowing the assembly and disassembly of protein complexes in a dynamic way. The structural organization of YabA offers a simple explanation for how it forms heterocomplexes with DnaA and DnaN. The flexible C-terminal moieties provide YabA with the dynamic capacity to interact (i) with more than one DnaA molecule, preventing the helical assembly of the latter at oriC, or (ii) after initiation with both DnaA and DnaN at the replication machinery. In the latter case, we anticipate that the two CTDs (say the AB' pair) at one end of the YabA tetramer could contact symmetrical binding surfaces on the ring-shaped DnaN dimer, with up to two DnaA monomers, interacting with the CTD pair (A'B) pair at the opposite end of the complex as depicted in Figure 6C. Future studies are required to gain insights into the architecture and stoichiometry of the different complexes YabA forms with its partners. Nevertheless, our experimentally informed structural model of the YabA tetramer sheds light on how YabA can mediate multiple interactions, and lays the foundation for further understanding of how YabA coordinates its actions to down-regulate replication initiation.

Phosphorylation of Y90 residue changes its affinity for DnaA and DnaN

This part of the project started with the identification by LC-MS of a second phosphosite involving the residue Y90 of YabA in a fashion that did not required the presence of ATP nor kinase. Although this result strongly suggested that this phosphorylation had occurred nonspecifically in E. coli, the localization of the tyrosine Y90 at the surface of the C-terminal domain, in close proximity of the determinants for interactions with DnaA and DnaN [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF], pressed us to investigate further the role of this residue in YabA function. We therefore constructed a Y90 non-phosphorylatable (YabAY90F) and a phosphomimetic (YabAY90E)

mutants in order to study the effect of the tyrosine phosphorylation in the interactions with its partners DnaA and DnaN and thereby the consequences in replication initiation control.

YabA is phosphorylated at Y90 residue in a YabT-independent manner LC-MS analyzes carried out at PAPPSO Platform (INRA, Jouy en Josas) by Céline Henry evidenced that when YabA is overexpressed in E. coli, the protein is very efficiently phosphorylated on the residue Y90 in a YabT independent manner (Figure 1.A). This tyrosine, highly conserved among YabA homologs (Figure S1, [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF], is located at the surface of the C-terminal domain of YabA (Figure 1.B). These observations, raise the question about the potential role of Y90 phosphorylation in the regulation of DNA replication.

YabA phosphorylation at Y90 is involved in replication initiation control

B. subtilis mutant strains carrying either non-phosphorylatable (YabAY90F) or phosphomimetic (YabAY90E) Y90 mutations at the yabA genomic locus were constructed, using the pop-in/out methodology (see supplementary material and methods). Using origin-proximal (ori) and terminus-proximal (ter) primer pairs, we monitored the ratio of origin-to-terminus (ori-to-ter) genomic sequences as a mean to detect replication initiation events. In vivo, YabA associates with DnaA and down regulates replication initiation. In the yabA null strain, the ratio of ori-toter copies was significantly higher compared to the wild-type, indicating over-initiation of replication, in agreement to previous studies (Figure 2) (Soufo et al., 2008;Murray and Koh, 2014;García-García et al., 2017). Both yabAY90E and yabAY90F mutants also showed an increased ori-to-ter ratio, as compared with the wild-type strain, the yabAY90E mutant being more affected than the yabAY90F mutant (Figure 2). These results indicate that the residue Y90 could also play a role in initiation control.

YabA phosphorylation at Y90 modulates the binding specificity to DnaA and DnaN

Previous studies indicates that also YabA down regulates initiation as part of a heterocomplex with DnaA and DnaN that localizes at the replication machinery (Noirot-Gros et al., 2006). We investigated whether the phosphorylation of YabA at residue Y90 could affect its binding properties, by yeast two-hybrid assay. We constructed translational fusions of the nonphosphorylatable (YabAY90F) and phosphomimetic YabA derivatives (YabAY90E and YabAY90D)

to the activating domain (AD) and binding domain (BD) of the yeast transcriptional factor GAL4.

These plasmids were transferred in the appropriate strains and a Yeast-two-hybrid mating assay was performed. Surprisingly, we found that the phosphorylation state of YabA at Y90 modulate a protein-interaction switch with its protein partners DnaA and DnaN. As shown in Figure 3.A, YabA interacted with both DnaA and DnaN. However, YabAY90F was able to support the interaction with DnaA but not with DnaN, while the YabAY90E and Y90D mutant derivatives were able to interact with DnaN but were defective for DnaA interaction.

Y90 phosphorylation might change the structural conformation of the protein

The next step was to purify the YabA Y90 derivative proteins in order to identify the kinase responsible for the phosphorylation at Y90 residue. For this purpose, we purified the YabAY90E and YabAY90F proteins by ammonium sulfate precipitation at 20%, followed with gel filtration We therefore compared the YabAY90E with the other YabA derivatives used in my studies (ie YabA, YabAY90F, T71A, T71D). After heating (5 min at 95°C), corresponding samples were loaded onto a SDS-PAGE at 15%. Surprisingly, we found that Y90E has a different behavior, as compared with the other YabA derivatives (Figure 5). The native YabA protein as well as the T71A and T71D derivatives always exhibited two distinct bands, with the large majority of the protein present in the lower migrating band. However, the migration of the large majority of the Y90E derivative corresponded to the upper band, when the Y90F derivative corresponded only to the lower band (Figure 5.A). The same pattern was obtained when migrating the samples with no prior heating (Figure 5.B). Moreover, the migration of YabAY90E was not likely to result from the introduction of a negative charge, since T71D behaved like the native YabA protein. We concluded that this behavior was probably resulting from a conformational change. Indeed, the introduction of a

Cloning procedures

Polymerase chain reaction (PCR):

PCR reactions were performed according to manufacturer's recommendations. TaKaRa Ex Taq DNA Polymerase (TaKaRa, clontech) was used to amplified short fragments (100 bp to 1 kb); Q5

High-Fidelity DNA Polymerase (NEB) for large fragments (> 1 kb). Oligonucleotides were designed using the Clone Manager 9 PE and purchased from Eurofins MWG. 100 pmol/μL aliquots were stored at -20 °C. Oligonucleotides used in this study are given in Table x.

Agarose gel electrophoresis of DNA fragments:

Agarose gel electrophoresis was performed in 0.8 % (w/v) agarose gels containing ethidium bromide (1 µg ml -1 ) in 1X TBE buffer. DNA samples were mixed at a 6:1 ratio with DNA loading buffer (30 % (v/v) glycerol, 0.25 % (w/v) bromophenol blue, 0.25 % (w/v) xylene cyanol) prior to loading. The voltage used for electrophoresis was 120 V. SmartLadder (Eurogentec) was used as a market size and the gels were revealed by UV at 254 nm.

DNA purifications:

The PCR products were purified using the kit Wizard SV gel and PCR clean-up System (promega)

or the QIAquick PCR Purification Kit (QIAGEN), according to manufacturer's recommendations.

Plasmids were purified from E. coli strains using a QIAprep Spin Miniprep Kit (QIAGEN), according to manufacturer's recommendations.

General strategy for sub-cloning DNA fragments into plasmids in E. coli:

PCR-generated DNA fragments were purified as described above; restriction digestions were performed according to manufacturer's recommendations (restriction sites are indicated in table 3), then inactivated by heating at 65°C for 20 min. Restricted fragments were then purified and mixed with the appropriate plasmid restricted with the same enzymes. Ligation was carried out overnight at RT using T4 Ligase (promega), followed by dialysis against ddH2O for 30 min before transformation in competent DH10B E. coli grown under selective pressure. The clones were checked by PCR, sequenced and finally saved as glycerol stock at -80 °C.

DNA sequencing:

Following sub-cloning in E. coli and cloning into B. subtilis, the complete plasmid or chromosomal DNA regions were sequenced to ensure the complete absence of unwanted mutations. For this, primers upstream and downstream of the inserted area were used. Sequencing of PCR products was performed by the sequence facility of Eurofins MWG and according to their recommendations.

Manipulation in B. subtilis

Chromosomal DNA purifications:

B. subtilis genomic DNA was isolated using the LiCl method as follow: 2 ml of culture was

Transformation of B. subtilis:

To make B. subtilis competent for the uptake of DNA, 2 ml of an overnight preculture was washed and resuspended in 1 ml of Solution I. 700 µL of this resuspension were taken to inoculate 10 ml of Solution I. After 4.5 h of incubation at 37°C, 1.5 ml were used to inoculate 10 ml of Solution II and further incubated at 37°C for 1. Ori/Ter ratios by qPCR DNA was isolated from cultures at exponential phase (OD=0.3-0.4) as described in the DNA purification section. Q-PCRs were carried out in triplicates of 25 µl each, with three dilutions of chromosomal DNA (10 -3 ng/µL, 10 -4 ng/µL and 10 -5 ng/µL). 5 µL diluted DNA were used as template and added to a mixture of 12.5 µL ABsolute Blue qPCR SYBR®Green ROX Mix (Thermo Scientific) and 0.3 µL primers mix (3µM each forward and reverse) in a 96-well PCR plates.

Primers pair for Origin proximal sequence (OriL3F and OriL3R) and Terminus proximal sequence (TerL3R and TerL3R) were used in separated reactions and tested for an efficiency greater than or equal to 90%. For list of primers see

Yeast-two-hybrid

Derivatives YabAY90F, YabAY90E and YabAY90D proteins were expressed as fusions to the GAL4 binding domain BD or activating domain AD from the vectors pGBDU-C1 and pGAD-C1, respectively (Figure 7). The bait (pGBDU) constructs were introduced in the Saccharomyces cerevisiae strain PJ69-4a and the prey (pGAD) constructs in strain PJ69-4α. The correct DNA sequence of all cloned fragments was confirmed by DNA sequencing. Binary interactions were tested by combinational mating of the strains a and  expressing the BD and AD fusions as previously described (Noirot-Gros et al, 2006). In brief, the mating were grown in rich YEPD medium and the diploïds obtained were selected for an interaction phenotype by growing them in synthetic complete medium (SC) lacking the appropriate amino acids (Leu, Trp, His) or nucleotides (Ade, Ura). An interaction between the two fusion proteins encoded by the inserts of the pGBDU and pGAD vectors was revealed by growth of the corresponding S. cerevisiae diploid strain.

Protein purifications

The yabA sequences carrying mutations Y90F and Y90E were amplified from, pGBDU-yabAY90F, pGBDU-yabAY90E bait plasmids with yabA-NdeI and yabA-XhoI primers sets and cloned into NdeI and XhoI sites of pSMG201, which allow cloning of ORF under control of the T7 promoter.

E. coli strain ER2566 was then transformed with the resultant plasmids. Cells were cultured in LB medium supplemented with 30 μg ml -1 kanamycin. When the OD600 reached 0.

III. DISCUSSION AND PERSPECTIVES

In all living organisms, protein phosphorylation is a widely conserve strategy to modulate aspects of their functionalities. In eukaryotes, the interplay between the cell cycle and the other cellular processes mostly involves phosphorylation of proteins by Ser/Thr/Tyr Hanks-kinases (Hanks et al., 1988). Protein phosphorylation also has emerged as one of the major post translational modifications in bacteria, involved in regulating a high number of physiological processes. Phosphorylation of various components of the DNA replication machinery, as well as of proteins involved in the replication cycle has been discussed in a peer-reviewed publication part of this thesis (García-García et al., 2016).

We showed in B. subtilis that YabA is specifically phosphorylated by the Ser/Thr kinase YabT and we also revidenced new functions of YabA beyond its role as a replication controller. YabA plays a major role in down regulating initiation replication through interaction with the initiator protein DnaA and the clamp polymerase DnaN. It has been suggested to function by tethering DnaA to the replication machinery via a ternary complex with DnaN during most of the cell cycle (Noirot-Gros et al., 2006, Soufo et al., 2008, Katayama et al., 2010). This model is in agreement with the YabA structure studies presented as complementary results in this thesis [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. YabA is a structural hub protein able to interact with other protein partners, AcuB (an enzyme involved in acetoin utilization) or TlpA (a chemotaxis protein) indicating it might be multifunctional. YabA possesses an unique tri-dimentional structure composed of N-terminal four helix-bundle tetramer connected to four monomeric C-terminal domains through flexible linkers, which makes YabA able to physically interact with more than one protein at a time, thus providing a suitable platform to integrate intracellular signals to replication initiation [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF].

YabT-mediated phosphorylation of YabA is not involved in replication control but rather modulates differentiation processes

We established that YabA is phosphorylated by YabT at the threonine 71. In vitro phosphorylation assays with the threonine 71 mutated proteins, non-phosphorylatable and phosphomimetic, led us to conclude that this residue is specifically phosphorylated by YabT. The threonine 71 is located within the flexible linker of YabA, a 14 residues long linking region which connects the N-terminal domain with the C-terminal domain and confers a high degree of flexibility to the YabA structure.

R III. DISCUSSION AND PERSPECTIVES

204

YabT is a transmembrane kinase devoid of the classical extracellular sensing domain but containing a DNA-binding motif essential for its activation (Bidnenko et al., 2014). This Ser/Thr kinase is mainly expressed at early stage of sporulation as well as in biofilm conditions (Nicolas et al., 2012). It has been previously established that YabT phosphorylates the general DNA recombinase RecA to enforce chromosome integrity during spore development (Bidnenko et al., 2014). In vitro, YabT is also able to phosphorylate RacA, another DNA-related protein involved in DNA anchoring to the cell pole (Shi et al., 2014b). We here evidenced that YabA is also phosphorylated by YabT. Nevertheless, functional analysis using phosphomimetic and nonphosphorylatable derivatives of YabA revealed that YabA phosphorylation affect neither the replication initiation control nor the interaction with the different protein partners. These results provide evidence that the substitutions at T71 did not affect the overall structure of YabA and that YabA derivative proteins can fully exert its function in replication control.

Since YabT is expressed during sporulation, we further investigated a possible role for YabA phosphorylation under these conditions. YabT is conserved in many but not all Bacilli, however it is absent in E. coli and non-sporulating bacteria, corroborating the idea that YabT mechanism evolves for specific task during spore development in this genus. The ∆yabA strain showed a severe defect in spore production, suggesting that YabA is indeed involved in this differentiation pathway. Interestingly, we evidenced an enhanced sporulation in the phosphomimetic yabT71D mutant, which correlates with an increase in expression from the spo0A promoter. These results suggest increase Spo0A~P intracellular levels in the phosphomimetic strain. By contrast, the non-phosphorylatable yabAT71A strain exhibited the same level of sporulation than the wildtype strain, also the number of sporulating cells was significantly reduced in the yabAT71A strain at early stages of the sporulation process (stage T2). These results indicate that phosphorylation of YabA at T71 enhances the commitment of B. subtilis cells to sporulation, leading to an increase in spore production, while the absence of phosphorylation only delays the entry to sporulation without affecting the final spore yield. Therefore YabA phosphorylation plays a role during sporulation which is relevant, since YabT is expressed during this physiological condition.

Sporulation in B. subtilis is a strictly regulated process linked to the phosphorylation of the master regulator Spo0A, modulated by a multicomponent phosphorelay system [START_REF] Vishnoi | Triggering sporulation in Bacillus subtilis with artificial two-component systems reveals the importance of proper Spo0A activation dynamics[END_REF]. Sporulation is an energy consuming process which becomes irreversible after engulfment of the prespore by the mother cell. (Fujita et al., 2005), whereas low and intermediate Spo0A~P levels can trigger biofilm formation, cannibalism and competence. To gain deeper insight into the impact of YabA phosphorylation onto the Spo0A~P levels, we tested the capacity of our YabA mutant strains to form biofilm. Biofilm mass formation was similar for the wild-type, non-phosphorylatable yabAT71A and ∆yabT strains, while ∆yabA and phosphomimetic yabAT71D strains were defective for biofilm formation. Interestingly, the yabAT71D strain exhibited a smooth phenotype indicative of a defect in EPS production. However, the ∆yabA strain displayed a hyper-wrinkled morphotype, which has been previously associated with a hyper-production of polymer matrix and thereby de-regulation of the EPS synthesis pathway. Our transcriptome data does not support this hypothesis and reveals a diguanylate cyclase (DgcW) highly expressed in ∆yabA strain, which could explain the hyper-wrinkled phenotype.

Our results pointed to a regulatory role of YabA during sporulation and biofilm formation triggered by YabT-mediated phosphorylation. We therefore highlight a multifunctional role of YabA as well as its role in integrating physiological signals that connect chromosomal replication initiation control with cell development.

Transcriptomic analysis highlight a complex regulatory network controlled by YabA phosphorylation

In order to further investigate the regulatory network that could be targeted by YabT-mediated phosphorylation of YabA in the B. subtilis life-style decision, we examined the effect of YabA phosphorylation on global gene expression and transcriptional responses at the onset of sporulation. Both the wild-type and the non-phosphorylatable yabAT71A strains show equivalent pattern of gene expression in the three conditions tested, weather both yabA deletion and yabAT71D phosphomimetic mutation deeply impact gene expression landscape.

Moreover, transcriptomic analysis support the phenotypes associated with our strains. The phosphomimetic strain exhibits an enhanced sporulation phenotype that could be explained by an increase in the expression of the sporulation-specific sigma factor sigF and the subsequent spo genes. In contrast, deletion of yabA caused a down-regulation of these genes in agreement with its delayed sporulation phenotype. The delayed entry to sporulation observed in the nonphosphorylatable yabAT71A mutant could be explain by a slightly down-regulation of sigH and sigF observed at T1. However, the expression pattern at T3 was similar to wild-type, supporting R III. DISCUSSION AND PERSPECTIVES 206 that the absence of YabA phosphorylation only delays the entry in sporulation and that this defect is rapidly solved by other regulatory mechanisms that trigger sporulation.

The mechanisms underlying this novel role of YabA remain unknown. Our results suggest that YabA phosphorylation correlates with high cellular levels of Spo0A~P, leading to sporulation stimulation. A consequence of elevated levels of activated Spo0A is the prevention of the expression of the anti-repressor sinI, which leads to the full repression of the EPS operon and the amyloid fibers (TasA) by the master regulator SinR (Chai et al. 2008[START_REF] Deloughery | Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR[END_REF].

This could explain the inhibition of biofim formation mediated by the phosphomimetic form of YabA. Nevertheless, transcriptomic analysis showed an activation of sinI expression, leading to the over-expression of the subsequent biofilm genes in the phosphomimetic strain.

Interestingly, we found that only the first part of the EPS operon was up-regulated, whereas the second part was down-regulated. The mechanism that control full expression of the EPS operon is an EAR element (eps-associated RNA), located between the second (epsB) and third (epsC) gene of the EPS operon [START_REF] Irnov | A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales[END_REF]. In the absence of this antitermination mechanism, EPS transcription is arrested at the epsF gene, resulting in an exopolysaccharidedeficient phenotype equivalent to that observed in the phosphomimetic mutant. Two models have been proposed in the litterature: (i) constitutive antitermination could result from cellular cofactors that assemble with EAR and the transcription elongation complex to promote processive antitermination and (ii) processive antitermination may require an unknown signal in addition to the EAR element. Up to date, the EAR cofactors and the molecular mechanism of its action remain unknown, but our data suggest that YabA phosphorylation could play a role in this regulation.

Phenotypic and transcriptomic studies presented in this work showed that regulation and function of YabA may be more complex than previously thought. Some highly impacted genes encode proteins members of the Rap-Phr family of Quorum Sensing (QS) system. The behavior of bacterial communities is often regulated by QS signaling pathways. B. subtilis utilizes peptides as QS signals, thereby eliciting a variety of behaviors, including the initiation of developmental processes (such as sporulation and biofilm formation), as well as horizontal gene transfer through transformation or conjugation (Perego and Hoch., 1996). Most of the rap promoters are activated by the quorum response transcription factor, ComA, which is activated by the ComX pheromone at high cell density. RapA, RapE and RapI dephosphorylate Spo0F~P, a component of the phosphorelay, therefore playing a major role in negatively regulating sporulation [START_REF] Jiang | Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis[END_REF][START_REF] Parashar | Conformational change-induced repeat domain expansion regulates rap phosphatase quorum-sensing signal receptors[END_REF][START_REF] Singh | Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling[END_REF]. We found that rapA-R III. DISCUSSION AND PERSPECTIVES 207 phrA, rapE-phrE and rapI-phrI are strongly down regulated in the phosphomimetic strain, suggesting an acceleration of the phosphorelay that could lead to a fast accumulation of Spo0A~P then to a faster entry into sporulation. RapI also controls the transfer of the mobile genetic element ICEBs1 [START_REF] Auchtung | Identification and characterization of the immunity repressor (ImmR) that controls the mobile genetic element ICEBs1 of Bacillus subtilis[END_REF][START_REF] Bose | Regulation of horizontal gene transfer in Bacillus subtilis by activation of a conserved site-specific protease[END_REF]. ICEBs1 gene expression is repressed by the element's repressor ImmR. Upon production of active RapI, ImmR is inactivated and ICEBs1 gene expression is de-repressed. We observed all genes from the ICEBs1 locus were down-regulated in the yabAT71D in all conditions tested. By contrast, deletion of yabA triggered up-regulation of these genes during the stationary phase. These results

suggest that YabA phosphorylation could be necessary to repress the conjugation genes during the sporulation process. The nucleoid-associated protein Rok is also involved in the repression of ICEBs1 (Smits and Grossman, 2010). Recently, interactions between the replication initiation protein DnaA and Rok have been identified using ChIP-seq assays (Seid et al., 2017). One of these regions is located downstream of phrI and upstream of yydM. PhrI is the response regulator of RapI and is organized in an operon with rapI. Interestingly, these genes are strongly repressed in the yabAT71D strain, suggesting a possible link between YabA phosphorylation and gene regulation, which may be mediated by the association of YabA with both Rok and DnaA.

However, Rok as well as DnaA are transcriptional regulators that are repressed by Spo0A (Molle et al., 2003). In the phosphomimetic strain, high levels of activated Spo0A could then repress dnaA and rok, leading to the de-regulation of genes controlled by Rok and DnaA.

Further work is needed to deciphering the regulatory role of YabA phosphorylation during sporulation and biofilm formation. One future lead could be to identify the potential interacting partners of YabA~P that controls the differentiation process. It could be interesting to test if the different forms of YabA are able to interact with transcriptional regulators involved in sporulation and/or biofilm, such as Spo0A, Rok, and ComA in a yeast-two-hybrid matrix.

Furthermore, in vitro gel shift assays using purified YabA derivatives and Rok will be useful to detect if YabA or YabA~P could be associated with DNA in a Rok-dependent manner since YabA alone appears incapable of binding DNA (Merrikh and Grossman, 2011). Thin Layer Chromatography (TLC) assays revealed an unusual ATP and GTP hydrolysis activity of YabA, leading to the accumulation of uncharacterized products. This unusual activity seems to be modulated by phosphorylation of the YabA T71 residue. Intermediate products were produced when ATP was hydrolyzed, suggesting a gradual degradation. Interestingly, only one final product was evidenced when GTP was used as the substrate. These results suggest that GTP may be the real substrate of YabA. Identification of the products resulting from the YabAdependent hydrolysis of GTP will help us to understand this unusual activity. Mass spectrometry (MS) appears an analytical tool of choice for the analysis of metabolites resulting from the nucleotide hydrolysis. MS analysis will be performed in collaboration with the « Plateforme Métabolisme-Métabolome » (Université Paris-Sud, le Moulon).

R III. DISCUSSION AND PERSPECTIVES 209 Unfortunately, we did not succeed in convincingly demonstrate a binding of ATP or GTP to YabA using Differential Scanning Fluorimetry (DSF). The elongated tetrameric conformation and the high flexibility of YabA hinder the interpretation of the results showing multiple transitions. A good alternative will be to purify the C-terminal monomeric part of YabA protein containing the linker region that carries the T71 residue but not the N-terminal part. We will avoid the tetramerization of YabA and will get a compacted folded protein adapted to DSF analysis.

Nevertheless, the tetramerization of YabA could be a prerequisite for the GTP binding activity of YabA. Alternatively, we could use Biacore-based assays to characterize YabA GTP binding activity. Biacore is the commercial name of a device that measures protein-protein or proteinligand interactions and affinity constants. This technology is based on surface plasmon resonance (SPR), one molecule is attached to the sensor surface and the other is free in solution.

YabA could be attached to the sensor surface and the GTP free in solution.

YabA could be linked to biofilm formation via the c-di-GMP signaling pathway

Furthermore, our functional analysis pointed to a potential role of YabA in the c-di-GMP signaling transduction pathway, known to regulate biofilm formation in many bacteria. Using engineered B. subtilis strains (c-di-GMP null and highly synthetizing c-di-GMP strains), we detected a potential diguanylate cyclase activity when we overexpressed YabA in a c-di-GMP null strain. Interestingly, two populations with distinct c-di-GMP-dependent GFP activities were observed. The first population (smaller cells) exhibited a low level of expression of the GFP protein indicative of a high level of c-di-GMP, similar to that observed in cells expressing the diguanylate cyclase DgcP. The second population (longer cells) exhibited a high level of GFP correlated to low levels of c-di-GMP, comparable to that observed in c-di-GMP null cells. In this second population, the elongation effect may result from a defect in replication control, linked to an over-repression of DNA replication initiation due to YabA overexpression.

Since no c-di-GMP binding signature is present in YabA, it is no likely to act as a diguanylate synthase. One hypothesis would be that YabA could trigger indirectly a yet unknown DGC enzyme. However, all DGC encoding genes are supposed to be deleted in the c-di-GMP null strain. This would imply that the YabA-mediated c-di-GMP increase observed in our assay results from a not yet identify enzyme. The second hypothesis is linked to the GTP hydrolysis observed in our previous studies. YabA could act directly on decreasing the intracellular GTP pool through GTP hydrolysis. This is not in agreement with our experimentations, where overexpression of YabA induces an increase in intracellular levels of c-di-GMP in part of the cells. One explanation to reconciliate our observations that i) overexpressing YabA leads to higher levels of c-di-GMP in the cells and ii) YabA possesses a GTP hydrolysis activity, would be that by lowering the intracellular pool of GTP, YabA would trigger the expression of a not yet characterized DGC activity. This could be modulated by the phosphorylation of YabA since we observed that YabAT71D stimulates GTP hydrolysis in vitro. However, the expression of YabAT71D in vivo is associated with a defect in biofilm formation, which is indicative of a low intracellular concentration of c-di-GMP.

To corroborate the role of YabA phosphorylation in modulating intracellular level of c-di-GMP, YabAT71D should be expressed in the c-di-GMP null strain to test if the c-di-GMP intracellular levels remain low. Most importantly, the identification by MS of the metabolic products resulting from the GTP hydrolysis is now critical to provide testable hypothesis on YabA regulatory role during sporulation and biofilm formation.

Taken together, our results strongly support a multifunctional role of YabA in cell physiology, beyond its known role as a negative regulator of replication initiation. Further studies will be needed to decipher the complex regulatory role of YabA during B. subtilis cells differentiation.

YabA is phosphorylated in a YabT-independent manner at its C-terminal domain and this phosphorylation modulates its affinity for DnaA and DnaN

LC-MS analyzes showed that when overexpressed in Escherichia coli, YabA is phosphorylated on the Tyrosine 90. The ser/thr kinase YabT was not expected to phosphorylate a tyrosine residue.

However, ser/thr/tyr kinases are commonly found in eucaryotes and recently, a dual specificity kinase has been identified in B. anthracis [START_REF] Arora | Unveiling the novel dual specificity protein kinases in Bacillus anthracis: identification of the first prokaryotic dual specificity tyrosine phosphorylation-regulated kinase (DYRK)-like kinase[END_REF]. However, we established that the phosphorylation at Y90 occurred in a YabT independent manner. The Y90 residue is located in the C-terminal domain of YabA, which is involved in binding to the initiation protein DnaA and the clamp polymerase DnaN [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. This residue is highly conserved among YabA homologs, suggesting that Y90 phosphorylation could be relevant for the regulation replication initiation. As we showed, Y90 phosphorylation leads to replication defaults, the ori-to-ter ratios were significantly higher compared to the wild-type in a non-phosphorylatable yabAY90F and phosphomimetic yabAY90E strains. YabA down regulates initiation as part of a heterocomplex with DnaA and DnaN that localizes at the replication machinery (Noirot-Gros et al., 2006). Our The kinase that triggers phosphorylation of this tyrosine residue remains unknown. The discovery of this kinase will be the next step to further study the role of YabA phosphorylation in DNA replication control. B. subtilis encodes two BY-kinases, PtkA and PtkB (EpsB). PtkB is involved in the production of extracellular polysaccharides during biofilm development [START_REF] Elsholz | Self-regulation of exopolysaccharide production in Bacillus subtilis by a tyrosine kinase[END_REF][START_REF] Gerwig | The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis[END_REF]. However, PtkA is a promiscuous kinase expressed during various growth conditions in B. subtilis, which has been shown to phosphorylate and thereby regulate the activity of a large number of target proteins. The consequences of PtkA-dependent phosphorylation vary considerably depending on its substrate. The protein targets of the PtkA kinase include Ugd and TuaD, involved in teichuronic acid biosynthesis [START_REF] Mijakovic | Transmembrane modulatordependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases[END_REF];

as well as the aspartate dehydrogenase Asd, which converts aspartyl-phosphate to a semialdehyde (Jers et al., 2010). A number of substrates phosphorylated and activated by PtkA are involved in single-stranded DNA-metabolism; these include the ssDNA-binding proteins SsbA and SsbB (Mijakovic et al., 2006) and the ssDNA-specific exonuclease YorK (Jers et al., 2010). The fatty acid-regulated transcriptional factor FatR is also phosphorylated by PtkA and this phosphorylation plays a key role in FatR interaction with its DNA binding site [START_REF] Derouiche | Interaction of bacterial fatty-acid-displaced regulators with DNA is interrupted by tyrosine phosphorylation in the helix-turn-helix domain[END_REF]. PtkA also influences localization of a number of protein substrates (Jers et al., 2010), is involved in biofilm formation [START_REF] Kiley | Post-translational control of Bacillus subtilis biofilm formation mediated by tyrosine phosphorylation[END_REF][START_REF] Gao | The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis[END_REF] and in the regulation of the cell cycle [START_REF] Petranovic | Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication[END_REF]. The transmembrane activator of PtkA, necessary for substrate phosphorylation, is TkmA (Mijakovic et al., 2006). However, MinD and SalA represent two alternative anchoring points for PtkA, directing its localization and activity toward different substrates throughout the cell cycle function (Shi et al., 2014b). Deletion of PtkA induces a pleiotropic phenotype with impaired DNA replication [START_REF] Petranovic | Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication[END_REF], suggesting that its cellular role is highly complex. Seeing the diversity of PtkA protein substrates, early on it could be imagined that YabA could be another substrate. The understanding of the role of Y90 and its potential phosphorylation remains an avenue to elucidate unsuspected levels of regulation by YabA during replication. 2. YabA is phosphorylated by the serine/threonine kinase YabT at the residue T71 located within the flexible linker.

IV. CONCLUSIONS IV. CONCLUSIONS

3. YabT-mediated phosphorylation of YabA is not involved in replication controls but rather modulates Bacillus subtilis differentiation processes, suggesting a multifunctional role of YabA.

4. YabA exhibits an unusual ATP/GTPase activity modulated by its phosphorylation state at T71.

5.

YabA could be linked to biofilm formation via the c-di-GMP signaling pathway.

6. YabA is phosphorylated in a YabT-independent manner at Y90 located at its C-terminal domain.

7. Phosphorylation of Y90 residue modulates its binding affinities for DnaA and DnaN, therefore affecting replication initiation control.

V. BIBLIOGRAPHY ANNEX 1. se fixer à l'ADN en interférant avec la propriété de liaison coopérative de DnaA sur l'ADN. YabA inhibe donc la formation d'oligomères hélicoïdaux de DnaA à oriC (Merrikh et Grossman, 2011 ;Scholefield et Murray, 2013).

YabA est une protéine très conservée chez les Bacilli, bactéries Gram-positif à faible pourcentage en bases G et C. Sa structure 3D révèle une architecture en deux domaines: Un domaine Nterminal adoptant un repliement de type superhélice et un domaine C-terminal globulaire structuré autour d'un atome de Zinc (Noirot-Gros et al., 2006 ;[START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. In vivo, YabA forme un tétramère grâce à des interactions entre les superhélices N-terminales, connecté aux domaines C-terminaux monomériques par une séquence déstructurée hyper flexible [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. YabA serait un hub structural interagissant simultanément avec plusieurs protéines et constituerait une plate-forme d'intégration entre différents signaux intracellulaires et l'initiation de la réplication.

B. subtilis est un organisme modèle développemental capable de se différencier en spores ou de former des biofilms en réponse à des signaux environnementaux. Le couplage entre la prise de décision développementale et le statut de réplication chez B. subtilis n'est pas entièrement compris. Chez B. subtilis, de multiples processus de régulation contribuent à réguler la réplication de l'ADN au début de la sporulation (Veening et al., 2009). Ces mécanismes ciblent essentiellement la protéine initiatrice majeure DnaA et modulent son activité [START_REF] Zakrzewska-Czerwinska | Regulation of the initiation of chromosomal replication in bacteria[END_REF], Katayama et al. 2010, Skarstad and Katayama 2013). Pendant la croissance végétative, DnaA se lie à l'origine de réplication oriC et forme des assemblages oligomériques, ce qui conduit au déroulement de l'ADN duplex et permet le chargement ultérieur de la machinerie de réplication. Plusieurs facteurs anti-coopératifs régulent l'oligomérisation de DnaA sur oriC au cours des différents modes de vie de la bactérie [START_REF] Jameson | Control of initiation of DNA replication in Bacillus subtilis and Escherichia coli[END_REF]. Pendant la croissance végétative, trois protéines régulatrices, YabA, DnaD et Soj, régulent négativement l'initiation de la réplication en interagissant avec DnaA, ce qui inhibe sa liaison coopérative à oriC (Merrikh and Grossman 2011[START_REF] Bonilla | The primosomal protein DnaD inhibits cooperative DNA binding by the replication initiator DnaA in Bacillus subtilis[END_REF], Scholefield and Murray 2013). Dans les cellules engagées dans la sporulation, les protéines SirA et Soj inhibent le chargement de DnaA à l'origine de réplication, inhibant l'initiation de nouveaux cycles de réplication (Rahn- Lee et al. 2011, Jameson et al. 2014, Duan et al. 2016).

Chez B. subtilis, la décision d'entrer en différenciation (sporulation ou formation de biofilms) implique la phosphorylation d'un régulateur transcriptionnel clé, Spo0A. La concentration intracellulaire de Spo0A-P est le facteur déterminant qui induit l'expression de plusieurs groupes de gènes nécessaires à la sporulation ou à la production de la matrice du biofilm. En réponse à certains signaux environnementaux, une cascade de phosphorylation (ou phosphorelais) est activée et aboutit à l'accumulation de Spo0A-P dans la cellule. Un mécanisme bien caractérisé permettant de coupler l'initiation de la sporulation avec la réplication de l'ADN, implique la protéine Sda. Sda induit la réduction du taux de Spo0A-P dans la cellule en inhibant l'activité d'une des kinases du phosphorelais, KinA (Burkholder et al. 2001, Veening et al. 2009). Au cours de la croissance végétative, l'expression du gène sda est positivement régulée par DnaA, qui agit également comme régulateur transcriptionnel de nombreux gènes impliqués dans le stress de réplication et la sporulation (Burkholder et al. 2001, Breier andGrossman 2009). Des études récentes ont établi que l'activation de la plus grande partie du régulon DnaA est médiée par Sda (Smith et Grossman 2015, Seid et al., 2017, Washington et al., 2017).

Chez les eucaryotes, la connexion du cycle cellulaire avec les autres processus cellulaires implique principalement la modification post-traductionnelle (PTM) de protéines, notamment leur phosphorylation par des kinases de type Hanks Ser / Thr / Tyr (Hanks et al., 1988). La phosphorylation des protéines joue un rôle clé dans la régulation de nombreux aspects du cycle cellulaire en intégrant les signaux environnementaux et cellulaires. La phosphorylation de protéines impliquées dans la réplication et la réparation de l'ADN a été identifiée chez diverses bactéries (Shi et al., 2014, Garcia-Garcia et al., 2016). Chez B. subtilis, la phosphorylation de la protéine de liaison à l'ADN simple brin SsbA sur un résidu tyrosine module sa liaison à l'ADN (Mijakovic et al., 2006). L'hélicase DnaC est un substrat de la kinase de type Hanks PrkD (Shi et al., 2014). Chez B. subtilis, la phosphorylation des résidus sérine ou thréonine est catalysée par les trois Ser / Thr kinases de type Hanks, PrkC, PrkD et YabT. YabT est une kinase transmembranaire dépourvue du domaine senseur extracellulaire caractéristique des Hanks kinases. YabT contient un motif de liaison à l'ADN essentiel à son activation (Bidnenko et al., 2013). YabT est exprimée en carence en glucose, en sporulation et en phase stationnaire (Nicolas et al., 2012). YabT phosphoryle notamment la recombinase RecA, ce qui permet de contrôler l'intégrité du chromosome pendant la sporulation (Bidnenko et al., 2013). YabT, en phosphorylant le facteur d'élongation EF-Tu, inhibe en outre la synthèse protéique (Pereira et al. 2015).

Dans cette étude, grâce à des expériences de phosphorylation in vitro et des analyses de Les mécanismes sous-jacents à ce nouveau rôle de YabA restent inconnus. Nos résultats suggèrent que la phosphorylation de YabA est corrélée avec des niveaux cellulaires élevés de Spo0A-P, ce qui conduit à une stimulation de la sporulation. Un taux élevé de Spo0A-P induit la répression du gène sinI, codant l'anti-répresseur de SinR, ce qui conduit à la forte répression de l'opéron eps (codant les enzymes permettant la synthèse des exopolysaccharides du biofilm) et de tasA (TasA formant des fibres amyloïdes) par le régulateur transcriptionnel majeur SinR (Chai et al., 2008 ;[START_REF] Deloughery | Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR[END_REF]. Cette répression pourrait expliquer l'inhibition de la formation de biofilms par la forme phosphomimétique de YabA. Cependant, l'analyse transcriptomique de la souche phosphomimétique montre une activation de l'expression de sinI, ce qui conduit à la surexpression des gènes de biofilms subséquents. De façon intéressante, seule la première partie de l'opéron eps est transcrite plus efficacement dans cette souche, la seconde partie de l'opéron étant moins exprimée. Le mécanisme qui contrôle la transcription de l'opéron eps est un élément EAR (ARN associé à eps), localisé entre le second (epsB) et le troisième gène (epsC) de l'opéron eps [START_REF] Irnov | A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales[END_REF]. En absence de ce mécanisme conjugaison (Perego et Hoch., 1996). La plupart des promoteurs rap-phr sont activés par un système de QS ComA-ComX, le facteur de transcription ComA étant lui-même activé par la phéromone associée ComX à haute densité cellulaire. Par ailleurs, RapA, RapE et RapI déphosphorylent Spo0F-P, un composant du phosphorelais, jouant ainsi un rôle majeur dans la régulation négative de la sporulation [START_REF] Jiang | Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis[END_REF][START_REF] Parashar | Conformational change-induced repeat domain expansion regulates rap phosphatase quorum-sensing signal receptors[END_REF][START_REF] Singh | Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling[END_REF].

R VI. ANNEXES 257 Nos études transcriptomiques ont montré que rapA-phrA, rapE-phrE et rapI-phrI sont fortement réprimés dans la souche phosphomimétique, ce qui suggère une accélération du phosphorelais qui pourrait conduire à une accumulation rapide de Spo0A-P, donc à une entrée plus rapide dans la sporulation. RapI contrôle également le transfert de l'élément génétique mobile ICEBs1 [START_REF] Auchtung | Identification and characterization of the immunity repressor (ImmR) that controls the mobile genetic element ICEBs1 of Bacillus subtilis[END_REF][START_REF] Bose | Regulation of horizontal gene transfer in Bacillus subtilis by activation of a conserved site-specific protease[END_REF]. L'expression de l'opéron ICEBs1 est réprimée par un répresseur transcriptionnel spécifique, ImmR, notamment au cours de la sporulation.

Lorsque RapI est produit, ImmR est inactivé et l'expression de l'opéron ICEBs1 est déréprimée.

Nous avons observé que tous les gènes du locus ICEBs1 étaient régulés à la baisse dans le mutant yabAT71D dans toutes les conditions testées. Par contre, la délétion de yabA déclenche l'expression de ces gènes pendant la phase précoce de sporulation. Ces résultats suggèrent que la phosphorylation de YabA permettrait la répression des gènes ICEBs1 de conjugaison pendant le processus de sporulation. La protéine Rok associée aux nucléoïdes est également impliquée dans la répression de ICEBs1 (Smits et Grossman, 2010). Récemment, des tests ChIP-seq ont montré l'existence d'interactions, au niveau de certaines régions ADN, entre la protéine d'initiation de la réplication DnaA et Rok (Seid et al., 2017). L'une de ces régions est localisée en We constructed YabA phosphomimetic (yab-AT71D) and non-phosohorylatable (yabA-T71A) mutants to (i) confirm the requirement of T71 for YabT-mediated phosphorylation in vitro and (ii) perform in vivo and in vitro functional studies. We show in vivo that the phosphorylation of YabA is not involved in initiation control, but rather modulates bacillus developmental processes. We found that YabA phosphorylation inversely regulates sporulation and biofilm formation highlighting the multifunctional role of YabA as well as its role in integrating physiological signals to connect chromosomal replication initiation control with cell development.

Our results support a role of YabT-mediated phosphorylation of YabA in Bacillus subtilis life-style decision making through the modulation of Spo0A-P intracellular levels. We established that YabA phosphorylation correlates with high cellular levels of Spo0A-P, leading to sporulation stimulation and preventing biofilm formation. Additionally, thin layer chromatography (TLC) analysis and In-Gel assays showed that YabA possess an atypical "ATP / GTPase" activity. This unusual activity seems to be modulated by phosphorylation of the YabA T71 residue. Our functional analysis pointed to a potential role of YabA in the c-di-GMP signaling transduction pathway, known to regulate biofilm formation in many bacteria. This suggests a complex regulatory role of YabA during development, involving signaling crosstalk. LC-MS analyzes showed that when overexpressed in Escherichia coli, YabA is phosphorylated on the residue Y90 in a YabT independent manner. Y90 belongs to the interaction C-terminal domain, which contacts DnaA and DnaN. We found that Y90 was involved YabA-mediated replication initiation control.

We provided evidence that phosphorylation state of YabA at Y90 can potentially modulates a protein-interaction switch with its protein partners DnaA and DnaN in a yeast-two-hybrid-based assay. Although we did not identified a kinase responsible for the phosphorylation of YabA at Y90 in B. subtilis, this finding hint at the possibility of a YabA-mediated control of initiation modulated by phosphorylation in this bacteria.

Thus, all of these in vitro and in vivo observations suggest the existence of different modes of regulation of YabA activity by phosphorylation, involving threonine and tyrosine residues. This study established that YabA, apart from its role during replication initiation, plays a key regulatory role in B. subtilis development.

Figure 1 .

 1 Figure 1. Schematic representation of the distinct cell types that differentiate in the communities of Bacillus subtilis. The cell types were classified according to the master regulator that triggers their differentiation. Arrows indicate the process of differentiation (Adapted from López, D. and Kolter, R., 2010).

Figure 2 .

 2 Figure 2. Comparison of the B. subtilis and E. coli origins of replication (A) B. subtilis origin of replication: DnaA-boxes are shown in blue, the dnaA gene in red, DNA unwinding elements in green and Spo0A-boxes in purple. (B) The E. coli origin of replication: strong DnaA-boxes are shown in dark blue, weak DnaA-boxes in light blue, the DNA unwinding element in green and binding sites for accessory proteins integration host factor (IHF) and Fis in orange and red, respectively(Jamenson and Wilkinson, 2017)

Figure 3 .

 3 Figure 3. Origin loading and activation of replicative helicases. (A) In Gram-negative E. coli, the initiator DnaA recognizes the bacterial replication origin oriC and melts a specific origin sequence to form an open complex in the presence of ATP. Then, DnaB-DnaC complex is loaded on each separated single-stranded DNA. The binding of primase DnaG with DnaB helicase and the primer synthesis induces removal of DnaC from DnaB. Dissociation of DnaC requires ATPase activity. Subsequent loading of DNA replicative polymerase Pol III and β-clamp triggers replication elongation. (B) In Gram-positive B. subtilis, DnaA binds and melts oriC sequence, followed by loading of DnaD and DnaB orderly onto the origin region. DnaC helicase and DnaI subsequently assemble to the origin in the presence of ATP. The remodeling includes removal of DnaD, DnaB and DnaI and the assembly of primase DnaG, two DNA polymerases, DnaE and PolC, and β-clamp activates helicase and replication fork movement (Li and Araki, 2013).

Figure 4 .

 4 Figure 4. DnaA organization domains. (A) Schematic of DnaA showing domain architecture and structures. Domain I (PDB code: 4TPS) is shown in blue, domain II in gray, domain III (PDB code: 1L8Q) in green and domain IV (PDB code: 1JLV) in red. (B) DnaA Domains III-IV bound to a non-hydrolysable ATP analogue (PDB code: 2HCB) form a spiral structure that is thought to mimic DnaA oligomerisation at the origin. A repeating pattern of DnaA protomers is shown in light blue, gold, coral and cyan (Figure adapted from Jameson and Wilkison, 2017).

  YabA and DnaN Binding to DnaA molecules, reducing the number available for initiation DnaD Direct binding to DnaA complexed with oriC Soj Direct binding to DnaA complexed with oriC SirA Direct binding to DnaA and inhibition of its binding to oriC during sporulation Spo0A Inhibition of DnaA binding to oriC (in vitro) DnaA DnaA box binding in the dnaA promoter region In the Gram positive and spore forming bacterium B. subtilis, no regulator has been identified which affects the conversion of DnaA-ATP to DnaA-ADP. Moreover, there is no concrete evidence that only the ATP-bound form, and not the ADP-bound form of B. subtilis DnaA is active in initiation. Replication initiation control is fulfilled by various proteins, which bind to DnaA and modulate its activity during the different life styles of the bacilli. The main regulatory protein I. INTRODUCTION 13

Figure 5 .

 5 Figure 5. YabA-Aim and YabA-Nim proteins are impaired for localization and cross-complementation can restore their localization at midcell. (A) Localization of YabA-wt, YabA-Nim and YabA-Aim. Overlay of fluorescence signals from GFP-YabA (green) and FM5-95 membrane dye (red) in living cells expressing YabA mutant proteins that have lost their ability to interact with DnaN (YabA-Nim) or with DnaA (YabA-Aim). (B) Restoration of YabA-Nim localization by YabA-Aim. YabA-Nim and YabA-Aim proteins fused to CFP (blue) were coexpressed with a wild-type YabA fused to YFP (yellow). YabA-Nim and YabA-Aim proteins fused to CFP (blue) or YFP (yellow), respectively, were also coexpressed in the same cells. (Noirot-Gros et al., 2006).

Figure 6 .

 6 Figure 6. Mechanism of YabA action (A) Replisome tethering model. YabA tethers DnaA to the replisome via interaction with both DnaA and DnaN, titrating DnaA away from the replication origin. (B) Cooperative inihibition model. YabA (Y) inhibits the cooperative binding of DnaA (A) at the origin during replication. When DnaN (N) is released after replication, YabA binds DnaN, realeasing DnaA.[START_REF] Jameson | Control of initiation of DNA replication in Bacillus subtilis and Escherichia coli[END_REF]

Figure 7 .

 7 Figure 7. Cell-cycle dependent localization of YFP-YabA. (A) Fluorescence microscopy showing YFP-YabA localization patterns in exponentially growing B. subtilis cells. Overlays of YFP-YabA signals (green) with origin-CFP (red) in the upper panel and with DnaX-mCherry (red) in the lower panel. Triangles indicate positions of YFP-YabA foci: white triangles no co-localization with origin or replication machinery, yellow triangles co-localization. (B) Different localization pattern of YFP-YabA, dependent on cell length. YFP-YabA (green) localization compared to the origin of replication or the replication machinery (DnaX) in red. White line, cell borders; scale bars, 2 μm (Schenk et al., 2017).

Figure 8 .

 8 Figure 8. (A) Pathway of the Soj activity cycle and (B) Model of monomeric Soj regulation of DnaA during DNA replication initiation.DnaA is stably bound to its high-affinity sites during most of the cell cycle. At initiation, additional DnaA molecules are recruited through a domain I interaction (step 1). At this point, Soj could interact with DnaA to prevent the structural transition necessary for it to form a helix on doublestranded DNA (step 2). This nucleoprotein complex facilitates the unwinding of the DUE and the loading of a compact helix onto ssDNA (step 3). Soj could interact with this newly recruited DnaA preventing the bending of the α-helix linking domains III and IV[START_REF] Scholefield | Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA[END_REF].

Figure 9 .

 9 Figure 9. Flow cytometry profiles of DBC-deleted strains. (A) The positions from which DBCs were deleted in the Δ6 strain are indicated on the B. subtilis circular chromosome. (B-K) Numer of origins/cell as determined by flow cytometric profiles of wild-type and mutant strains; (B) 168 (wild-type), (C) ΔDBC[gcp-ydiF], (D) ΔDBC[yqeG-sda], (E) ΔDBC[ywlC-ywlB], (F) ΔDBC [ywcI-vpr], (G) ΔDBC[yydA-yycS], (H) ΔDBC[thdF-jag], (I) Δ6, (J) Δ5, and (K) ΔDBC[yydA-yycS] ΔDBC[ywcI-vpr] (Okumura et al., 2012).

Figure 10 .

 10 Figure 10. Protein-protein interaction map centered on DNA replication process. Primary baits are indicated by a double blue line except PriA and DnaD, which were omitted. The dashed line symbolizes a bacterial replisome. The arrows are oriented from the bait to the prey. Functional categories are represented with colors: orange, DNA replication/recombination/repair; dark blue, mobility and chemotaxis; light blue, signal transduction; light green, transcription; pink, protein synthesis; dark green, metabolism of carbohydrates and amino acids; purple, cell division; and yellow, unknown (Noirot-Gros et al., 2002)

Figure 11 .

 11 Figure 11. Sporulation cycle.Sporulation begins when a sporangium divides asymmetrically to produce two compartments (stage II): the mother cell and the forespore, which are separated by a septum. Next, the mother cell engulfs the forespore, and following membrane fission at the opposite pole of the sporangium, a double-membrane bound forespore is formed (stage III). Coat assembly begins just after the initiation of engulfment and continues throughout sporulation (stage IV). The peptidoglycan cortex between the inner and outer forespore membranes is assembled during late sporulation (stage V, VI). In the final step, the mother cell lyses to release a mature spore into the environment (stage VII). Spores are capable of quickly germinating and resuming vegetative growth in response to nutrients (Modified from[START_REF] Mckenney | The Bacillus subtilis endospore: assembly and functions of the multilayered coat[END_REF].

Figure 12 .

 12 Figure 12. Sporulation signaling cascade in Bacillus subtilis. A simplified representation of the sporulation cascade in Bacillus subtilis showing the master transcriptional regulator Spo0A and the major sporulation-specific sigma factors up-and downstream of Spo0A. (Modified from Al-Hinai et al., 2015).

Figure 13 .

 13 Figure 13. Replication cycle-dependent expression of Sda. (A) Typical still frames from a time-lapse experiment of strain JWV159 (Psda-gfp-sda, DnaN-mCherry) after nutritional downshift. Arrows indicate a cell that shows a pulse of GFP-Sda fluorescence concurrently with the first appearance of a DnaN-mCherry focus (B) Single-cell trajectory of a typical lineage from strain JWV159. A red dotted line indicates the first appearance of a replisome, and the gray-shaded area represents the time a DnaN-mCherry focus is present.After cell division, one of the two resulting siblings is arbitrarily selected for further analysis. The increasing cell cycle length as time progresses is likely due to reduced nutrient availability(Veening et al., 2009)

Figure 14 .

 14 Figure 14. Spo0A, LexA and DnaA modulate sda transcription. (A) Schematic of the sda promoter and consensus transcription factor-binding sites. (B) Model of the Sda checkpoint network. Arrows and perpendiculars indicate positive or negative activities, respectively. Activation of the SOS response deactivates LexA (slashed lines), thereby relieving LexA-dependent repression of Psda (Figure adapted fromVeening et al., 2009)

Figure 15 .

 15 Figure 15. Phenotypic consequences of SirA induction and DnaA depletion in liquid CH medium. Time (in minutes) after induction or depletion is indicated. Membranes were stained with the fluorescent dye FM4-64 (red) and DNA was stained with DAPI (false-colored green). Examples of anucleate cells (white carets) and guillotined nucleoids (yellow carets) are indicated(Wagner et al., 2009)

Figure 16 .

 16 Figure 16. Structure of the SirA-DnaA DI . Ribbon diagram of the SirA-DnaA DI complex with SirA shown in light green and DnaA DI shown in blue(Jameson et al., 2014).

Figure 17 .

 17 Figure 17. Overview of endospore development.Sporulation is initiated by high levels of Spo0A~P, leading to a block of matrix formation. After asymmetric septation, transcriptional differentiation is directed by dedicated sigma-factors, eventually leading to spore maturation and mother cell lysis[START_REF] Slager | Hard-Wired Control of Bacterial Processes by Chromosomal Gene Location[END_REF]

Figure 18 .

 18 Figure 18. Cell-cycle-mediated pulsing controls onset of sporulation. The pulsatile profile of the Spo0A∼P concentration during the cell cycle (bottom graph, bottom panel) is made possible by the asymmetrical placement (top left) and therefore copy number development of spo0F and kinA (bottom graph, top panel; red and orange lines, respectively), which are involved in the sporulation phosphorelay, leading to phosphorylation of Spo0A (top right) (Slager and Veening, 2016).

Figure 19 .

 19 Figure 19. The five stages of biofilm development. Stage 1: Planktonic (free motile cells bacteria adhere to the biomaterial surface. Stage 2: Cells aggregate, form micro-colonies and excrete extracellular polymeric substances (EPS) (orange). Stage 3: A biofilm is formed. It matures and cells form multi-layered clusters. Stage 4: Three-dimensional growth and further maturation of the biofilm, providing protection against host defense mechanisms and antibiotics. Stage 5: The biofilm reaches a critical mass and disperses planktonic bacteria and spores, to colonize other surfaces (adapted from Vlamakis et al., 2013).

Figure 20 .

 20 Figure 20. Regulatory networks governing biofilm formation. Schematic of the complex regulatory pathways that control gene transcription during (A) planktonic growth and (B) biofilm. Rounded rectangles indicate proteins, triangles indicate open reading frames (ORFs), arrows indicate activation, T-bars indicate repression. Dashed arrows or T-bars indicate indirect activation and repression respectively. Green represents active gene transcription with a green arrow indicating translation, dark blue represents absence of gene transcription, red indicates a transcriptional repressor and orange indicates a protein-protein interaction. Light blue indicates a protein that is able to bind to DNA to activate transcription. Pink structure represents a flagellum, with the curved arrow indicating rotation and the cross indicating inhibition of flagellar rotation. Vertical rectangles labelled with "signal input" indicate sensor kinases for the Spo0A pathway. Faded shading indicates parts of the pathway that are inactive (Modified from Cairns et al., 2014).

Figure 21 .

 21 Figure 21. Schematic representation of the respective signal transduction pathways leading to the production of surfactin and the extracellular matrix via quorum sensing. The quorum-sensing pheromone ComX triggers the production of surfactin by phosphorylation of the master regulator ComA, via activation of the membrane histidine kinase ComP (left). The quorum-sensing molecule surfactin induces the phosphorylation of Spo0A via activation of the membrane histidine kinase KinC. Spo0A induces the expression of SinI, which antagonizes the repressor SinR and causes derepression of genes involved in matrix synthesis (right) (López et al., 2009b).

Figure 22 .

 22 Figure 22. Gradual increase in the level of Spo0A~P defines which developmental pathways are activated in B. subtilis. Sliding, biofim development or production of resistant spores are induced at certain levels of Spo0A~P protein in the bacterial and mediated by the differential activation of histidine kinases (Adapted from[START_REF] Kovács | Bacterial differentiation via gradual activation of global regulators[END_REF]

Figure 23 .

 23 Figure 23. Schematic representation of c-di-GMP-mediated regulation of motility and biofilm formation in bacteria. C-di-GMP is generated from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE) to the linear form, 5′phosphoguanylyl-guanosine (pGpG), and ultimately to two molecules of guanosine monophosphate (GMP). In many bacteria, c-di-GMP levels reciprocally regulate the transition between motility and biofilm formation (Adapted from Chen et al., 2012).

Figure 24 .

 24 Figure24. Protein phosphorylation reaction using ATP as phosphate donnor (A) Catalytic cycle of protein phosphorylation performed by a protein kinase. First, ATP binds to the active site, followed by the binding of substrate. The γ-phosphate of the ATP is then transferred to the substrate. Subsequently, the phosphorylated substrate is released by the kinase, and ADP disassociates to complete the cycle. (B) The substrate is attracted to the catalytic site of the protein kinase, with the hydroxyl (OH) group of the to-be-phosphorylated residue (Ser, in this example) bound to a negatively-charged residue of the kinase. The γ-phosphate of the ATP co-factor binds to a magnesium ion, which is stabilized by negatively-charged residues of the kinase. (C) A nucleophilic attack is performed by the oxygen of the substrate OH group on the γ-phosphorous of ATP. (D) The reaction proceeds with the breaking of the phosphoester bond between the γ-phosphate and ADP, resulting in the phosphorylated residue (P-Ser) and ADP (Modified from[START_REF] Ubersax | Mechanisms of specificity in protein phosphorylation[END_REF].

Figure 25 .

 25 Figure 25. Schematic representation of the major families of bacterial kinases with their respective signaling cascades. The components of the signaling cascade are color coded. External signals and target phosphorylation are represented by red arrows. Kinase domains are white, and substrates are gray. Source of phosphate is indicated next to each kinase. Specific signal outputs, or consequences of phosphorylation, are indicated below each system. Putative oligomerisation of kinases is indicated with gray shadows. Hanks-type, BY, and two-component kinases are classical ATP-dependent kinases, whereas the components of the PTS use phosphotransfer, originating by PEP-dependent EI autophosphorylation(Mijakovic and Macek, 2012).

Figure 26 .

 26 Figure 26. Schematic representation of Eukaryotic-like serine/threonine kinases (eSTK).Eukaryotic-like serine/threonine kinases are either cytoplasmic or inserted in the membrane. Their kinase domain contains the 12 conserved signatures defined by Hanks. Both of them possess a variety of additional subdomains (illustrated by the blue and white striped ovals) that regulate their activity or that are proposed to influence their cellular localization and/or to favor their interaction with their substrates. They are able to be autophosphorylated on, but not exclusively, their activation loop (Adaptated from[START_REF] Garcia-Garcia | Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria[END_REF]

  Walker A and B motifs usually found in ATPases and GTPases constitute the ATP-binding site. BY-kinases also possess an additional Walker B motif located between the A and B motifs and termed A'. A much less conserved C-terminal sequence-motif with several clustered tyrosines represents the autophosphorylation site. The exact length of the tyrosine cluster and the position of autophosphorylated tyrosines can vary considerably among BY-kinases.

  These activator domains are typically transmembrane, with the C-terminus pointing to the cytosol and interacting with the catalytic domain. The catalytic and activator domains of BYkinases can exist separately (encoded by two different genes) or in a single polypeptide chain. If one examines the distribution of these two architectural prototypes in different bacterial phyla, one can conclude that the two-protein prototype is very common in firmicutes, whereas the single-protein prototype is most widely represented in proteobacteria[START_REF] Jadeau | BYKdb: the bacterial protein tyrosine kinase database[END_REF] Shi

Figure 27 .

 27 Figure 27. Schematic representation of Bacterial tyrosine-kinases (BY-kinases). BY-kinases are either found in the form of a single-membrane protein (in red) or two separate polypeptides (in orange and red). Interaction between the two polypeptides is required for kinase activation. The cytoplasmic part constitutes the catalytic site, which possess three Walker motifs (A, A' and B)(yellow boxes) that are required for ATP binding and hydrolysis. They are able to autophosphorylate on several tyrosine residues (red circle) in a C-terminal motif termed tyrosine cluster (YC, green circle) (Adaptated from[START_REF] Garcia-Garcia | Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria[END_REF].

Figure 28 .

 28 Figure 28. Schematic illustration of regulations by Ser/Thr phosphorylation during a Bacillus subtilis spore life.The sporulation steps are presented in the green panel and the spore revival steps in the red panel. In the schematic spore, the core is in yellow, the cortex in green and the coat in gray. The two types of spore signal receptors are represented: the GerA, B, and K in sticks, the STPK PrkC in balls and stick (transmembrane domain). The two-component cascade leading to Spo0A phosphorylation is presented in the framed rectangle and regulations by STPKs are listed in the white rectangles. Other possible cross-regulations by two-component systems, BY-kinases, or other phosphorylations are not represented here in order to not overload the drawing(Pompeo et al., 2016)

Figure 29 .

 29 Figure 29. YabT phosphorylates RecA and participates in a sporulation checkpoint. (A) Expression profile of yabT adapted from Nicolas et al. (2012). (B) YabT and RecA are involved in checking chromosome integrity during spore formation. YabT (in red) is synthesized 2-3 h after the onset of sporulation. It localizes to the septum, where it encounters and binds DNA. Activated YabT phosphorylates RecA (green), whichs forms a mobile focus moving along the chromosome. If nonreparable damage is encountered, the RecA focus is maintained 6 h after initiation of sporulation and this is incompatible with the formation of a mature spore. If no irreparable damage is encountered, RecA focus is dissassembled, and sporulation proceeds normally[START_REF] Cousin | Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation[END_REF]

Figure 30 .

 30 Figure 30. PrkC senses muropeptides and triggers spore germination. (A) Expression profile of prkC adapted from(Nicolas et al., 2012). (B) Regulatory loop of spore germination depending on PrkC. Muropeptides derived from peptidoglycan of growing bacteria signal the return of favorable growth conditions. YocH generates muropeptides by cleaving the peptidoglycan and these fragments are bound by PASTA domains of PrkC. The activated kinase then phosphorylates the cellular substrates, including EF-G, which presumably stimulates protein synthesis and germination. PrkC also activates yocH transcription in a phosphorylation-dependent manner, and this reinforces the forward-feeding regulatory loop[START_REF] Cousin | Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation[END_REF].

Figure 31 .

 31 Figure 31. Genetic arrangement of the tkmA-ugd operon. Promoters and predicted transcription terminators in the intergenic regions between ywqB and tkmA and between ptkA and ptpZ are indicated by arrows and stem-loops, respectively. A previously characterized small ORF with unknown function in the intergenic region of ywqB and tkmA is highlighted in yellow. The protein products encoded by individual genes in the tkmA-ugd operon are noted below the diagram[START_REF] Gao | The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis[END_REF].

Figure 32 .

 32 Figure 32. TkmA and TkmB are involved in regulating the BY-kinase PtkB and biosynthesis of EPS. (A) Yeast-two-hybrid showing reciprocal interactions between PtkA and its cognate modulator TkmA (fused to either AD or BD) (left) and interactions among components of the two BY-kinase systems: PtkA/TmkA (fused to BD) and PtkB/TkmB (fused to AD) (right). (B) A proposed model for how both TkmB and TkmA can be involved in regulating the BY-kinase PtkB and biosynthesis of EPS. TkmB and PtkB were shown to regulate biosynthesis of EPS via a feedback mechanism by TkmB sensing the presence of self-produced EPS molecules. The roles of TkmA and PtkA in regulating other protein targets such as SSB, Ugd, etc were reported previously.The putative signal for TkmA is unknown. In this model, TkmA is predicted to crosstalk with PtkB and thereby contributes to the regulation of EPS biosynthesis. (Adaptated from Shiet al., 2014 and[START_REF] Gao | The Bacterial Tyrosine Kinase Activator TkmA Contributes to Biofilm Formation Largely Independently of the Cognate Kinase PtkA in Bacillus subtilis[END_REF].

Figure 33 .

 33 Figure 33. Protein substrates of B. subtilis BY-Kinases. Network of BY-kinases and their substrates in B. subtilis. Proteins are color-coded: BY-kinases are shown in red, kinase activators in green and substrates in yellow. Phosphorylation reactions are indicated with red arrows. Substrate physiological role is indicated next to substrate whose activity is affected by phosphorylation. Substrates whose subcellular localization is affected in the ΔptkA strain are grouped together[START_REF] Mijakovic | Protein-tyrosine phosphorylation in Bacillus subtilis: a 10-year retrospective[END_REF]

FIGURE 1 |

 1 FIGURE 1 | PPI network centered on ser/thr (yellow nodes, bleu edges) and tyr (red nodes, black edges) kinases, phosphatases and modulators of B. subtilis reveals potential regulations by phosphorylation of various DNA-related pathways. Proteins are represented as nodes connected by edges. Potential substrates, defined as connected by both a kinase and cognate phosphatase, are represented by diamonds. HTH-containing proteins, connected by more than one kinase, are represented by squares; In vitro characterized phospho-proteins are labeled in red. Proteins found phosphorylated in other bacteria are indicated by an asterix. Other interactions between the proteins are illustrated by light gray edges (fromMarchadier et al., 2011; Shi et al., 2014b). In vitro validated kinase-substrate phosphorylation is indicated by dashed lines.

FIGURE 2 |

 2 FIGURE 2 | PPI profile similarities between the protein modulators TkmA (anchor) and MinD (scaffold) in B. subtilis. Tyr and Ser/Thr phosphorylation pathways are filled in red and yellow, respectively. Cell division pathway is indicated in green.

  bacteria, phosphorylation of various components of the DNA replication machinery, as well as of proteins involved in the replication cycle, has been identified (García-García et al., 2016; I.INTRODUCTION-Chapter 5.4). The bacterial SSB proteins (single strand binding proteins) coordinate DNA replication and repair through their binding to DNA in association with the replisome. B. subtilis SSB proteins, SSBA and SSBB, are phosphorylated onto a tyrosine residue.

  Bacteria developed various regulatory strategies to ensure only one initiation event per chromosome per cell cycle and to coordinate chromosome replication with cell division. The Gram-positive bacterium Bacillus subtilis is a developmental model organism capable of differentiating into spores or forming biofilms in response to environmental signals. How B.

  . During vegetative growth, DnaA binds to the replication origin oriC and forms high-order oligomeric assemblies leading to unwinding of the duplex DNA for subsequent loading of the replication machinery. Several anti-cooperativity factors regulate DnaA oligomerization at oriC during the different life-styles of the bacterium (Jameson and Wilkinson 2017). During vegetative growth, three regulatory proteins, YabA, DnaD and Soj, down-regulate replication initiation by binding DnaA to prevent its cooperative binding at origin sequences (Merrikh and Grossman 2011, Bonilla and Grossman 2012, Scholefield and Murray 2013

Figure 1 .

 1 Figure 1. YabA is phosphorylated by the Ser/Thr kinase YabT at the residue T71 localized within the flexible inter-domain region (A) In vitro phosphorylation assay of YabA, YabAT71A (nonphosphorylatable) and YabAT71D (phosphomimetic) in the presence of YabT. Presence of purified proteins is indicated with +/-above each line. (B) Mass spectrometry detection of the phosphorylation site on YabA. In vitro phosphorylation reaction of YabA was carried out in the presence of YabT as described in materials and methods, only with non-radioactive ATP. Identification of the phosphorylation site was performed by LTQ-Orbitrap and Q exactive and validated by MaxQuand (Fig. S1). The result shows an 80 Da molecular weight increment (corresponding to phosphate) at the position Threonine 4 of the peptide that corresponds with the Threonine 71 of the protein. (C) Mapping of the YabA T71 phosphosite within the flexible hinge region and YabA tetramer model showing the possible sites susceptible to phosphorylation.

Figure 2 .

 2 Figure 2. Phosphorylation of the YabA T71 residue is not involved in the replication initiation control. (A) Ori/ter values determined by qPCR on total DNA extraction of unsynchronized cells grown on LB media at 37 °C up to OD ~ 0.3. The asterisks indicate the results is statistically different compare to the control (P<0.001 where n ≥3). The error bars represent standard error of the mean. (B) YabA and YabA phoshomutants localization. Fluorescence signals from GFP-YabA* (green) in living cells expressing YabA, YabAT71A or YabAT71D proteins.

Figure 3 .

 3 Figure 3. YabA phosphorylation enhances sporulation (A) Quantification heat resistant spores of wild type B. subtilis and strains ΔyabA, yabAT71A, yabAT71D, ΔyabT and double mutants (ΔyabT-yabAT71A and ΔyabT-yabAT71AD) in Difco sporulation media DSM at T18. Spores counts are expressed as % of total number of viable cells. Error bars represent standard deviation from 3 biological replicates. (B).Percentages of sporulating cells observed by microscopy at stage T2 after induction of sporulation by the resuspension method. Error bars represent standard deviation from 5 biological replicates. Over 400 cells were counted for each strain and replicate (left). Effect of YabT overexpression using a pDG148-flag-yabT plasmid inducible by IPTG. Sporulation was carried out by the resupension method as previously and expression of YabT was induced by adding 1 mM of IPTG at T0 (resuspension). Percentage of sporulating cells was calculated at T2 (right).

Figure 4 .

 4 Figure 4. Expression of spo0A increases in yabAT71D strain. (A) Schematic representation of spo0A regulatory loop. When phosphorylated, Spo0A is accumulated and activates its own expression. (B) Expression of spo0A was measured after induction of sporulation by the resuspension method using a Pspo0A-gfp transcriptional fusion. Bacillus strains expressing the yabA wild type or mutated derivatives are indicated by colors.

  ∆yabA mutant. Taken together, these results highlighted a regulatory role of YabA phosphorylation at residue T71 during the early sporulation program. The stimulation of the expression of the early sporulation spoIIA gene indicates a positive activation of this σ Hdependent promoter. These results provide additional evidence for enhanced levels of Spo0A~P mediated by YabA phosphorylation.

Figure 5 .

 5 Figure 5. Effect of YabA phosphorylation at T71 on sporulation early developmental program. (A) Illustration of sigma factors cascade controlling the sporulation process. (B) Luciferase was placed under the control of P-spoIIA promoter, which drives the expression of spoIIA, a early-stage sporulation gene whose expression is directly regulated by σ H , and P-spoIIID promoter, which drives the expression of spoIIID, a middle-stage sporulation gene whose expression is directly regulated by σ E . Bacillus strains expressing the yabA wild type or mutated derivatives are indicated by colors.

Figure 6 .

 6 Figure 6. YabA phosphomimetic (T71D) shows a defect in biofilm formation. (A) Growth curves of B. subtilis strains in MSgg media (B) Measurement of optical density at 600 nm of re-suspended biofilm cells. Error bars represent 3 independent experiments with 4 biological replicates (C) Effect of YabT overexpression using pDG148-flag-yabT plasmid inducible by IPTG in biofilm formation. Biofilms were carried out as previously, with the only difference of adding 0.5 mM or 1 mM of IPTG to MSgg media to induce yabT expression. Error bars represent of the standard error of the mean over 5 biological replicates. (** P<0.01 and ***P<0.001).

  Our results highlight the existence of a role of the phosphorylation of the replication initiation controller YabA in B. subtilis cell fate decision, through the modulation of Spo0A~P intracellular levels. YabA phosphorylation is carried out by the Ser/Thr kinase YabT, expressed at the early stage of sporulation and during biofilm formation. We established that YabA phosphorylation correlates with high cellular levels of Spo0A~P, leading to sporulation stimulation. A consequence of elevated levels of activated Spo0A is the prevention of the expression of the anti-repressor sinI leading to full repression of the EPS operon by the master regulator SinR(Chai

Figure 7 .

 7 Figure 7. Structure and morphology of air-liquid biofilms. (A) 3D-volumes of the wild-type, ΔyabA, yabAT71A, yabAT71D, ΔyabT and doubles mutants (ΔyabT-yabAT71A and ΔyabT-yabAT71D) derivatives of CCBS185 strain. 3D-volumes were imaged and analyzed by Imaris software. Biovolumes based on total volume (μm3) per area (μm2) are plotted. Error bars represent of the standard error of the mean (n≥ 3). (B) Colony morphology of wild-type B. subtilis, and strains ΔyabA, yabAT71A, yabAT71D, ΔyabT and doubles mutants (ΔyabT-yabAT71A and ΔyabT-yabAT71D). Representative images of colonies incubated in MSgg plates for 4 days at 30 °C.

  partially overlapping DNA fragments containing parts of yabA-T71A gene and the flanking regions. Primers pair yabA fwd + YabAT71DR and YabAT71DF + yabA rev were used for yabA-T71D. The insertion cassette encoding the Lambda CI repressor and the phleomycin-resistance gene was flanked by mutagenized yabA fragments in direct orientation by joining PCR using R II. RESULTS 107 primers yabA fwd and yabA rev. The amplified fragment was used to transform CCBS185 cells with selection on phleomycin. The obtained clones were controlled for the loss of neomycin resistance due to CI, which inhibits the transcription from the Pr promoter. Finally, homologous regions allow a recombination process leading to the excision of the cassette and cells were isolated by counter-selection for neomycin-resistance. CCBS185 cells were transformed with the chromosome from the strain BMR26(Bidnenko, Shi et al. 2013) to obtain the yabT deletion marked with the spectinomycin-resistance in CCBS185 background.

  6, IPTG was added to 0.5 mM to induce expression of the protein and the culture was incubated overnight at 18°C. All subsequent steps were carried out at 4°C. Cells were pelleted and resuspended in buffer A (50 mM Tris pH 8, 1 M NaCl) and were disrupted by sonication and finally centrifuged at 40.000 rpm for 1h. Proteins from the supernatant were precipitate by adding ammonium sulfate at a final concentration of 20%. After centrifugation, the pellet containing YabA protein was collected and resuspended in buffer B (50 mM Tris pH 8, 0.5 M NaCl). The soluble fraction was concentrated and injected onto a Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated in buffer C (50 mM Tris pH 8, 400 mM NaCl). Franctions containing YabA, YabA-T71A or YabA-T71D were pooled, dialyzed and stored at -20 °C in presence of 50 % glycerol (Fig.S1).Identification of the phosphorylated residue by mass spectrometryIn vitro phosphorylation reaction of YabA was carried out in the presence of YabT essentially as described below, with the only difference of using non-radioactive ATP. A typical 30 µL reaction contained 40 µM of YabA, 0.8 µM of YabT, 5 mM MgCl2 and 50 mM Tris-HCl pH 7,4 and 50 µM ATP. After SDS-PAGE, in-gel digestion was performed as described previously by addition of modified trypsin (Promega) dissolved in 50 mM NH4CO3. The resulting peptides were extracted successively with 2% trifluoroacetic acid (TFA) and 50% acetonitrile (ACN) and then with ACN.Vacuum-dried peptide extracts were resuspended in 30 μl of 0.05% TFA, 0.05% HCOOH, and 2% ACN. A Q Exactive (Thermo Fisher Scientific) coupled to Eksigent nano LC (AB-sciex) was used for the nano-LC-MS/MS analysis. 4 µl were injected on the nanoLc-Ultra system (Eksigent) chain.Sample was loaded at 7.5 µl / min on the precolumn cartridge (PepMap 100 C18, 5 µm, 120 Å, 5 mm Dionex) and desalted with 0.1% HCOOH. Then, peptides were separated with a gradient of acetonitrile on the reverse phase column (C18 Biosphere, 3µm, 120 Å, 75 µm i.d, 15 cm,). Eluted peptides were analysed on-line with a Q Exactive mass spectrometer (Thermo Electron) using a nanoelectrospray interface. Ionization (1.5 kV ionization potential) was performed with stainless steel emitters(30 µm i.d.; Thermo Electron). Peptide ions were analysed using Xcalibur 2.1 with the following data-dependent acquisition steps:(1) full MS scan (mass-to-charge ratio (m/z) 400 to 1400) and (2) MS/MS. Step 2 was repeated for the 8 major ions detected in step 1. Dynamic exclusion was set to 40 s. Lock mass option was chosen "best", MS resolution 70000 at m/z 400, auto gain control was 3e6, maximum injection time 250 ms. For MS2, the resolution was 17500 at m/z 400, auto gain control was 2e5, maximum injection time 120 ms, isolation window m/z = 3, normalized collision energy: 25, underfill ratio 0.5 %, intensity threshold 2.5 e4. Charge state: 2.3.4. Dynamic exclusion 40 seconds.

In vitro phosphorylation assay

 assay Phosphorylation reactions were performed in a total volume of 30 µl, in the presence of 40 µM of YabA, YabAT71A or YabAT71D and 1,6 µM of YabT. In addition to the appropriate proteins, the reaction mixture contained 50 µM [γ-32 P] ATP (20 µCi/mmol), 5 mM MgCl2 and 50 mM Tris-HCl pH7,4. Reactions were incubated at 37°C for 1,5 hours and stopped by adding loading buffer for SDS-PAGE. Proteins were separated by electrophoresis on denaturing 15% polyacrylamide gels. After drying the gels, radioactive signals were visualized using STORM phosphoimager.Isolation of chromosomal DNA and Q-PCRDNA was isolated from cultures at exponential phase (OD=0.3-0.4) in order to determinate the Ori/Ter ratio by qPCR. 2 ml of culture was centrifuged and rinced with 10 mM Tris-HCl (pH 8), 10 mM EDTA (pH 8) and 300 mM NaCl. The pellet was resuspended in 200 µL Lysis buffer(50 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl and 10 mg/ml lysozyme). After incubation for 10 min at 37°C, was added 10 µL sarkozyl 30 % were added to complete cell lysis. The samples were incubated at 65°C for 20 min followed by lithium chloride precipitation to precipitate proteins and other contaminants by salting out. 0.8 volume of 2-propanol was added before R II. RESULTS 110 centrifugation at 16000 rpm. The pellet was then resuspended in 200 µL double-distilled H2O (ddH2O) and 200 µL of 10 M LiCl4. Samples were incubated at -20°C for 20 min and the DNA was precipitate by addition of ethanol and sodium acetate. Precipitated DNA was resuspended in 50 µL of ddH2O. Q-PCRs were carried out in triplicates of 25 µl each and using three dilutions of chromosomal DNA (1, 0.1 and 0.01 pg µL -1 ). 5 µL diluted DNA was used as template and added to a mixture of 12.5 µL ABsolute Blue qPCR SYBR®Green ROX Mix (Thermo Scientific) and 0.3 µL primers mix (3µM each forward and reverse) in a 96-well PCR plates. Primers pair for Origin proximal sequence (OriL3F and OriL3R) and Terminus proximal sequence (TerL3R and TerL3R, Table 2) were used in separated reactions and tested for an efficiency greater than or equal to 90%. Reactions were carried out with a Mastercycler®ep realplex (Eppendorf) with the following program: 15 min at 95°C, (15 sec at 95°C, 1 min at 60°C)x40 cycles, 15 sec at 95°C, 15 sec at 60°C, 20 min to reach 95°C, 15 sec at 95°C. Results were analyzed with Realplex program (Eppendorf) and quantified by ΔΔCt method.Yeast two-hybrid and three-hybrid assaysFull length YabA and derivatives YabA-T71A and YabA-T71D, full length DnaA and DnaN proteins were expressed as fusions to the GAL4 binding domain BD or activating domain AD from the vectors pGBDU-C1 and pGAD-C1, respectively. pGBDU and pGAD derivative constructs were introduced by transformation into PJ694-(α) and (a) haploid strains, respectively. Binary interactions were tested by combinational mating of the strains expressing the BD and AD fusions as previously described(Noirot-Gros et al, 2006). Interacting phenotypes were tested by the ability of the diploid cells to grow on selective media SD-LUH and SD-LUA.Construction of the GFP Fusions and Epifluorescence MicroscopyThe N-terminal tagged GFP-YabA proteins were conditionally expressed from the xyloseinducible promoter Pxyl, at the ectopic amyE locus in the B. subtilis chromosome. Primers yabAapa and yabA-R-salI, carrying ApaI and SalI restriction sites, respectively, were used to PCR amplify the wild-type and mutant yabA genes from the corresponding bait vectors. The PCR fragments and pSG1729 were digested with ApaI + SalI. E. coli DH10B cells were transformed by the ligation mixtures, and single transformants were verified by sequencing. The gfp-yabA constructs were then integrated into the amyE locus of the B. subtilis ∆yabA strain (JJS142) by transformation, using spectinomycin (60 μg ml -1 ) as a selection. The presence of the integrated gfp fusions were verified by PCR amplification from the amyE flanking region and by the inability of the strain to produce amylase. For induction of the GFP-tagged YabA proteins, cells from overnight cultures grown at 37°C in LB supplemented with spectinomycin were diluted to OD600 = 0.01 in the same medium supplemented with xylose to 0.5% until the OD600 reached 0.3-0.5.

3. 5

 5 ml of MSgg media at OD600=0.1. Cultures were maintained in a growth chamber at 28°C and 70% humidity for 48 hours. Pellicles were brought up to the top of the wells by slowly adding MSgg media and peeled of onto a 2.5 cm diameter circular cover slide. The cover slides with intact biofilm pellicles were mounted onto an Attofluor Cell Chamber and stained with the FilmTracer FM 1-43 Green Biofilm dye (Thermo Fisher Scientific). Stained biofilms were observed using a spinning disc confocal microscope (Nikon Eclipse Ti-E coupled with CREST X-LightTM confocal imager; objectives Nikon CFI Plan Fluor 10X, DIC, 10x/0.3 NA (WD = 16 mm); excitation was performed at 470 nm and emission recorded at 505 nm). The three-dimensional biofilm images were quantified using the IMARIS x64 9.0.2 software (XTension biofilm plug-in).

Figure S2 .

 S2 Figure S2. MaxQuant validation of YabA phosphorylation at T71 : Spectra view from MaxQuant that confirms the position T71 with a very good score of Localization probability (0.9, over a maximum of 1).

Figure S3 .

 S3 Figure S3. YabA T71 mutants are not affected in their ability to interact with YabA partners. (A) Interaction between YabA non-phosphorylatable (T71A) and phosphomimetic (T71D) derivatives with DnaA and DnaN detected by Yeast-two-hybrid. All genes were expressed in fusion with both the binding domain (BD) and activating domain (AD) of GAL4 and tested for interaction in a yeast two-hybrid matrix assay. (B) YabAT71A and YabAT71D mutant baits were still proficient for interaction with the YabA partners AcuB and TlpA in a yeast two-hybrid assay.

Figure S5 .

 S5 Figure S5. YabT-mediated phosphorylation of YabA at 71 enhances sporulation. (A) A sample of sporulating cells observed at T2 with FM4-64 to stain membranes and DAPI to stain DNA. (B) Effect of YabT overexpression using a pDG148-yabT plasmid inducible by IPTG in WT, yabAT71A and yabAT71D backgrounds. (C) Kinetics of sporulation of wild-type B. subtilis and strains yabAT71A and yabAT71D in resuspension medium from stage T0 to T6. Sporulation was followed by microscopy and the percentage of sporulating cells was calculated for each time. (D) Expression of YabT was confirmed by western blot, using ANTI-FLAG M2 monoclonal antibodies (Sigma-Aldrich; dilution 1:10.000) as primary antibody and revealed by the secondary goat peroxidase-coupled anti-mouse IgG antibodies (Sigma-Aldrich; dilution 1:20.000), from equivalent amounts of total cells lysates of B.subtilis expressing YabT.

Figure S6 Figure S6 .

 S6S6 Figure S6

  but rather modulates B. subtilis differentiation processes such as sporulation and biofilm formation, suggesting a multifunctional role of YabA. We established that YabA phosphorylation correlates with high cellular levels of Spo0A~P, leading to sporulation stimulation. In order to further investigate the regulatory network that could be targeted by YabT-mediated phosphorylation of YabA in the B. subtilis life-style decision, we wished to determine the effect of YabA phosphorylation on global gene expression and the transcriptional responses at the onset of sporulation. For this propose, we established a collaboration with Jean-Yves Coppee, Odile Sismeiro, Rachel Legendre and Hugo Varet from the Institute Pasteur (Transcriptome and EpiGenome platform).

Figure 1 .

 1 Figure 1. Bioanalyzer profiles of total RNA. (A) Bioanalyzer electrophoresis showing 23S, 16S and traces of multimeric forms. (B) Electropherograms. The RNA Integrity Number (RIN) is shown next to each total RNA profile.

Figure 2 .

 2 Figure 2. MA-plot of the dataset. MA-Plot of the data for comparison with the wild-type strain in each condition. Red dots represent significantly differentially expressed features. A p-value adjustment was performed and the level of controlled false positive rate was set to 0.05.

Figure 3 .

 3 Figure 3. Heatmap showing the expression pattern of genes involved in (A) sporulation and (B) biofilm (Next page). Differences and similarities between mRNA levels are shown, violet indicates decreased levels, and orange indicates increased levels. Expression pattern of yabAT71D at T3 is indicated with a red arrow.

Figure 4 .

 4 Figure 4. Heatmap showing the expression pattern of high impacted genes. Differences and similarities between mRNA levels are shown, violet indicates decreased levels, and orange indicates increased levels.

  This part was done at the Institut Pasteur-Transcriptome and EpiGenome platform. In bref, the ribosomal RNA (rRNA) was removed by depletion using the RiboZero rRNA removal kit (Illumina), adapted to bacteria. For library preparation, rRNA depleted samples were submitted to different steps in order to obtain a double-stranded amplified cDNA carrying adapters, allowing compatibility with the Illumina sequencers, using the TruSeq Stranded RNA Library Prep kit (Illumina). cDNA libraries were checked for concentration and quality on DNA chips with the Bioanalyser Agilent before loading on the flowcell. Samples normalized to 1nM in 0.1N NaOH were denaturated 5' at RT and finally diluted at 10 pM for loading on the flowcell. The sequencing was performed on the HiSeq 2500 Illumina sequencing machine and it was a SRM V4 run (number of cycles: 65 and number of index bases read: 7). Statistical analysis of the data was performed using the R software, Bio conductor packages including DESeq2 and the PF2 tools package (version 1.4.7) developed at PF2 (Institut Pasteur). Normalization and differential analysis were carried out according to the DESeq2 model and package (version 1.16.0).

Figure 1 .

 1 Figure 1. YabA exhibits differences activities. In vitro auto-phosphorylation assay of YabA, nonphosphorylatable YabAT71A and YabA3M containing 3 mutated residues (T69A, T71A, T74A). All reaction contained [γ-32 P] ATP, 5 mM MgCl 2 and 50 mM Tris-HCl pH 7.5.

Figure 2 .

 2 Figure 2. ATPase activity detected in the purified YabA and derivaties proteins using In-gel assay following CN-PAGE. (A) 20 μg of purified YabA, YabA non-phoshorylatable (T71A) and YabA phosphomimetic (T71D) proteins were resolved on CN-PAGE (4-16%) gel and stained with coomassie brilliant blue. Several YabA oligomers were observed (B) In-gel ATPase assay for 20 μg of purified YabA and YabA derivatives proteins showing white bands due to the formation of lead phosphate upon ATP hydrolysis. ST: is the NativeMark™Unstained Protein Ladder (Ref: LC0725 TermoFisher) which serves as positive and negative control, the 242 kDa protein shows an ATPase activity.

Figure 3 .

 3 Figure 3. Unusual ATP/GTP hydrolysis by YabA and derivatives proteins using TLC. (A) Separation of nucleotides generated from [α-32 P] ATP (left) or [α-32 P] GTP (right) by YabA and phospho-mutants (YabAT71A and YabT71D) at different times (15, 30, 60, 90 and 120 min). (B) TLC showing GTP hydrolysis by two YabA proteins purified using different approaches: YabA 3 was purified by ammonium sulfate precipitation followed by gel filtration as describe materials and methods, YabA 3 was purified as describe Felicori et al., 2015. (C) SDS-PAGE showing purified YabA proteins. Line 1 and 2 represent YabA-6xHis, lines 3 and 4 YabA after cleaving the 6xHis tag, 5 and 6 YabA purified by ammonium sulfate precipitation. Red arrows represent proteins used for TLC assay.

  gradually heated from 25°C to 95°C, the fluorescence increases gradually, until the protein is completely unfolded. The melting point (Tm) of the protein of interest is inferred from the fluorescence curve (Figure 4.A). Addition of a compound that binds and thermally stabilizes the protein induces a shift in the melting point, and the new curve of fluorescence allows the calculation of the new Tm of protein-ligand complex (Figure 4.B). R II. RESULTS 150 We utilized a StepOnePlus RT-PCR instrument, using a solution volume of 25 µL in 96 well plates. The reactions contain 0.5 to 1 mg/ml of the protein of interest and 5X SYPRO orange (SYPRO orange is provided as a 5000X stock solution by Invitrogen) as manufacturer recommendations, thus in the presence or absence of the indicated ligand. Non-hydrolyzable ATP and GTP (Adenosine 5′-[γ-thio] triphosphate tetralithium salt and Guanosine 5′-[γ-thio] triphosphate tetralithium salt) were used at different concentrations. Purified DnaA was used as a positive control, since the interaction between DnaA and ATP is well characterized (see I.INTRODUCTION-Chapter 2.1.2).As expected, thermostabilization was readily observed for DnaA using 200 µM of non-hydrolyzable ATP (Figure5). Indeed, the Tm of DnaA increased in temperature (Tm=13°C) in the presence of ATP (Tm DnaA = 45°C and ATP-DnaA= 58°C), meaning that DnaA binds ATP in these conditions. By contrast, no Tm variation was evidenced when DnaA was kept in presence of GTP. These results are in agreements with published data.

Figure 4 .

 4 Figure 4. Differential Scanning Fluorimetry (DSF) principle. (A) Representation of the dye SYPRO orange binding upon protein unfolding. (B) Thermal shift in the apparent melting temperature of a protein upon the addition of stabilizing ligand.

Figure 5 .

 5 Figure 5. Differential Scanning Fluorimetry of DnaA in presence of ATP or GTP. Thermal denaturation curves obtained for DnaA and DnaA plus ATP (ATP-DnaA) or DnaA plus GTP (GTP-DnaA). Normalized and derivative views are showed upper and lower, respectively. DnaA shows average Tm= 45°C and ATP-DnaA, average Tm = 58°C.

Figure 6 .

 6 Figure 6. Differential Scanning Fluorimetry of YabA in presence of ATP or GTP. Thermal denaturation curves obtained for YabA in red and YabA plus ATP in green (left) or YabA plus GTP (right). Normalized and derivative views are showed upper and lower, respectively. Derivative views showing several transition states.

Figure 7 .

 7 Figure 7. Riboswitch based system which responds to changes in c-di-GMP levels Schematic representation of the riboswitch-GFP reporter (Left). In response to ligand binding (c-di-GMP) to a riboswitch aptamer, changes occur in the expression platform that result in regulation of downstream open reading frames, in this case by increasing the frequency of transcriptional termination upstream of the GFP. This riboswitch is integrated in a c-di-GMP null strain, highly fluorescent (Δdgcs, Δpde) or in a non-fluorescent strain, highly synthetizing intracellular c-di-GMP (dgc ++ , Δpde) (right).

Figure 8 .

 8 Figure 8. Expression of YabA induces a DGC activity (A) PDE assay showing normalized fluorescence data with pdhE (as max GFP-positive control) in the NP400 background. (B) DGC assay showing normalized fluorescence data with NP401 (as max GFP) in the NP401 background. Cells were grown at in LB for 5 h and GFP fluorescence. Error bars from 4 independent experiments with 2 biological replicates.

Figure 9 .

 9 Figure 9. YabA shows two population in which only one induces a DGC activity. Riboswitch-based assessment of in vivo activity for the parent strain NP401(c-di-GMP null), DgcP and YabA. Decreased in GFP fluorescence relative to that of the c-di-GMP null strain are indicative of an active diguanylate cyclase. Two population of GFP intensity are present in YabA.

  ATPase and GTPase activities of YabA, YabAT71A and YabAT71D were tested in reaction mixtures containing 4,8 mM protein, 50 µM [-32 P]ATP or [-32 P]GTP (20 µCi/mmol), 5 mM MgCl2 and 50 mM Tris-HCl pH 7.4. After 15, 30, 60, 90 and 120 min incubation at RT, aliquots of 4 µL were withdrawn and the reaction products were separated by thin layer chromatography (TLC) on polyethyleneimine cellulose sheets (CEL 300 PEI, Macherey-Nagel) using 0.3 M potassium phosphate (K2HPO4 and KH2PO4) buffer pH 7.4 as solvent. Radioactive signals on the dried sheets were visualized with a STORM Phosphoimager.Differential scanning fluorimetry (DSF)Thermal shift assays were performed by thermal denaturation in the presence of SYPRO orange (Invitrogen) using a StepOnePlus Real-Time PCR machine (Life Technologies) and thermal ramping (1°C per minute between 25 and 94°C). Measurements were performed in a 96-well plate in 25 μl containing 1 mg/ml of protein, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2 and 400 mM NaCl, in the presence or absence of the indicated concentrations of ligand. Data were processed using the StepOne Software v2.3 to generate the curves. The thermal melting point (Tm) was identified from the midpoint of each melting curve.

3 .

 3 Complementary results: YabA structure and architectural organization of its complex with DnaA and DnaN. Here I present a collaborative work that unravels the quaternary structure of YabA and provides insight into its interactions with both DnaA and DnaN, and thus the potential mechanisms employed by YabA to regulate the initiation of DNA replication. I participated to this work in deciphering the architectural organization of the DnaA:YabA:DnaN hererocomplex via yeast two-and three-hybrid assays. YabA negatively controls replication initiation through interactions with the initiator protein DnaA and the sliding clamp DnaN. YabA has been shown to interact with DnaA and DnaN via its C-terminal domain(Noirot-Gros et al., 2006). YabA structural organization has been predicted to be composed of a leucine zipper at its N-terminus and a putative zinc-binding domain at its C-terminus. YabA is conserved in low-GC Gram-positive bacteria and represents the prototype of a distinct family of replication controller proteins. Structural information was need for understanding how YabA regulates the initiation of replication.

  hybrid assays as well as in vivo subcellar localization studies. This study proposed a structural architecture of full-length structure of YabA, where the N-terminal domains form a tetrameric helical 'core' structure flanked by the monomeric C-terminal domains, one attached to each Nterminal domain via a flexible linker region.The full length YabA can form a ternary complex with DnaA and DnaN as revealed by yeast three hybrid assay, genetic and subcellular localization studies(Noirot-Gros et al., 2002). Moreover, the C-terminal domain of YabA has previously been shown to carry the determinants for interacting withDnaA and DnaN (Noirot-Gros et al., 2006). As the C-terminal domain has been characterized to be spatially independent of the N-terminal domain, this posed the question of whether or not these two domains were also functionally independently. Yeast two-and three hybrid experiments were carried out to assess whether or not an independent C-terminal domain could contact DnaA and DnaN, or if the N-terminal domain was also required for binding.Our data reveal that the C-terminal domain alone can interact with both DnaA or DnaN. However, in contrast to the full length, self-interacting YabA, one C-terminal domain is unable R II. RESULTS 162 to form a simultaneous interaction with both DnaA and to DnaN. These results determine a functional role for the N-terminal domain of YabA, which appears to provide a structural 'hub' from which both DnaA and DnaN can interact with separated C-terminal domains. Furthermore, the determinants for interaction with DnaA and DnaN were mapped in the Cterminal in silico model. They delimit interacting surfaces that partially overlap, thus indicating a mutually exclusive mode of interaction on the same C-terminal domain. YabA has previously been shown to form foci at the mid-cell during most of the cell-cycle, indicating localization with the replication machinery. Introduction of the mutations leading to the loss of interaction (LOI) with either DnaA or DnaN into YabA (YabADnaA and YabADnaN-LOI mutants) affects its localization in vivo. LOI with either DnaA or DnaN is associated with both loss of YabA localization and loss of initiation control. When YabA LOI mutants were expressed individually in the cell, they lose their ability to localize at the replication machinery. However, when YabA LOI mutants deficient for either interaction with DnaA or with DnaN were co-expressed in the cell, they complemented within a tetramer and restored the formation of YabA foci at the mid-cell. This provides evidence for the physiological role of a DnaA-YabA-DnaN complex, and highlights the requirement of the tetramerization of YabA to allow interaction with multiple partners in vivo.

Figure 1 .

 1 Figure 1. (A) Schematic representation of YabA domain organization with the NTD (Indian red) and CTD (green) protein constructs used in the study. Bars indicate the position of Zn binding residues. (B) Web logo showing conservation of amino acid residues in the C-terminal region of YabA. Conserved histidines and cysteines are indicated by arrows. (C) Atomic Absorption Spectroscopy of YabA in solution. Zn measurements by AAAF. (D) Circular dichroism spectroscopy. Spectra were recorded for YabA (blue), YabA 1-58 (red) and YabA 70-119 (green). All three spectra exhibit features characteristic of a folded protein, with increasing molar ellipticity with decreasing wavelength below 205 nm and minima over the wavelength range 205-225 nm indicating a preponderance of ␣-helices. (E) Molecular mass measured from SEC-MALS analysis.

Figure 2 .

 2 Figure 2. Structure of the YabA-NTD tetramer. (A) Ribbon representation of the two chains of YabA 1-62 in the asymmetric unit A (ice blue) and B (light green) with the N-and C-terminal residues labelled. (B and D) The YabA 1-62 tetramer. Each chain is shown as a ribbon either colored ramped as shown in the key (B) or colored by chain (D) with an accompanying surface rendering. (C) Ribbon representation of molecules A' and B illustrating the tweezer arrangement of these chains. (E) The four helix bundle region. The chains are displayed as ribbons colored by chain A (ice blue) B (light green) A' (lemon) and B' (coral). The C␣ and side chains of apolar residues in the interior are colored by residue type, light green for Val and Ile and lawn green for Leu to emphasize the leucine zipper. Polar interactions between the chains are shown as dashed lines and residues forming the prominent interchain interactions referred to in the text are labeled. (F) Stereo image of the coiled coil region of chains B and A'. The C␣ and the side chains of residues from the two chains are displayed with the carbon atoms colored according to the chain and with nitrogens in blue, oxygens in red and sulphurs in yellow. Polar interactions between the chains are shown as dashed lines.

Figure 3 .

 3 Figure 3. Homology model of the YabA-CTD monomer. (A) Structural sequence alignment of YabA model with the identified homologs EEA1 (pdb code 1JOC)[START_REF] Dumas | Multivalent endosome targeting by homodimeric EEA1[END_REF] and profilin (1YPR) using the Joy algorithm[START_REF] Mizuguchi | JOY: protein sequence-structure representation and analysis[END_REF], highlighting the structural similarities between YabA model and it's templates. Alpha helix (red); 3 10 helix (maroon); beta strand (blue); solvent accessible residues (lower case); solvent inaccessible residues (upper case); hydrogen to bond main-chain amide(bold), hydrogen bond to main-chain carbonyl (underline). Secondary structures of YabA are indicated on top and correspond to the crystal structure (residues 1-62) and the homology model (76-119). The residues involved in Zn coordination are indicated by orange dots. (B) Cartoon representation of YabA CTD model colored as in (A). Side chains of the residues participating in Zn binding are indicated as ball and sticks. (C) Surface representation of YabA-CTD revealing surface-exposed residues important for interaction with DnaN (left) and DnaA (right) identified in a previous study[START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF].

Figure 4 .

 4 Figure 4. (A) Averaged scattering curve of the YabA protein coming from frames recorded on the main peak from SEC. The values of the radius of gyration Rg and the maximum dimension, Dmax, of the scattering particle are calculated from Guinier extrapolation (first box image) and autocorrelation function p(r) determination (second box image). (B) Bead model of YabA compatible with the SAXS data. (C) Averaged scattering curve of the YabA protein and the fitting curve calculated and superimposed with CRYSOL, coming from the model produced by Dadimodo under SAXS constraint. The logarithm of scattering intensity I is displayed as a function of the momentum transfer q = 4sin/, where 2 is the scattering angle and = 1.033 Å is the wavelength. (D) YabA structure model.

Figure 5 .

 5 Figure 5. Surface representation of YabA showing key residues involved in interactions with partner proteins. Mapping of mutational data onto the structural model with interacting surfaces specified by color; DnaA (Blue) DnaN (red). Previous data is indicated in italic. Zn is illustrated by a cyan sphere. Residues with no interacting phenotypes are labeled in gray.

Figure 6 .

 6 Figure 6. Cellular localization of YabA-WT and -LOI mutants by fluorescence monitoring of signals from CFP-(cyan) or YFP-(yellow) YabA fusions in living cells. (A) Impaired localization of YabA LOI mutants in DnaA (N85D and R105E) and in DnaN (E107T and F111Y) compared to YabA-WT. (B) Restoration of localization of YabA DnaN -LOI mutants by cross-complementation with a YabA-DnaA -LOI mutant. The co-expression in the same cell of the YabA-DnaN mutants F111Y or E107T fused to CFP (blue) with a YabA-DnaA N85D fused to YFP (yellow) restored the ability of each YabA mutant derivative to localize as a focus at the cell centre. The co-localization of foci indicates that they are part of a DnaA/YabA/DnaN complex, where the YabA tetramer is potentially composed of a mixture of YabA-DnaN (proficient for binding to DnaA) and YabA-DnaA (proficient for binding to DnaN) mutants. (C) Model illustrating cross complementation between YabA-DnaA (CTDs in green with yellow stars) and YabA DnaN (CTDs in green with blue stars) for the binding and the cellular localization of DnaN (purple doughnut) and DnaA (orange). The YabA NTD's tetrameric core is represented in Indian red.

FigureFigure S6 :

 S6 Figure S1: YabA multiple sequence alignment. The algorithm Jnet implemented through Jalview version 2 software (1) was used to search for similarities to the YabA sequence in UniRef90 (2). An alignment was constructed with PSI-BLAST (3). In the alignment, sequence names are composed of UniRef90 + SwissProt identifier + protein length. A histogram of amino acid residue conservation in the consensus sequence is shown below.

Figure 1 .

 1 Figure 1. Phosphorylation of YabA at Y90 residue in a YabT-independent manner. (A) Identification of the phosphorylated residue by mass spectrometry (LTQ-Orbitrap and Q exactive). The result shows an 80 Da molecular weight increment (corresponding to phosphate) at the position Y2 of the peptide that corresponds with the Tyrosine 90 of the protein. (B) Mapping of the phosphorylated residues on the structural model of YabA CTD. The phosphorylated residue is shown in pink. Interaction surfaces for DnaA and DnaN are shown in red and blue, respectively.

Figure 2 .

 2 Figure 2. Phosphorylation of YabA at Y90 residue is involved in replication initiation control. Ori-to-ter values determined by qPCR on total DNA extraction of unsynchronized cells. Cells were grown on LB at 37 °C up to OD ~ 0.3. Error bars represent 3 independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Figure 3 .

 3 Figure 3. Y90 phosphorylation modulates its binding ability to DnaA and DnaN. (A) Interaction between YabA non-phosphorylatable (Y90F) and phosphomimetic (Y90E or Y90D) derivatives with DnaA and DnaN detected by Yeast-two-hybrid. (B) YabA Y90 phospho-mutants are impaired for localization. Fluorescence signals from GFP-YabA* (green) in living cells expressing YabA mutants that have lost their ability to interact with DnaN (YabAY90F) or with DnaA (YabAY90E). BF is shown in grey, GFP in green and DNA in pink.

Figure 4 .

 4 Figure 4. YabY90F and YabAY90E protein purification. SDS-PAGE showing YabAY90F (A) of YabAY90E (B) purification by precipitation with ammonium sulfate 20 % and 30% (band at ~14 kDa). The soluble and insoluble (ins.) parts containing the proteins was separated by centrifugation and loaded in the SDS-PAGE.The proteins then were injected in a Superdex 200 10/300 GL gel filtration and all the fractions were loaded in a SDS-PAGE. Finally fractions from 11 to 14 were pooled and dialyzed (red rectangle).

  centrifuged and rinced with 10 mM Tris-HCl (pH 8), 10 mM EDTA (pH 8) and 300 mM NaCl. The pellet was resuspended in 200 µL Lysis buffer[START_REF] Jameson | Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA[END_REF] mM EDTA, 100 mM NaCl and 10 mg/ml lysozym. After incubation for 10 min at 37°C 10 µL sarkozyl 30 % were added to complete cell lysis. The samples were incubated at 65°C for 20 min, followed by lithium chloride precipitation to precipitate proteins and other contaminants by salting out, 0.8 volume of 2propanol was added before centrifugation at 16000 rpm. The pellet was then resuspended in 200 µL double-distilled H2O (ddH2O) and 200 µL of 10 M LiCl4. Samples were incubated at -20°C for 20 min and the DNA was precipitated with ethanol and sodium acetate. DNA was finally resuspended in 50 µL of ddH2O. Concentration of DNA in solution was determined by the use of a NanoDrop spectrophotometer (Thermo Scientific). DNA solutions were assumed to be largely free of contaminating proteins if the ratio of absorbance at 260:280 nm was > 1.6.

  5 h. At this time, B. subtilis cells were competent. 500 µL of competent cells were mixed with 1 µg of chromosomal of plasmid DNA, incubated 20 min at 37°C and plated on selective medium. Plates were incubated O/N and 4 colonies per transformation were checked by colony PCR.

Figure 6 .

 6 Figure 6. Pop-In/Pop-Out market-less point mutation method. Schematic overview of the Pop-In/Pop-Out method for mutant construction on the yabA gene. Upstream (oligonucleotides yabAfwd + a) and downstream (yabArev + b) fragments of yabA are PCR-amplified, introducing homologous sequences to the extremities of the cI-phleo cassette (which is also amplified using oligonucleotides Phleo5' + Phleo 3').The three fragments then are assembled in a joining PCR and used to transform strain CCBS185 (upp::Pλneo). Selection of the pop-in colonies is carried out by its resistant to pheomicyn. Finally eviction of cI-phleo is achieved by recombination of homologous regions at both sides of the cassette and the popout colonies are selected for neomycin-resistance.

  6, IPTG was added to 0.5 mM to induce expression of the protein and the culture was incubated overnight at 18°C. All subsequent steps were carried out at 4°C.The cells were pelleted and resuspended in buffer A (50 mM Tris pH 8, 1 M NaCl), disrupted by sonication and centrifuged at 40.000 rpm for 1h. The supernatant was used to precipitate the proteins by adding ammonium sulfate at final concentration of 20%. After centrifugation, the pellet containing the YabA protein was collected and resuspended in buffer B (50 mM Tris pH 8, 0.5 M NaCl). The soluble fraction was concentrated and injected onto a Superdex 200 10/300 GL gel filtration column(GE Health-care)

Figure 7 .

 7 Figure 7. Yeast-two-hybrid (Y2H) principle. Y2H is based on transcription factor gene that produces two domain protein (BD and AD), which are essential for transcription of the reporter gene. Two fusion proteins have to be prepared: BD+Bait and AD+Prey. None of them is usually sufficient to initiate the transcription (of the reporter gene) alone. When both fusion proteins are produced and Bait part of the first interact with Prey part of the second, transcription of the reporter gene take place.

3 .

 3 Our results highlight the multi-functional nature of YabA and reveal a novel function that might provide B. subtilis cells a useful cross talk between the different cellular processes, for fine tuning cell fate decision and to better respond to environmental changes. However the mechanisms underlying this novel role of YabA remain to be further characterized. R III. DISCUSSION AND PERSPECTIVES 208 YabA exhibits an unusual ATP/GTPase activity modulated by its phosphorylation state Our In-gel ATP assays revealed an ATPase activity associated to the different YabA bands. This ATPase activity was also found associated with the non-phosphorylatable (YabAT71A version) and is stimulated in the presence of the phosphomimetic YabAT71D protein. Interestingly, under native gel electrophoresis, YabA and YabA phospho-mutant proteins migrated as a major band associated with upper ladder bands that might correspond to different oligomerization levels and/or different conformation of the YabA protein. However, YabAT71D migrated faster suggesting that the repliement adopted by YabAT71D is different from that adopted by YabA and YabAT71A. The replacement of the threonine 71 residue located in the flexible region by an aspartate residue (YabAT71D) introduced a supplementary negative charge, which could alter the migration of the protein and/or modify the conformation of the protein and affect its oligomerisation state and could therefore explain the different migration pattern observed for YabAT71D. This observation is in agreement with our hypothesis that phosphorylation of T71 residue affects the overall intrinsic flexibility of YabA and modulates the affinity to binding protein partners. Interestingly, Yeast-two-hybrid experiments showed that T71 modifications do not affect the ability of YabA to interact with DnaA, DnaN, AcuB or TlpA. In vivo, YabA derivatives are fully able to perform replication initiation control. However through its flexible linkers, YabA could adopt different conformations which facilitate the interaction with yet unidentified proteins. A good approach to discover new protein partners of YabA targeted by YabT-mediated phosphorylation, will be a Yeast-two-hybrid genetic screening using YabAT71A and YabAT71D as a baits. This technique will provide new insight into the complex regulatory network mediated by YabA in B. subtilis life-tyle decision.

  results support that the phosphorylation state of YabA at Y90 could potentially modulate the protein-interaction switch with its protein partners DnaA and DnaN. As a consequence of this modification, Y90 mutants exhibit impaired localization with the replication machinery, which requires YabA dual interaction with DnaA and DnaN(Noirot-Gros et al., 2006; Felicori et al., R III. DISCUSSION AND PERSPECTIVES 211 2016). Therefore modification of Y90 induces both loss of YabA localization and loss of initiation control by modulation of its binding ability to DnaA and DnaN.

Figure A1 .

 A1 Figure A1. Heatmap showing the expression pattern of genes involved in DNA replication and cell division. Differences and similarities between mRNA levels are shown, violet indicates decreased levels, and orange indicates increased levels

1 .

 1 spectrométrie de masse, nous avons montré que le contrôleur d'initiation de réplication YabA est spécifiquement phosphorylé par YabT sur une thréonine, localisée dans la région flexible de liaison. Nous avons construit des mutants phosphomimétique de YabA (YabAT71D) et non R VI. ANNEXES 254 phosphorylable (YabAT71A) pour i) confirmer le rôle de T71 dans la phosphorylation et ii) réaliser des études fonctionnelles. La phosphorylation médiée par YabT de YabA n'est pas impliquée dans le contrôle de la réplication, mais module plutôt les processus de différenciation. Nous avons établi que YabA est phosphorylée par YabT sur la thréonine 71. Les tests de phosphorylation in vitro des protéines mutées YabAT71A et YabAT71D nous ont permis de démontrer que T71 est spécifiquement phosphorylé par YabT. La thréonine 71 est située dans le linker flexible de YabA, région de 14 résidus reliant le domaine N-terminal au domaine C-terminal et conférant un degré élevé de flexibilité à la structure de YabA. L'analyse fonctionnelle montre que la phosphorylation de YabA n'affecte ni le contrôle de l'initiation de la réplication ni l'interaction avec ses différents partenaires protéiques. Les substitutions phosphoablative ou phosphomimétique du résidu T71 n'affectent donc pas la structure globale de YabA, et les protéines YabAT71A et YabAT71D exercent pleinement leur rôle dans le contrôle de la réplication. YabT étant exprimée pendant la sporulation, nous avons testé le rôle potentiel de la phosphorylation de YabA dans ces conditions. YabT est conservée chez les bacilles sporulant et est absent d'E. coli et des bactéries non sporulées, ce qui suggère que la phosphorylation médiée par YabT serait un mécanisme évolutif lié au développement des spores. La sporulation de la souche ΔyabA est très fortement altérée, ce qui suggère que YabA est effectivement impliqué dans cette voie de différenciation chez B. subtilis. De façon surprenante, nous avons mis en évidence que la sporulation du mutant phosphomimétique yabT71D est plus efficace. Cette augmentation est corrélée à une augmentation de l'expression du promoteur spo0A, ce qui suggère une augmentation des niveaux intracellulaires de Spo0A-P.La souche yabAT71A non phosphorylable présente le même niveau final de sporulation que la souche sauvage, mais le nombre de cellules sporulantes à T2 (premiers stades du processus de sporulation) est significativement réduit. Ces résultats suggèrent que la phosphorylation de YabA sur le résidu T71 favorise l'entrée en sporulation des cellules, ce qui conduit à une augmentation de la production de spores. En revanche, l'absence de phosphorylation de YabA retarde l'entrée en sporulation mais n'affecte pas le rendement final en spores. Par conséquent, la phosphorylation de YabA joue un rôle dans les phases précoces de la sporulation, ce qui est pertinent, puisque YabT est exprimée à ce stade.La sporulation chez B. subtilis est un processus strictement régulé lié à la phosphorylation du régulateur principal Spo0A, modulé par un système de phosphorelais multi-composants(Narula R VI. ANNEXES 255 et al., 2013). C'est est un processus fortement consommateur d'énergie, qui devient irréversible après l'engloutissement du prépore par la cellule mère. Les cellules peuvent également adopter d'autres solutions pour survivre, comme le cannibalisme, la compétence ou la formation de biofilm. La concentration intracellulaire de Spo0A-P est déterminante pour ces choix développementaux. Seules les cellules exprimant un niveau élevé de Spo0A-P sont capables d'initier la sporulation(Fujita et al., 2005). Des niveaux faibles ou intermédiaires de Spo0A-P permettent le déclenchement du cannibalisme, de la compétence et de la formation de biofilm.Afin d'appréhender l'impact de la phosphorylation de YabA sur les niveaux de Spo0A-P, nous avons testé la capacité de nos souches à former un biofilm. La formation de biofilm est similaire pour le type sauvage, non-phosphorylable yabAT71A et ∆yabT, les souches ∆yabA et phophomimétique yabAT71D étant incapables de former des biofilms. YabA joue un rôle majeur dans la réplication, et nous avons montré que sa phosphorylation module inversement la sporulation et la formation de biofilm. Nos résultats soulignent donc la multifonctionnalité de YabA et son rôle dans la signalisation cellulaire connectant l'initiation de la réplication et la différenciation. Nos résultats suggèrent que la phosphorylation YabTdépendante de YabA affecte la différenciation en modulant le taux intracellulaire de Spo0A-P. En effet, la phosphorylation de YabA est corrélée à un taux élevé de Spo0A-P, ce qui stimule la sporulation et inhibe la formation de biofilm 2. L'analyse transcriptomique met en évidence un réseau de régulation complexe contrôlé par la phosphorylation de YabA. Afin d'étudier le réseau régulateur ciblé par la phosphorylation YabT-dépendante de YabA, nous avons examiné l'effet de la phosphorylation de YabA sur l'expression génique globale et les réponses transcriptionnelles au début de la sporulation. La souche non-phosphorylable yabAT71A et la souche de type sauvage présentent un profil global d'expression génique équivalent dans les trois conditions testées (phase exponentielle, stades T1 et T3 de la sporulation). La délétion de yabA et la mutation phosphomimétique yabAT71D perturbent profondément l'expression des gènes. L'analyse transcriptomique conforte les phénotypes associés à nos souches. La souche phosphomimétique présente un phénotype de sporulation amélioré qui pourrait être lié une augmentation de l'expression du facteur sigF, spécifique du stade T2 de la sporulation et des gènes spo subséquents. Par contre, la délétion de yabA provoque l'inhibition de l'expression de ces gènes, ce qui pourrait expliquer le retard de sporulation de cette souche. L'entrée en sporulation tardive du mutant non phosphorylable R VI. ANNEXES 256 yabAT71A peut être imputée à une légère inhibition de l'expression des gènes sigH et sigF observée à T1.

  d'antiterminaison, la transcription de l'opéron eps s'arrête au niveau du gène epsF, ce qui confère un phénotype déficient en exopolysaccharides équivalent à celui observé chez le mutant phosphomimétique. Jusqu'à présent, les cofacteurs EAR et le mécanisme moléculaire de son action restent inconnus, mais nos données suggèrent que la phosphorylation de YabA pourrait jouer un rôle dans cette régulation. Les études phénotypiques et transcriptomiques présentées dans ce travail montrent que la régulation et la fonction de YabA sont probablement plus complexes qu'on ne le pensait. Certains gènes hautement affectés par la phosphoryation de YabA codent pour des protéines appartenant à la famille Rap-Phr du système Quorum Sensing (QS). Le comportement des communautés bactériennes est souvent régulé par les voies de signalisation QS. Chez B. subtilis, différents système de QS sont impliqués dans l'adaptation, notamment dans l'initiation de la sporulation et la formation de biofilm et le transfert horizontal de gènes par transformation ou

  aval de phrI et en amont de yydM. Ces deux gènes sont fortement réprimés dans la souche yabAT71D, ce qui suggère un lien possible entre la phosphorylation de YabA et la régulation de ces gènes. Cette régulation pourrait être médiée par l'association de YabA, peut-être sous sa forme phosphorylée, avec Rok et DnaA. Cependant, Rok et DnaA sont des régulateurs transcriptionnels dont l'expression est réprimée par Spo0A(Molle et al., 2003). Dans la souche phosphomimétique, des niveaux élevés de Spo0A activée pourraient alors réprimer dnaA et rok, conduisant à la dérégulation des gènes contrôlés par Rok et DnaA. D'autres travaux sont donc nécessaires pour déchiffrer le rôle régulateur de la phosphorylation de YabA pendant la sporulation et la formation de biofilm. Cependant, nos résultats soulignent la nature multifonctionnelle de YabA et révèlent une nouvelle fonction qui pourrait fournir aux cellules de B. subtilis une interconnexion entre les différents processus cellulaires. Ce nouveau réseau de régulation affinerait la prise de décision de la cellule face à différentes formes de différenciation, et lui permettrait de mieux répondre aux changements environnementaux.

3 . 4 . 5 .

 345 YabA présente une activité ATP / GTPase atypique modulée par son état de phosphorylation Par ailleurs, nos expériences de chromatographie sur couche fine (TLC) et de "In-Gel" suggèrent que YabA possède une activité "ATP/GTPasique" atypique modulée par la phosphorylation de R VI. ANNEXES 258 YabA sur T71. Nos tests de "In-Gel" ont révélé une activité ATPase associée aux différente oligomères de YabA. Cette activité ATPase est également associée à la protéine nonphosphorylable (version YabAT71A) et elle est fortement stimulée dans la protéine phosphomimétique YabAT71D. De façon intéressante, après électrophorèse en gel de polyacrylamide natif, YabA et ses versions phospho-mutantes migrent sous forme d'une bande majeure associée à des bandes d'échelles supérieures qui pourraient correspondre à différents niveaux d'oligomérisation et / ou une conformation différente de la protéine YabA. La protéine YabAT71D migrent plus rapidement, ce qui suggère que le repliement adopté par YabAT71D est différent de celui adopté par YabA et YabAT71A. Le remplacement du résidu thréonine 71 localisé dans la région flexible par un résidu aspartate (YabAT71D) introduit une charge négative supplémentaire, qui pourrait altérer la migration de la protéine et / ou modifier la conformation de la protéine et affecter son état d'oligomérisation, ce qui pourrait expliquer les différents modèles de migration observés pour YabAT71D. Les essais de chromatographie sur couche mince (TLC) ont révélé une activité d'hydrolyse inhabituelle de l'ATP et du GTP de YabA, conduisant à l'accumulation de produits non caractérisés. Cette activité inhabituelle semble être modulée par la phosphorylation du résidu YabA T71. Des produits intermédiaires sont produits lorsque l'ATP est hydrolysé, ce qui suggère une dégradation progressive. Un seul produit final ayant été mis en évidence en présence de GTP, il semble que YabA utilise le GTP comme substrat. Ces résultats suggèrent que le GTP pourrait être le véritable substrat de YabA. YabA pourrait être un rôle potentiel sur des voies de signalisation dépendant du c-di-GMP Nos analyses fonctionnelles révèlent un rôle potentiel de YabA sur des voies de signalisation dépendant du c-di-GMP, qui contrôle la formation du biofilm chez de nombreuses bactéries. En utilisant des souches de B. subtilis modifiées (souches incapables de synthétiser le c-di-GMP ou au contraire accumulant de fortes concentrations de c-di-GMP), nous avons détecté une activité diguanylate cyclase potentielle lorsque YabA est surexprimée dans la souche ne synthétisant pas de c-di-GMP. Dans cette souche, deux populations possédant des activités GFP dépendant ducdi-GMP distinctes sont observées. La première population (cellules plus petites) présente un faible niveau d'expression de la protéine GFP, ce qui indique un taux élevé de c-di-GMP similaire à celui observé dans les cellules témoins synthétisant du c-di-GMP. La seconde population (cellules plus longues) présente un taux élevé de GFP corrélé à de faibles taux de c-di-GMP, comparable à celui observé dans les cellules ne synthétisant pas de c-di-GMP. Dans cette seconde population, l'effet d'élongation peut résulter d'un défaut de contrôle de la réplication, lié à une sur-répression de l'initiation de la réplication de l'ADN provoquée par la surexpression de YabA. Nos résultats suggèrent que YabA est capable de promouvoir la synthèse de c-di-GMP chez B. subtilis. Aucune signature de liaison c-di-GMP n'étant présente dans YabA, il est peu probable qu'elle agisse comme une diguanylate synthase. Notre première hypothèse est que YabA pourrait induire indirectement la synthèse d'une enzyme DGC encore inconnue, puisque tous les gènes codant des diguanylate synthases ont été délétés dans la souche utilisée. La seconde hypothèse est liée à l'hydrolyse du GTP observée dans nos études in vitro précédentes. YabA, en catalysant l'hydrolyse du GTP, agirait indirectement sur le pool de c-di-GMP en diminuant le pool de GTP intracellulaire. L'ensemble de nos résultats suggère fortement un rôle multifonctionnel de YabA dans la physiologie cellulaire. Au-delà de son rôle connu en tant que régulateur négatif de l'initiation de la réplication, YabA intégrerait différentes voies de signalisation grâce à sa phosphorylation YabT-dépendante. YabA est phosphorylée indépendamment de YabT dans son domaine C-terminal, et cette phosphorylation module son affinité pour DnaA et DnaN. Enfin, des analyses de LC-MS montrent que lorsqu'elle est surexprimée chez Escherichia coli, YabA est phosphorylée sur la tyrosine 90 indépendamment de la présence de YabT. Le résidu Y90 est localisé dans le domaine C-terminal de YabA, domaine impliqué dans la liaison à la protéine d'initiation DnaA et à la polymérase clamp DnaN (Felicori et al., 2016). Ce résidu est hautement conservé parmi les homologues de YabA, ce qui suggère que la phosphorylation Y90 pourrait être pertinente pour l'initiation de la réplication de régulation. Nous avons montré in vivo que la phosphorylation de Y90 conduit à des défauts de réplication, les ratios ori-to-ter étaient significativement plus élevés par rapport au type sauvage dans les souches non-phosphorylatable yabAY90F et phosphomimétiques yabAY90E. Une étude doublehybride chez la levure montre que la phosphorylation de Y90 module l'interaction de YabA avec ses partenaires DnaA et DnaN. Nos résultats montrent que l'état de phosphorylation de YabA sur Y90 modulerait l'interaction avec ses partenaires protéiques DnaA et DnaN. La modification du résidu Y90 altère la colocalisation de YabA avec la machinerie de réplication, cette colocalisation nécessitant une double interaction de YabA avec DnaA et DnaN. La kinase R VI. ANNEXES 260 impliquée n'a pas encore été identifiée, mais ces résultats suggèrent l'existence d'un contrôle de l'initiation de la réplication lié à la phosphorylation de YabA par une tyrosine kinase chez B. subtilis. En conclusion, l'ensemble de mes travaux suggère l'existence de différents niveaux de régulation de l'activité de YabA par phosphorylation sur thréonine et tyrosine. YabA, outre son rôle dans l'initiation de la réplication, joue un rôle majeur dans la différenciation de B. subtilis. Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Analyse fonctionnelle de la phosphorylation de YabA, un régulateur de la réplication chez Bacillus subtilis Mots clés : YabA, phosphorylation, sporulation, biofilm, transduction de signal. Résumé: Les bactéries ont besoin d'adapter leur cycle cellulaire et leur taux de croissance aux changements environnementaux et nutritionnels. L'initiation de la réplication est ainsi strictement coordonnée aux autres processus cellulaires afin de transmettre un chromosome conforme. Chez B. subtilis, bactérie modèle des Gram + , la protéine YabA joue un rôle majeur en réprimant l'initiation de la réplication, ceci en formant un complexe avec la protéine initiatrice DnaA et la clamp polymérase DnaN. YabA interagit en outre avec d'autres protéines partenaires et serait donc multifonctionnelle. Sa structure 3D révèle une architecture en deux domaines: Un domaine N-terminal adoptant un repliement de type superhélice et un domaine C-terminal globulaire structuré autour d'un atome de Zinc. In vivo, YabA est un tétramère par interaction entre les superhélices, connecté aux domaines C-terminaux monomériques par une séquence déstructurée hyper flexible. YabA serait un hub structural interagissant simultanément avec plusieurs protéines et constituerait une plateforme d'intégration entre différents signaux intracellulaires et l'initiation de la réplication. La phosphorylation est une modification posttraductionnelle modulant l'activité de nombreuses protéines en réponse à certains signaux cellulaires. Grâce à des expériences de phosphorylation in vitro et des analyses de spectrométrie de masse, nous avons montré que YabA est phosphorylée par la kinase de type Hanks YabT sur une thréonine, localisée dans la région flexible de liaison interdomaines. YabT est une kinase activée par l'ADN, exprimée en carence en glucose, en sporulation et en phase stationnaire. Nous avons construit des mutants phosphomimétique de YabA (yabA-T71D) et non phosphorylable (yabA-T71A) pour i) confirmer le rôle de T71 dans la phosphorylation et ii) réaliser des études fonctionnelles. Nous avons montré in vivo que la phosphorylation de YabA n'est pas impliquée dans l'initiation de la réplication, mais module des programmes de différenciation. La phosphorylation de YabA module inversement la sporulation et la formation de biofilm, ce qui souligne sa multifonctionnalité et son implication dans la signalisation cellulaire connectant l'initiation de la réplication et la différenciation. Nos résultats suggèrent que la phosphorylation YabTdépendante de YabA affecte la différenciation en modulant le taux intracellulaire de Spo0A-P. En effet, la phosphorylation de YabA est corrélée à un taux élevé de Spo0A-P, ce qui stimule la sporulation et inhibe la formation de biofilm. Par ailleurs, nos expériences de chromatographie sur couche fine (TLC) et de "In-Gel" suggèrent que YabA possède une activité "ATP/GTPasique" atypique modulée par la phosphorylation de YabA sur T71. Nos analyses fonctionnelles révèlent un rôle potentiel de YabA sur des voies de signalisation dépendant du c-di-GMP, qui contrôle la formation du biofilm chez de nombreuses bactéries. Ces résultats suggèrent que YabA joue un rôle complexe pendant la différenciation en intégrant différentes voies de signalisation. Enfin, des analyses de LC-MS montrent que lorsqu'elle est surexprimée chez Escherichia coli, YabA est phosphorylée sur la tyrosine 90, qui appartient au domaine d'interaction Cterminal. Une étude double-hybride chez la levure montre que la phosphorylation de Y90 module l'interaction de YabA avec ses partenaires DnaA et DnaN. In vivo, la phosphorylation de Y90 module l'initiation de la réplication. La kinase impliquée n'a pas encore été identifiée, mais ces résultats suggèrent l'existence d'un contrôle de l'initiation de la réplication lié à la phosphorylation de YabA chez B. subtilis. En conclusion, l'ensemble de nos études suggère l'existence de différents niveaux de régulation de l'activité de YabA par phosphorylation sur thréonine et tyrosine. YabA, outre son rôle dans l'initiation de la réplication, joue un rôle majeur dans la différenciation de B. subtilis. Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Title : Functional analysis of phosphorylation of the replication controller YabA in Bacillus subtilis Keywords : YabA, phosphorylation, sporulation, biofilm, signal transduction Abstract: Upon environmental or nutritional changes, bacteria must adjust their cell cycle with their growth rate. Most particularly, DNA replication initiation events must be controlled and coordinated with cell physiology to ensure faithful chromosome inheritance. In Bacillus subtilis, a model of Gram-positive bacteria, YabA plays a major role in down regulating initiation replication through interaction with the initiator protein DnaA and the clamp polymerase DnaN. However, YabA is a structural hub protein able to interact with other protein partners, indicating it might be multifunctional. Through its unique overall tridimensional structure composed of a Nterminal four helix-bundle tetramer connected to four monomeric C-terminal domains by a highly flexible linker, YabA is capable to physically interact with more than one protein at a time, thus providing a suitable platform to integrate intracellular signals to replication initiation. Phosphorylation is the most prevalent post translational modification that modulates protein activities in response to cellular signals. Using in vitro phosphorylation and mass spectrometry we demonstrated that YabA is phosphorylated by the Hanks-type serine/threonine kinase YabT at a threonine residue localized within the flexible interdomain region. YabT is a kinase activated by DNA and up-regulated during glucose starvation, sporulation and stationary phase.

  . To gain rapid and comprehensive access to this knowledge, several databases have been developed. These are either part of global /sporeweb.molgenrug.nl/), respectively. The database SubtiWiki has been developed to collect and integrate all available layers of informations on B. subtilis to render them accessible

	efforts	such	as	BsubCyc	(https://bsubcyc.org/)	and	SubtiList
	(http://genolist.pasteur.fr/SubtiList/), or specialized to specific scientific problems such as
	regulation	or	sporulation;	DBTBS	(http://dbtbs.hgc.jp)	and	SporeWed
	(http:/

Table 2 . Negative regulatory systems involved in replication initiation

 2 (Modified from Katayama et al.,

	2010)

Table 3 :

 3 Serine/threonine kinases and phosphatases in B. subtilis.

	Name	Specificity Substrates	Regulated processes
	KINASES
	PrkC	Ser/Thr	EF-G (FusA) 1 , YezB 2 , CpgA 2 ,	Sporulation, germination,
			EF-Tu 2 , YwjH 3 , GlnA 3 , Icd 3 ,	biofilms and cell division
			AlsD 3 , HPr 3 , AbrB 6 , GpsB 20 ,
			YvcK 21
	PrkD (YbdM)	Ser/Thr	DegS 4 , DnaC 5 , AbrB 6	Competence, social behavior,
				DNA replication
	YabT	Ser/Thr	RecA 5,7 , RacA 5 , AbrB 6 , EF-Tu 8 DNA repair, sporulation
	PrkA	Ser	60 kDa protein 9	Sporulation (synthesis of the
				sigma K transcription factor)
	SpoIIAB	Ser	SpoIIAA 10	Sporulation (Control of sigma F
				activity)
	RsbT	Ser	RsbRA 11 , RsbS 11	Stress (Control of sigma B
				activity)
	RsbW	Ser	RsbV 12	Stress (Control of sigma B
				activity)
	PHOSPHATASES
	PrpC	Ser-P/Thr-P	YezB 2 , CpgA 2 , EF-Tu 2 , EF-G	Sporulation, germination,
			(FusA) 13 , PrkC 13 , HPr 14 ,	biofilms (antagonist of PrkC-
			YvcK 21	dependent phosphorylation)
	SpoIIE	Ser-P	SpoIIAA-P 15 , RecA 5 , RacA 5	Sporulation (antagonist of
				SpoIIAB-and YabT-dependent
				phosphorylation)
	RsbX	Ser-P	RsbS 16 , RsbR 17	Sporulation
	RsbU	Ser-P	RsbV-P 16	Stress (Control of sigma B
				activity)
	RsbP	Ser-P	RsbV-P 18	Stress (Control of sigma B
				activity)
	KINASE/PHOSPHORYLASE
	HprK/P	Ser/Ser-P	HPr-P 19 , Crh 19	Carbon metabolism
	Phosphorylation events have been reported in the following: 1 (Shan and Dworkin, 2010); 2 (Abalon et al.,
	2009); 3 (Pietack et al., 2010); 4 (Jers et al., 2011); 5 (Shi et al., 2015b); 6 (Kobir et al., 2014); 7 (Bidnenko et al.,
	2013); 8

TABLE 1 |

 1 Phosphorylation of proteins involved in DNA-dependent machineries and processes in bacteria.

	Pathway	Target	KO	Function	Tyr-P	Ser/Thr-P
			MUTL	K03572	DNA Mismatch repair factor	MUTL_BACSU; B1XQB2_SYNP2	B1XQB2_SYNP2
			MUTS	K03555	DNA Mismatch repair protein	B1XM45_SYNP2
			MUTM	K10563	FPG/DNA-N glycosylase	B1XQB2_SYNP2	B1XQB2_SYNP2
			UVRB	K03702	UvrABC system protein B		UVRB_STRP2
			UVRD	K03657	ATP-dep. DNA helicase (PcrA)	PCRA_BACSU; O26013_HELPY	O26013_HELPY ; Q3K0V8_STRA1
	DNA repair and recombination		RUVX RECA RECG MFD RECQ REP DINI RECT EX1	K07447 K03553 K03655 K03723 K03654 K03656 K12149 K07455 K01141	Holliday junction resolvase SOS repair/DNA processing ATP-dependent DNA helicase Transcription-repair factor ATP-dependent DNA helicase ATP dependent DNA helicase DNA-damage-inducible DNA-Binding protein Exodeoxyribonuclease I (sbsB)	RUVX_ECO57 RECA_BACSU; RECA_ECO57 B1XL29_SYNP2 DINI_ECO57 RECT_ECOLI Q8X8T9_ECO57; Q9HW85_PSEAE	RECA_BACSU; RECA_THET8 RECG_MYCTO Q04N58_STRP2; B1XL29_SYNP2 B1XQJ2_SYNP2 Q3K4P8_PSEPF Q9HW85_PSEAE
			EX3	K01142	Exodeoxyribonuclease III	EX3_ECOLI
			EX7L	K03601	Exodeoxyribonuclease VII (A)		EX7L_THETN
			EX7S	K03602	Exodeoxyribonuclease VII (B)		EX7S_STRA1
			RADA	K04485	DNA repair protein (radA, sms)	Q8XB28_ECO57, B1XJC1_SYNP2	B1XJC1_SYNP2
			SSBB		Single-stand binding protein B		SSBB_BACSU
			SSBA	K03111	Single-stand binding protein A	SSBA_BACSU;	Q04JK9_STRP2; SSBA_BACSU
			DPO1	K02335	DNA polymerase I	DPO1_BACSU; B1XL22_SYNP2	B1XL22_SYNP2; DPO1_ECOLI
	DNA replication		LIGA DNAA DPO3 DNAB	K01972 K02313 K02337 K02314	DNA ligase A DNA replication initiator Replicative DNA polymerase PolC Replicative DNA helicase	HOLE_ECO57 B1XQ85_SYNP2	DNLJ_LISMO; DNLJ_ECOLI DNAA_STRCO B1XQ85_SYNP2; DNAC_BACSU
			DPO3A	K02337	DNA polymerase III (θ su)	HOLE_ECO57
			TUS	K10748	DNA replication terminus protein	TUS_ECO57
			GYRB	K02470	DNA gyrase β subunit	GYRB_ECO57	GYRB_BACSU
			TOP1	K03168	DNA topoisomerase 1	TOP1_MYCTO; Q8X7C5_ECO57	TOP1_MYCPN
	Nucleoid structure and	DNA condensation	PARC NAP DBH1 DPS DBHB	K02621 K09747 K03530 K04047 K03530	DNA topoisomerase 4 suA Nucleoid associated protein DNA-binding protein HupA DNA protection during starvation DNA-binding protein HupB	DPS_ECOLI; DPS_ECO57	PARC_PSEAE B1XLF8_SYNP2; DBH1_BACSU; Q92A74_LISMO; DBH1_STRCO; B1XQQ7_SYNP2 DPS_ECOLI; DPS_CAMJE; DPS_LISMO; Q3K7V3_PSEPF DBHB_ECOLI; Q6N5M1_RHOPA
			HNS	K03746	DNA-binding protein H-NS	HNS_ECOLI; HNS_ECO57	HNS_ECOLI
			STPA	K11685	DNA-binding protein StpA		CNU_ECO57, STPA_ECOLI
			SMC	K03529	Chromosome partition protein		Q84EC7_SYNP2; SMC_MYCTO
	Chrom.	segregation	PARA FTSK	K03496 K03466	ATPase, ParA-family DNA translocase FtsK	FTSK_ECO57; FTSK_ECOLI	B1XQR5_SYNP2 FTSK_MYCTO
	Restriction	modification	B1XQZ6 ECO57IR HSDM	K01156 K00571 K03427	Type III R/M-Helicase Type IIS R/M-methyltransferase Type I restriction enzyme M	O26046_HELPY O25953_HELPY	B1XQZ6_SYNP2 Q04K98_STRP2 O33298_MYCTU
	DNA transcription		RPOB RPOC	K03043 K03046	RNA polymerase β subunit RNA polymerase β' subunit	RPOB_BACSU; RPOB_ECO57 RPOC_BACSU; RPOC_ECO57	RPOB_ECOLI; RPOB_MYCTO RPOB_CLOAB; RPOB_STRP2; RPOB_BACSU RPOC2_SYNP2; RPOC_CLOAB; RPOC_BACSU; C4WZY6_KLEPN RPOC_CLOAB; RPOC_STRP2
							(Continued)
	Frontiers in Microbiology | www.frontiersin.org

TABLE 2 |

 2 DNA-binding proteins targeted for phosphorylation in B. subtilis.

	Acc	Name	Function	PK	PP	P-sites	Val	Orth	References
	TYROSINE PHOSPHORYLATION
	P23477	ADDB	ATP-dependent deoxyribonuclease (B)	PtkB			-		1
	P49850	MUTL	DNA mismatch repair factor	PtkB	PtpZ		a,d	Y	1
	O34996	DPO1	DNA polymerase I	PtkB	PtpZ		a,d	T	1
	O34580	UVRD	ATP-dependent DNA helicase (PcrA)	PtkB			d	Y,S,T	1
	P16971	RECA	SOS repair factor/DNA processing	PtkA			b # ,d	Y	1,2,3
	P37870	RPOB	RNA polymerase β subunit	PtkB	PtpZ	Y 695	a,c,d	Y,S,T	1,4
	P37871	RPOC	RNA polymerase β' subunit			Y 338	D		4
	P37455	SSBA	Single-Stand Binding protein A	PtkA		Y 82	b* # ,c,d	S	5,6
	O31903	YORK	Putative SPBc2 ss-DNA exonuclease	PtkA		Y 3/11/168/220/368/473	d		6,7
	SERINE/THREONINE PHOSPHORYLATION
	P17867	CISA	Site-specific DNA recombinase	YabT	SpoIIE		a		1
	P16971	RECA	SOS repair factor/DNA processing	YabT	SpoIIE	S 2	a,b,c,d	Y	1,2,3
	P45870	RACA	Chromosome-anchoring protein	YabT	SpoIIE		a, b #		1
	O08455	SBCE	DSB repair	YabT	SpoIIE		a		1
	P46344	YQFF	Putative Phosphodiesterase	YabT	SpoIIE		a		1
	P37469	DNAC	Replicative DNA helicase DnaB-like	PrkD			b # ,d	Y	1
	P08821	DBH1	DNA-binding protein HU-1			T 4 , T 65 , S 74	c,d	S,T	6
	P05652	GYRB	DNA gyrase subunit B			S 400	c,d	Y	2
	P94590	SSBB	Single-stand DNA binding protein B	PtkA		Y 82 ,T 52	c,d	S	4
	P37871	RPOB	RNA polymerase β subunit			S 314	c,d	S,T,Y	4
	P37871	RPOC	RNA polymerase β' subunit			S 339	c		4

  Cys carboxyamidomethylation and Met oxidation were set to static and possible modifications, respectively. Precursor mass was 10 ppm and fragment mass tolerance was 0.02 Th. A refinement search was added with similar parameters except that semitrypsic peptides, possible N-ter protein acetylation and phosphorylation of serine threonine or tyrosine were searched for the phosphoproteins. /pappso.inra.fr/bioinfo/xtandempipeline) according to: (1) A minimum of two different peptides was required with a E value smaller than 0.05, (2) a protein E value (calculated as the product of unique peptide E values) smaller than 10-4. To take redundancy into account, proteins with at least one peptide in common were grouped. This allowed to group proteins of similar function. Within each group, proteins with at least one specific peptide relatively to other members of the group were reported as sub-groups. For phosphoproteomic data, only one peptide is required with a E value smaller than 0.01 and the protein E value (calculated as the

	For data of proteomic, only peptides with an E value smaller than 0.1 were reported. Identified
	proteins	were	filtered	and	grouped	using	XTandem	Pipeline
	(http:/
									1)
	(http://www.thegpm.org/TANDEM/) and the B. subtilis strain 168 database was downloaded
	from Uniprot Database site (http://www.uniprot.org/, version 2012, 4195 protein entries). This
	database was merged and in conjunction with reverse and contaminant databases, were
	XTandem	pipeline	(version	3.3.0)	developed	by	PAPPSO	platform

searched by Xtandem CYCLONE (version 2011.12.01.1, http://www.thegpm.org/tandem/) using (http://pappso.inra.fr/bioinfo/). Enzymatic cleavage was declared as a trypsin digestion with R II. RESULTS 109 one possible miss-cleavage. product of unique peptide E values) smaller than 10-2. Identified phosphopeptides were filtered and grouped using XTandem Pipeline confirmed with MaxQuant (http://www.maxquant.org/) and manually validated.

  2 ml of MSgg medium[5 mM potassium phosphate (pH 7), 100 mM MOPS (pH 7), 2 mM MgCl2, 700 μM CaCl2, 50 μM MnCl2, 50 μM FeCl3, 1 μM ZnCl2, 2 μM thiamine, 0.5% glycerol, 0.5% glutamate, 50 μg mL -1 tryptophan, 50 μg mL -1 phenylalanine](Branda et al., 2001) in glass tubes.

Table S1 .

 S1 TABLES Strains used in this study

		Strains E. coli
	Strain	Genotype	Source/Construction
	M15	pREP4-groESL	Amrein et al., (1995)
	Strain DH10B	F-endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Strains B. subtilis Genotype hsdRMS-mcrBC) λ-Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-	Source/Construction Grant et al., (1990)
	CCBS185	168 trpC2 ∆upp (Pr-neo) F-λ-fhuA2 [lon] ompT lacZ::T7 gene 1 gal sulA11 Δ(mcrC-	Laboratory stock, RLC lab.
	JJS42 ER2566	168 trpC2 ∆upp ∆yabA 210::Tn10)(TetS) endA1 [dcm] mrr)114::IS10 R(mcr-73::miniTn10-TetS)2 R(zgb-	Noirot-Gros et al., (2006) NEB
	TGG158 M15-yabT	168 trpC2 ∆upp yabAT71A (YabA-Non-phosphorylatable) M15 qQE-30-yabT	This study, see text for details This study, M15 x pQE-30-yabT
	TGG148 ER2566-YabA	168 trpC2 ∆upp yabAT71D (YabA-Phosphomimetic) ER2566 pSMG251	This study, see text for details Laboratory stock
	TGG137 TGG160 ER2566-YabAT71A TGG161 ER2566-YabAT71D	168 trpC2 ∆upp ΔyabT::Spec 168 trpC2 ∆upp ΔyabT::Spec yabAT71A ER2566 pSMG251-YabAT71A 168 trpC2 ∆upp ΔyabT::Spec yabAT71D ER2566 pSMG251-YabAT71D	This study, see text for details This study, ER2566 x pSMG251-YabAT71A This study, TGG137 x TGG158 This study, TGG137 x TGG148 This study, ER2566 x pSMG251-YabAT71A
	TGG9 TGG12 Strain TGG36 PJ69-4α	Strains S.cerevisiae 168 trpC2 amyE::Pxyl-gfp-yabAT71A Genotype 168 trpC2 amyE::Pxyl-gfp-yabAT71D 168 trpC2 ∆upp yabAT71A amyE::Pxyl-gfp-yabAT71A MATα trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal180Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2:: GAL7-lacZ	This study, CCBS185 x pSG1729 yabAT71A Source/Construction This study, CCBS185 x pSG1729 yabAT71A This study, TGG158 x pSG1729 yabAT71A James et al., (1996)
	TGG38 TGG167 PJ69-4a	168 trpC2 ∆upp yabAT71D amyE::Pxyl-gfp-yabAT71D 168 trpC2 ∆upp x pDG148-3Flag-yabT MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal180Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2:: GAL7-lacZ	This study, TGG148 x pSG1729 yabAT71D yabT This study, CCBS185 x pDG148-3Flag James et al., (1996)
	TGG169 131 TGG173 305 5816	PJ69-α, pGAD-C1 : Leu2 Gal4AD 168 trpC2 ∆upp ΔyabT::Spec x pDG148-3Flag-yabT PJ69-α, pGAD-C1 : Leu2 Gal4AD-yabA 168 trpC2 ∆upp PspoIIA-luc PJ69-α, pGAD-C1 : Leu2 Gal4AD-yabAT71A	This study, TGG137 x pDG148-3Flag Laboratory stock yabT Laboratory stock This study, CCBS185 x luc fusion spoIIA This study
	TGG174 5712	168 trpC2 ∆upp PspoIIID-luc PJ69-α, pGAD-C1 : Leu2 Gal4AD-yabAT71D	This study, CCBS185 x luc fusion spoIIID This study
	TGG176 1534 TGG177 39 687	PJ69-α, pGAD-C1 : Leu2 Gal4AD-dnaA 168 trpC2 ∆upp yabAT71A PspoIIA-luc PJ69-α, pGAD-C1 : Leu2 Gal4AD-dnaN 168 trpC2 ∆upp yabAT71A PspoIIID-luc PJ69-α, pGAD-C1 : Leu2 Gal4AD-acuB	This study, TGG158 x luc fusion Laboratory stock spoIIA Laboratory stock This study, TGG158 x luc fusion spoIIID Laboratory stock
	TGG179 675	168 trpC2 ∆upp yabAT71D PspoIIA-luc PJ69-α, pGAD-C1 : Leu2 Gal4AD-tlpA	This study,TGG148 x luc fusion spoIIA Laboratory stock
	TGG180 121	168 trpC2 ∆upp yabAT71D PspoIIID-luc PJ69-a, pGBDU-C1 : Ura3 Gal4BD	This study, TGG148 x luc fusion spoIIID Laboratory stock
	TGG182 319	168 trpC2 ∆upp ∆yabA PspoIIA-luc PJ69-a, pGBDU-C1 : Ura3 Gal4BD-yabA	This study, JJS42 x luc fusion spoIIA Laboratory stock
	5818	PJ69-a, pGBDU-C1 : Ura3 Gal4BD-yabAT71A	This study
	TGG183 5812	168 trpC2 ∆upp ∆yabA PspoIIID-luc PJ69-a, pGBDU-C1 : Ura3 Gal4BD-yabAT71D	This study, JJS42 x luc fusion spoIIID This study
	TGG188 592	168 trpC2 ∆upp Pspo0A-gfp PJ69-a, pGBDU-C1 : Ura3 Gal4BD-dnaA	This study, CCBS185 x pBSBII-spo0A Laboratory stock
	TGG189 52	168 trpC2 ∆upp yabAT71A Pspo0A-gfp PJ69-a, pGBDU-C1 : Ura3 Gal4BD-dnaN	This study, TGG158 x pBSBII-spo0A Laboratory stock
	TGG190 3580	168 trpC2 ∆upp yabAT71D Pspo0A-gfp PJ69-a, pGBDU-C1 : Ura3 Gal4BD-acuB	This study, TGG148 x pBSBII-spo0A Laboratory stock
	TGG191	168 trpC2 ∆upp ∆yabA Pspo0A-gfp	This study, JJS42 x pBSBII-spo0A

Table S2 .

 S2 Oligonucleotides used in this study

			R II. RESULTS
	1729-R	GAATTCGATATCAAGCTTATCGATACCGTC	amyE N-terminal reverse
	Oligonucleotides for mutagenesis Sequence 5' to 3' ACATCCAGAACAACCTCTGC GAGCTCGAATTCACTGGCCGTCG TATGTAAGATTTAAATGCAACCG Oligonucleotides for yeast-two-hybrid CGACCTGCAGGCATGCAAGCT CGCGTTTGGAATCACTACAGG GGCTTCAGTGGAGACTGATA GAGCTCGAATTCACTGGCCGTCGCAAAAGCACAGAAGACAGAGCAAACTG TCAGAGGTTACATGGCCAAG CGACCTGCAGGCATGCAAGCTCTGTCTTCTGTGCTTTTGATTC AACGGTCCGAACCTCATAAC GAGCTCGAATTCACTGGCCGTCGCAAAAGACCAGAAGACAGAGCAAACTGAT ATAG GTCTCCGTCGACTAGGGCAC CGACCTGCAGGCATGCAAGCTCTGTCTTCTGGTCTTTTGATTCTTTTTTATCA AGCTTCTGAATAAGCCCTCG * Sequences homologous to the extremities of the cI-phleo cassette are shown in blue (5') or red (3') in Nom pDG148-forward Bpspac 161 Description phleo5' K7 phleo -forward pDG148-reverse Apspac 162 -complementary to phleo 5' phleo3' mADA1 pGAD-forward K7 phleo -reverse mBD1 pGBDU-forward -complementary to phleo 3' yabAT71AF mBD2 pGAD and pGBDU-reverse yabA-forward-mutation T71A yabAT71AR mAD1ext pGAD-exterior-forward yabA-reverse-mutation T71A yabAT71DF mBD1ext pGBDU-exterior-forward yabA-forward-mutation T71D yabAT71DR mBD2ext pGAD and pGBDU-exterior-reverse yabA-reverse-mutation T71D
	YabA Fwd the oligonucleotides used for mutagenesis. GTGTCCGCTTTAAGAAAGCG	forward yabA exterior-
	YabA Rev	ATGCAGATCGTTGGTATACCCG	yabA exterior-reverse
		Oligonucleotides for qPCR
			thdF-
	OriL3-F	CCCAGCATCTTGTAAGGTCAAT	4212889…4211510
			forward
			thdF-
	OriL3-R	TTATGTCAGCAACACACGTCAC	4212889…4211510
			reverse
			proH/yoxE -
	TerL3-F	GGGAGTGGTTGAATGTTCCTA	2017711...2016845
			forward
			proH/yoxE -
	TerL3-R	ATAATAGCGTGTCACCTCATCG	2017711...2016845
			reverse
		Oligonucleotides for cloning
	yabA-F-Eco	ATCCGGAATTCTTGGATAAAAAAGAGTTATTTGATACAG	yabA-forward-EcoRI
	yabA-R-SalI	ACGCGTCGACTATTTTTTATTTAAGAATGACAGACAG	yabA-reverse-SalI
	yabA-apa	ATGGGCCCTTGGATAAAAAAGAGTTATTTGATACAG	yabA-forward-ApaI
	yabA-NdeI	GGTAAAACATATGGATAAAAAAGAG	yabA-reverse-NdeI
	YabT-F-SalI	GATGTCGACATGATGAACGACGCTTTGA	yabT-forward-SalI
	YabT-R-SphI	GCCGCATGCTCAGATTAAGAAAAAGATAATATAGGCG	yabT-reverse-SphI
	OSMG 464	AATTAATACGACTCACTATAGGG	T7 promoter-forward
	OSMG 438	CAAAAAACCCCTCAAGACCC	T7 terminus-reverse
	OSMG 538	AGGCTCGAGCTATTTTTTATTTAAGAATGAC	yabA-reverse-XhoI
	1729-F	TCTAGAAAGGAGATTCCTAGGATGGGTACC	amyE N-terminal forward
			125

Table S3 .

 S3 Plasmids used in this study

		Yeast plasmids
	Plasmids	Relevant genotype	Resistant E. coli S. cerevisiae	source/construction
	pGAD-C1	Gal4AD LEU2 Amp	Amp R	L	James et al., (1996)
	pGBDU-C1	Gal4BD URA3 Amp	Amp R	U	James et al., (1996)
	pGAD-dnaA	Gal4AD-dnaA LEU2 Amp	Amp R	L	Laboratory stock
	pGBDU-dnaA	Gal4BD-dnaA URA3 Amp	Amp R	U	Laboratory stock
	pGAD-dnaN	Gal4AD-dnaN LEU2 Amp	Amp R	L	Laboratory stock
	pGBDU-dnaN	Gal4BD-dnaN URA3 Amp	Amp R	U	Laboratory stock
	pGAD-yabA	Gal4AD-yabA LEU2 Amp	Amp R	L	Laboratory stock
	pGBDU-yabA	Gal4BD-yabA URA3 Amp	Amp R	U	Laboratory stock
	pGAD-yabAT71A	Gal4AD-yabAT71A LEU2 Amp	Amp R	L	This work
	pGBDU-yabAT71A	Gal4BD-yabAT71A URA3 Amp	Amp R	U	This work
	pGAD-yabAT71D	Gal4AD-yabAT71D LEU2 Amp	Amp R	L	This work
	pGBDU-yabAT71D	Gal4BD-yabAT71D URA3 Amp	Amp R	U	This work
		Bacterial plasmids
	Plasmids	Relevant genotype	Resistant E. coli B.subtilis	source/construction
	pDG148	Pspac	Amp R	Kn R Phleo R	Stragier et al., (1988)
	pDG148F	Pspac:3xflag	Amp R	Kn R Phleo R	Laboratory stock
	pDG148-yabT	Pspac: yabT	Amp R	Kn R Phleo R	This work
	pDG148F-yabT	Pspac:3xflag-yabT	Amp R	Kn R Phleo R	This work
	pSG1729	bla amyE3′ spc Pxyl-′gfpmut1 amyE5′ -N-terminal fusion vector	Amp R	Spc R	Lewis et al., (1999)
	pSG1729-yabA	Pxyl-gfp-yabA	Amp R	Spc R	Noirot-Gros et al,. (2006)
	pSG1729-yabAT71A	Pxyl-gfp-yabAT71A	Amp R	Spc R	This work
	pSG1729-yabAT71D	Pxyl-gfp-yabAT71D	Amp R	Spc R	This work
	pSMG201	PT7	Kn R		Laboratory stock
	pSMG251	PT7-yabA	Kn R		Laboratory stock
	pSMG251-T71A	PT7-yabAT71A	Kn R		This work
	pSMG251-T71D	PT7-yabAT71D	Kn R		This work
	pQE-30-yabT	PT5-yabT	Amp R Cm R		Jers et al., (2010)
	pBSBII-spo0A	Pspo0A	Amp R	Spc R	Laboratory stock

Table 1 .

 1 Number of up-, down-and total number of differentially expressed features for each comparison.

		Exponential phase
	Strain vs WT	# genes up	# genes down	# total
	yabAT71A	1	0	1
	yabAT71D	742	639	1381
	∆yabA	693	602	1295
		Sporulation T1
	Strain vs WT	# genes up	# genes down	# total
	yabAT71A	16	85	101
	yabAT71D	303	273	576
	∆yabA	615	481	1096
		Sporulation T2
	Strain vs WT	# genes up	# genes down	# total
	yabAT71A	6	47	53
	yabAT71D	762	961	1723
	∆yabA	980	576	1556

Table 3 . Expression of genes bound by DnaA associated with Rok and affected in the yabAT71D strain.

 3

		Gene	YabAT71D
	yceB/yceC	yceC	-1,420
	dtpT/ycIG	dtpT	0,665
	phrI/yddM	phrI	-10,494
		yddM	-9,392
	ydjE/pspA	ydjE	0,423
	katA/ssuB	ssuB	2,432
	yhzC/comK	-	-
	yjaZ/appD	yjaZ	0,733
	yjcM/yjcN	yjcM	-2,157
		yjcN	-0,853
	ykuV/rok	ykuV	0,416
		rok	-0,711
	ppsA/dacC	ppsA	-0,650
	yobI	-	-
	yobI	-	-
	yosX/yosW	-	-
	yonX/yonV	yonX	1,054
	sunT	sunT	-1,112
	sunA/sunI	-	-
	yqgB/yqgA	yqgA	-1,059
	yqxI/cwIA	yqxI	-12,88
	yraN/yraM	yraN	-0,619
	araA/abnA	-	-
	argG/moaB	argG	-0,879
	iscS/braB	braB	-1,179
	malR/nupN	-	-
	yutK/yuzB/yutJ	-	-
	lytA/tagU	lytA	-2,486
		tagU	-0,443
	ggaB	-	-
	ggaA/tagH	-	-
	glyA/ywIG	glyA	-0,777
	ywiB/sboA	-	-
	yxkD/yxkC	-	-
	yxaJ/yxaI	yxaI	-1,183
	gntz/ahpC	-	-
	yydH	-	-
	yydD	-	-
	yydO/yydN	yydO	0,903
		yydN	-1,09
	yydM/yydL	-	-

  with the followed modifications: CN-gel was used instead of BN-gel, CN-gel was pre-incubated for 2 h in 270 mM glycine, 35 mM Tris-HCl pH 8.4 before incubating the gel in the buffer assay containing 270 mM glycine, 35 mM Tris, 14 mM MgSO4, 8 mM ATP, 0.2% Pb(NO3)2 , pH 8.4.[START_REF] Wittig | High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes[END_REF]. ATP hydrolysis correlated with the development of white lead phosphate precipitates. The reaction was stopped using 50% methanol for 30 min. The gel was then washed in water and scanned.Following scanning, the lead-phosphate precipitates were dissolved by overnight incubation in 10 % acetic acid, and the gel was finally restained with Coomassie dye.

Table 1 .

 1 Data collection, phasing and refinement statistics

	Native	Pt derivative

Table 1 .

 1 Strains used in this study

		Strains B. subtilis
	Strain	Genotype	Source/Construction
	CCBS185	168 trpC2 ∆upp (Pr-neo)	Laboratory stock, RLC lab.
	JJS42	168 trpC2 ∆upp ∆yabA	Noirot-Gros et al., (2006)
	TGG57	168 trpC2 ∆upp yabAY90F (YabA-non-phosphorylatable)	This study, see text for details
	TGG48	168 trpC2 ∆upp yabAY90E (YabA-phosphomimetic)	This study, see text for details
	TGG4	168 trpC2 amyE::Pxyl-gfp-yabAY90F	This study 168 ∆upp (Pr-neo) x pSG1729 yabAY90F
	TGG1	168 trpC2 amyE::Pxyl-gfp-yabAY90E	This study, 168 ∆upp (Pr-neo) x pSG1729 yabAY90E
		Strains E. coli
	Strain	Genotype	Source/Construction
		F-endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74
	DH10B	Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-	Grant et al., (1990)
		hsdRMS-mcrBC) λ-
		F-λ-fhuA2 [lon] ompT lacZ::T7 gene 1 gal sulA11
	ER2566	Δ(mcrC-mrr)114::IS10 R(mcr-73::miniTn10-TetS)2	NEB
		R(zgb-210::Tn10)(TetS) endA1 [dcm]
	ER2566-YabA	ER2566 pSMG251	Laboratory stock
	ER2566-YabAY90F	ER2566 pSMG251-YabAY90F	This study, ER2566 x pSMG251-YabAY90F
	ER2566-YabAY90E	ER2566 pSMG251-YabAY90E	This study, ER2566 x pSMG251-YabAY90E
		Strains S. cerevisiae
	Strain	Genotype	Source/Construction
	PJ69-4α	MATα trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal180Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2:: GAL7-lacZ	James et al., (1996)
	PJ69-4a	MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal180Δ LYS2::GAL1-HIS3 GAL2-ADE2 met2:: GAL7-lacZ	James et al., (1996)
	131	PJ69-α, pGAD-C1 : Leu2 Gal4AD	Laboratory stock
	305	PJ69-α, pGAD-C1 : Leu2 Gal4AD-yabA	Laboratory stock
	5842	PJ69-α, pGAD-C1 : Leu2 Gal4AD-yabAY90F	This study
	5841	PJ69-α, pGAD-C1 : Leu2 Gal4AD-yabAY90E	This study
	5844	PJ69-α, pGAD-C1 : Leu2 Gal4AD-yabAY90D	This study
	1534	PJ69-α, pGAD-C1 : Leu2 Gal4AD-dnaA	Laboratory stock
	39	PJ69-α, pGAD-C1 : Leu2 Gal4AD-dnaN	Laboratory stock
	121	PJ69-a, pGBDU-C1 : Ura3 Gal4BD	Laboratory stock
	319	PJ69-a, pGBDU-C1 : Ura3 Gal4BD-yabA	Laboratory stock
	5848	PJ69-a, pGBDU-C1 : Ura3 Gal4BD-yabAY90F	This study
	5846	PJ69-a, pGBDU-C1 : Ura3 Gal4BD-yabAY90E	This study
	5850	PJ69-a, pGBDU-C1 : Ura3 Gal4BD-yabAY90D	This study
	592	PJ69-a, pGBDU-C1 : Ura3 Gal4BD-dnaA	Laboratory stock
	52	PJ69-a, pGBDU-C1 : Ura3 Gal4BD-dnaN	Laboratory stock

Table 2 .

 2 Plasmids used in this study

		Bacterial plasmids
	Plasmids	Relevant genotype	Resistant E. coli B.subtilis	source/construction
	pSG1729	bla amyE3′ spc Pxyl-′gfpmut1 amyE5′ -N-terminal fusion vector	Amp R	Spc R	Lewis et al., (1999)
	pSG1729-yabA	Pxyl-gfp-yabA	Amp R	Spc R	Noirot-Gros et al., (2006)
	pSG1729-yabAY90F	Pxyl-gfp-yabAT71A	Amp R	Spc R	This work
	pSG1729-yabAY90E	Pxyl-gfp-yabAT71D	Amp R	Spc R	This work
	pSMG201	PT7	Kn R		Laboratory stock
	pSMG251	PT7-yabA	Kn R		Laboratory stock
	pSMG251-Y90F	PT7-yabAY90F	Kn R		This work
	pSMG251-Y90E	PT7-yabAY90E	Kn R		This work
		Yeast plasmids
			Resistant
	Plasmids	Relevant genotype	E. coli	S. cerevisiae	source/construction
	pGAD-C1	Gal4AD LEU2 Amp	Amp R	L	James et al., (1996)
	pGBDU-C1	Gal4BD URA3 Amp	Amp R	U	James et al., (1996)
	pGAD-yabA	Gal4AD-yabA LEU2 Amp	Amp R	L	Laboratory stock
	pGBDU-yabA	Gal4BD-yabA URA3 Amp	Amp R	U	Laboratory stock
	pGAD-yabAY90F	Gal4AD-yabAY90F LEU2 Amp	Amp R	L	This work
	pGBDU-yabAY90E	Gal4BD-yabAY90E URA3 Amp	Amp R	U	This work
	pGAD-yabAY90F	Gal4AD-yabAY90F LEU2 Amp	Amp R	L	This work
	pGBDU-yabAY90E	Gal4BD-yabAY90E URA3 Amp	Amp R	U	This work

Table 3 .

 3 Oligonucleotides used in this study

	Nom	Sequence 5' to 3'	Description
		Oligonucleotides for mutagenesis
			K7 phleo -forward
	phleo5'	GAGCTCGAATTCACTGGCCGTCG	-complementary
			to phleo 5'
			K7 phleo -reverse
	phleo3'	CGACCTGCAGGCATGCAAGCT	-complementary
			to phleo 3'
	oY90F fwd	GAGCTCGAATTCACTGGCCGTCGTCTGTTTCAAGAAGGATTTCATATCTG	yabA-forward-mutation Y90F
	oY90F rev	CGACCTGCAGGCATGCAAGCTATGAAATCCTTCTTGAAACAGACGTGCCAG	yabA-reverse-mutation Y90F
	oY90E fwd GAGCTCGAATTCACTGGCCGTCGTCTGGAACAAGAAGGATTTCATATCTGCAATGTC	yabA-forward-mutation Y90E
	oY90E rev	CGACCTGCAGGCATGCAAGCTATGAAATCCTTCTTGTTCCAGACGTGCCAGATT	yabA-reverse-mutation Y90E

Table 4 .

 4 List of solution used for B. subtilis transformation

	*50 ml for each	SOLUTION I	SOLUTION IIII
	MM x10	5 ml	5 ml
	H2O	41.65ml	43.125 ml
	Glucose50%	0.5 ml	0.5 ml
	Mg2SO4 1M	0.3 ml	0.3 ml
	Yeast extract 5%	2 ml	1 ml
	Trp 1%	0.25 ml	0.025ml
	Casein hydrolisate	0.1 ml	0.025ml
	AAs If auxotrophies	0.25 ml	0.05ml

Table 3 .

 3 Reactions were carried out with a

	Mastercycler®ep realplex (Eppendorf) with the following program: 15 min at 95°C, (15 sec at
	95°C, 1 min at 60°C) x40 cycles, 15 sec at 95°C, 15 sec at 60°C, 20 min to reach 95°C, 15 sec at
	95°C. Results were analyzed with Realplex program (Eppendorf) and quantified by ΔΔCt method.
	Fluorescence Microscopy
	For localization of YabA derivatives, the N-terminally tagged GFP-YabA proteins were
	conditionally expressed from the xylose-inducible promoter Pxyl, from the ectopic amyE locus in
	the B. subtilis chromosome. Cells from overnight cultures grown at 37°C in LB supplemented
	with spectinomycin were diluted to OD600 = 0.01 in the same medium supplemented with xylose
	to 0.5% until the OD600 reached 0.2-0.4. Cells were rinsed in Minimal Medium (MM) and
	mounted on 1.2 % pads. Cells were stained with DAPI to visualize the nucleoid. Fluorescence
	microscopy was performed using Leica DMRA2. System control and image processing were
	performed using MetaMorph software.

  All of these in vitro and in vivo observations suggest the existence of different modes of regulation of YabA activity by phosphorylation, involving threonine and tyrosine residues. This

	study established that YabA, apart from its role during replication initiation, plays a key
	regulatory role in B. subtilis differenciation, highlighting a multitasking protein which exerts a
	variety of activities in different biological pathways, thus contributing to cellular network
	complexity. The main conclusions of this work are listed below.
	1. YabA possesses an unique tri-dimentional structure composed of N-terminal four
	helixbundle tetramer connected to four monomeric C-terminal domains through
	flexible linkers, which makes YabA able to physically interact with more than one protein
	at a time, thus providing a suitable platform to integrate intracellular signals to
	replication initiation.

Table Annex 3

 Annex . List of genes differentially expressed in yabAT71D (left) and ∆yabA (right) during exponential phase. Padj represents p-value adjusted.

	BSU16180 BSU25210 ANNEX 4.	flgB dnaG	1,626 0,480	9,94E+07 2,76E-02	BSU16340 BSU36250	fliZ ptkA	-0,953 6,23E-04 1,015 2,40E-06
	BSU16190 BSU34430	flgC racX	0,795 0,701	1,53E-02 4,46E-03	BSU16360 BSU04720	fliQ rsbW	-1,058 7,76E-04 -2,237 2,02E-04
	BSU35400 BSU38090	flgL vpr	0,482 0,514	1,35E-02 2,86E-02	BSU16270 BSU04740	fliK rsbX	-0,906 3,55E-03 -2,067 6,50E-04
	BSU35410 Cell division	flgK	1,031	8,84E+08	BSU16320 BSU04710	fliY rsbV	-0,893 4,85E-03 -1,839 1,89E-03
	T71D vs WT 0,5 flgM 1,734 divIB 0,470 Name log2FC fliE 0,788 divIC 0,349 fliD 0,768 ftsW 0,583 sigF 0,997 fliS 0,499 ftsA 0,494 sigH 0,506 fliW 0,558 minJ 0,495 sigX 0,386 motA 1,220 ftsH -0,793 1,37E+09 1,53E+06 9,11E-03 padj 1,90E-02 1,76E-02 2,14E-03 2,04E-02 1,12E-02 3,68E-02 4,15E-02 4,07E-04 2,50E-02 3,11E-02 3,10E-02 3,14E-03 Sporulation genes BSU35430 BSU15240 Id BSU16200 BSU00620 Sigma factors BSU35340 BSU14850 BSU23450 BSU35330 BSU15280 BSU00980 BSU35380 BSU35220 BSU23100 BSU13690 BSU00690 BSU13680 motB 0,926 5,34E-03 BSU35250 ftsX -0,719 1,86E-03 BSU13990 kinA 0,469 1,16E-02 BSU35230 swrAA 1,731 1,84E+09 BSU27990 minD -0,568 7,83E+09 BSU31450 kinB 0,594 8,96E-03 BSU06710 swrC -0,895 3,17E+09 BSU28030 mreB -0,667 1,91E-03 BSU13660 kinD 0,370 1,55E-02 autolytic genes BSU28020 mreC -1,167 1,91E+09 BSU13530 kinE 0,995 5,85E-04 BSU35640 lytA 0,869 7,97E-03 Kinases and phosphatases BSU12430 rapA -6,058 1,64E+01 BSU35630 lytB 0,596 3,59E-02 0 BSU12440 phrA -5,533 4,53E-03 BSU35620 lytC -0,617 1,94E-02 ImmR and PBSX BSU25830 rapE -6,113 1,46E+09 BSU35650 lytR 0,433 2,94E-02 BSU04820 immR -7,744 9,67E+04 BSU25840 phrE -11,272 1,05E-03 BSU28920 lytT -0,468 1,03E-02 BSU05010 rapI -6,984 3,03E+07 BSU36690 rapB 1,091 1,53E-04 competence/others BSU05020 phrI -8,937 2,22E+03 BSU37130 spo0F 0,805 2,83E-02 BSU31710 comQ 1,420 9,32E+07 BSU12560 xpf -4,371 2,81E-03 BSU23460 spoIIAB 0,948 2,90E-02 BSU35460 comFB 0,982 5,87E-04 others BSU23470 spoIIAA 1,009 2,97E-02 BSU35470 comFA 1,044 5,49E-03 BSU31250 tlpA 2,313 5,75E+05 Matrix genes BSU27660 comN 0,506 1,65E-02 BSU29740 ccpA -0,483 1,60E-02 BSU24600 sinI 0,709 3,42E-02 BSU31690 comP 0,420 2,09E-02 BSU12410 yjoA -15,355 5,61E-19 BSU24620 tasA 1,235 5,73E-04 BSU24630 sipW 1,419 1,35E-03 BSU31080 bslA 1,02 0,0024 BSU24640 yqxM 1,779 3,10E+08 BSU34380 slrR 1,721 7,95E+09 BSU38220 ywcC 0,472 3,53E-02 BSU34370 epsA 1,248 1,30E-03 BSU34360 epsB 1,027 3,17E-03 BSU34350 epsC 0,728 1,94E-02 BSU34320 epsF 1,351 3,92E-04 BSU34310 epsG -10,294 7,87E-01 BSU34300 epsH -10,215 3,55E+00 BSU34290 epsI -10,727 4,03E-02 BSU34280 epsJ -9,322 7,62E-04 BSU34265 epsK -11,290 2,41E-03 BSU34250 epsL -4,207 1,06E-05 BSU34240 epsM -2,797 4,26E+04 BSU34230 epsN -2,416 3,07E+06 BSU34220 epsO -1,454 1,66E-04 BSU25860 yqcG -10,386 8,22E-03 BSU25870 yqcF -10,763 6,03E-04 motility/flagelle BSU18090 parE -0,562 4,07E-03 BSU18100 parC -0,703 7,58E-03 BSU37080 rho -0,588 2,41E-02 BSU40900 ssbA -1,125 6,85E-03 BSU24240 recN -0,639 2,76E+09 BSU00210 recR -0,521 1,37E-02 BSU16940 recA -0,365 8,06E-03 BSU27620 recJ -0,658 6,11E-03 BSU40440 dnaC -0,846 2,47E+09 BSU00020 dnaN -0,929 2,45E+08 BSU00330 yabA -0,488 1,62E-03 Replication BSU13420 ykoW 0,767 4,87E-04 BSU09120 yhcK -0,793 5,76E-03 C-di-GMP BSU16170 codY -0,847 2,59E-03 BSU10300 aprE 0,640 3,60E-02 BSU03480 srfAA 0,770 1,68E+09 BSU03520 srfAD 0,717 6,08E+08 BSU03490 srfAB 0,776 4,67E+08 BSU03510 srfAC 0,817 1,58E+07 BSU35500 degS 0,674 5,16E-03 BSU35450 comFC 0,700 2,38E-02 BSU12420 yjoB -13,454 1,59E-11	BSU16350 BSU15760 Id BSU16310 BSU37240 BSU35360 BSU07880 Sigma factors BSU23450 autolytic genes ∆yabA vs WT 0,5 fliP -0,885 6,47E-03 prpC -0,45 4,23E-02 Name log2FC fliM -0,909 6,82E-03 ywiE -2,422 4,84E-03 padj hag -1,179 2,13E-12 yfkJ -1,803 9,15E-04 sigF High impated -0,947 1,87E-02 BSU16470 sigD BSU35780 lytD -0,867 3,20E-07 BSU12560 xpf 3,351 3,70E-12 -1,169 1,57E-05 BSU04730 sigB BSU31120 lytG 1,006 5,41E-05 BSU04730 sigB -2,291 1,17E-04 -2,291 1,17E-04 BSU01730 sigW BSU28930 lytS 0,443 1,06E-02 BSU24770 mgsR -3,301 6,58E-05 -0,749 1,33E-02 BSU13450 sigI 1,279 competence/others BSU05310 ydeR 12,022 2,89E-159 6,47E-03 BSU24720 comGB -1,569 1,19E-02 BSU05290 ydeP 7,89 7,62E-117 Sporulation genes BSU13530 kinE BSU03520 srfAD -1,274 5,02E-18 BSU05300 ydeQ 7,766 2,11E-116 -0,715 1,97E-02 BSU27930 spo0B 0,461 BSU03510 srfAC -1,155 6,30E-15 others 4,38E-02 BSU13640 spo0E BSU03490 srfAB -1,094 1,17E-11 BSU12410 yjoA -1,015 3,81E-03 -0,895 7,33E-03 BSU23530 spoIIM BSU03480 srfAA -0,939 6,42E-08 BSU12420 yjoB -1,1 6,38E-04 -1,212 3,19E-02 BSU12830 spoIISA 0,53 BSU10300 aprE -0,705 2,30E-02 2,31E-02 BSU24370 C-di-GMP spoIIIAG -1,968 4,50E-02 BSU16800 spoIIIE BSU29650 ytrP -1,017 1,22E-05 -0,692 1,51E-04 BSU00560 spoVT BSU09120 yhcK 0,642 2,57E-02 -0,569 1,54E-02 BSU16980 spoVS BSU22910 ypfA 0,704 2,48E-03 -0,654 2,74E-02 BSU28410 gerE Replication -0,684 6,71E-03 BSU10680 gerPE BSU00330 yabA -2,984 4,03E-99 -0,992 2,93E-02 BSU22200 cotD 1,175 BSU00010 dnaA 1,187 3,39E-11 2,79E-05 BSU24630 sipW 1,531 BSU31080 bslA -0,800 BSU24640 yqxM 1,411 BSU38229 slrA 0,652 BSU34380 slrR 1,589 BSU38220 ywcC 0,469 BSU34370 epsA 1,721 BSU34360 epsB 1,367 BSU34350 epsC 0,939 BSU04010 sipU 0,682 BSU16280 flgD BSU35400 flgL BSU35430 flgM BSU35410 flgK BSU16290 flgE BSU35340 fliD BSU16240 fliI BSU16230 fliH BSU35320 fliT -1,01 BSU35330 fliS BSU35000 hprK 0,341 4,25E-02 -0,775 5,75E-04 Kinases and phosphatases 1,57E-05 BSU35250 ftsX 0,789 4,79E-04 -0,685 4,27E-02 BSU35260 ftsE 1,163 4,62E-05 -0,726 2,15E-02 BSU27990 minD -0,405 6,68E-03 -0,667 8,91E-03 BSU15290 ftsZ -0,679 2,34E-04 -0,861 1,33E-02 Cell division -0,855 2,82E-04 BSU38090 vpr -0,499 3,91E-02 -0,768 2,46E-02 BSU10650 sbcC -0,83 4,21E-07 -0,996 2,61E-08 BSU29080 mutM -0,576 1,09E-03 -0,795 3,20E-02 BSU17050 mutL -0,394 3,28E-02 motility/flagelle BSU34430 racX -0,559 2,99E-02 4,57E-03 BSU24240 recN -0,387 1,66E-02 1,70E-03 BSU04330 mutT 0,724 4,16E-02 3,85E-05 BSU37160 rpoE 0,793 2,04E-05 2,85E-06 BSU17850 lexA 0,617 7,81E-03 3,65E-02 BSU00210 recR 0,635 1,36E-03 2,25E-04 BSU22310 recU 0,689 2,30E-02 8,73E-03 BSU25470 dnaK -0,583 2,84E-03 0,00118 BSU36310 ssbB 1,097 3,08E-03 0,0237 BSU00190 dnaX 0,995 1,44E-09 3,61E-04 BSU40440 dnaC 0,76 1,19E-04 Matrix genes BSU00020 dnaN 0,535 1,01E-02

Table Annex 4

 Annex . List of genes differentially expressed in yabAT71D (left) and ∆yabA (right) during sporulation stage T1. Padj represents p-value adjusted.

	BSU16360 BSU38180 ANNEX 5.	fliQ ywzA	-0,744 7,32E-02 2,193 1,25E-03	BSU34290 BSU04820	epsI immR	-1,964 3,25E-08 0,704 1,50E-02
	BSU16340 BSU07920	fliZ yfkE	-0,783 1,84E-02 2,198 1,10E-04	BSU34280 BSU12560	epsJ xpf	-2,016 4,54E-06 2,537 3,41E-12
	BSU16320 BSU07930	fliY yfkD	-0,76 2,158	4,39E-02 1,29E-04	BSU34265 BSU05310	epsK ydeR	-1,854 9,20E-06 10,949 2,36E-163
	BSU16310 BSU07850	fliM yfkM	-0,741 7,08E-02 2,052 6,46E-03	BSU34250 BSU05290	epsL ydeP	-1,868 1,69E-06 8,752 1,15E-228
	T71D vs WT T1 flgE -0,958 1,27E-02 yfkI 1,423 1,02E-02 Name log2FC padj flgD -0,998 1,40E-02 yfkH 1,123 2,92E-02 fliK -1,022 2,01E-03 sigH -0,339 3,94E-02 fliI -1,007 1,78E-03 sigD -0,707 3,60E-02 sigB 1,288 8,82E-02 Sporulation genes BSU16290 BSU07890 Id BSU16280 BSU07900 Sigma factors BSU16270 BSU00980 BSU16240 BSU16470 BSU04730 BSU12430 rapA -9,568 2,88E-35 BSU12440 phrA -10,152 3,31E-78 BSU25830 rapE -9,446 1,08E-18 BSU25840 phrE -10,135 6,25E-37 BSU36970 spoIIR 1,893 6,71E-04 BSU26390 spoIIIC -1,824 1,20E-04 BSU25770 spoIVCA -0,518 2,21E-02 Matrix genes BSU24600 sinI 0,772 1,86E-02 BSU24620 tasA 1,731 4,40E-07 BSU31080 bslA 1,159 BSU24640 yqxM BSU24630 sipW 1,858 1,27E-07 BSU34380 slrR 1,653 1,55E-07 BSU38229 slrA 0,458 3,26E-02 BSU34370 epsA 1,57 1,07E-05 BSU34360 epsB 1,478 3,81E-06 BSU34350 epsC 1,142 9,60E-05 BSU34340 epsD 1,156 1,02E-02 BSU34330 epsE 1,216 7,58E-03 BSU34320 epsF 1,811 7,59E-07 BSU34310 epsG -10,175 2,78E-17 BSU34300 epsH -9,977 1,86E-16 BSU34290 epsI -10,169 1,28E-17 BSU34280 epsJ -11,484 4,86E-18 BSU34265 epsK -11,675 8,86E-19 BSU34265 epsK -11,675 8,86E-19 BSU34250 epsL -2,481 3,24E-10 BSU34240 epsM -1,747 1,63E-04 BSU34230 epsN -1,829 4,07E-05 BSU34220 epsO -1,954 7,26E-08 BSU25860 yqcG -10,326 1,86E-45 BSU25870 yqcF -12,619 1,80E-09 motility/flagelle BSU35400 flgL -0,557 8,63E-03 BSU36720 ywmE 3,039 2,04E-04 BSU38730 cydD 4,099 7,04E-01 BSU38750 cydB 1,939 4,73E-02 BSU38740 cydC 2,982 1,40E+09 BSU24950 pstBB 2,379 2,04E+08 BSU24960 pstBA 3,472 8,44E+06 BSU24970 pstA 4,213 4,12E+04 BSU24980 pstC 4,034 6,05E+02 BSU24990 pstS 4,504 6,33E-01 others BSU12420 yjoB -9,612 3,42E+00 BSU12410 yjoA -6,085 6,47E-08 BSU24770 mgsR 2,215 2,30E-02 BSU05020 phrI -9,156 5,08E-12 BSU05010 rapI -9,648 1,14E-11 BSU12560 xpf -5,630 3,93E+09 BSU04820 immR -9,875 2,26E-02 others high impacted BSU07880 yfkJ 1,812 2,02E-03 BSU37240 ywiE 2,012 4,61E-02 Kinases and phosphatases 0 Cell division BSU16940 recA -0,344 3,03E-02 BSU24240 recN -0,468 7,63E-03 2,109 2,73E+03 BSU15870 recG -0,679 3,33E-03 0,00086 BSU00330 yabA -0,778 1,09E-07 Replication BSU04280 ydaK 1,099 4,40E-02 BSU14090 ykuI -0,488 3,33E-04 BSU29650 ytrP 0,515 8,37E-02 C-di-GMP BSU31710 comQ 0,665 9,01E-02 competence BSU35620 lytC -1,341 4,68E-08 BSU35630 lytB -1,224 4,78E-06 BSU35640 lytA -0,831 4,18E-02 autolytic genes BSU16220 fliG -0,723 7,21E-02 BSU16230 fliH -0,721 6,23E-02	BSU34240 BSU05300 Id BSU34230 BSU12420 BSU34220 BSU33950 Sigma factors BSU23450 motility/flagelle ∆yabA vs WT T1 epsM -2,096 1,28E-06 ydeQ 8,573 9,97E-180 Name log2FC epsN -2,033 1,68E-06 yjoB -1,15 6,33E-04 padj epsO -2,061 5,27E-09 cggR -2,783 4,35E-15 sigF -1,73 BSU38760 cydA -5,864 4,39E-13 1,85E-06 BSU15320 sigE BSU15330 sigG BSU01730 sigW BSU13450 sigI 1,051 BSU38700 sigY 0,642 BSU13990 kinA BSU27930 spo0B 0,551 BSU23470 BSU23460 BSU28060 spoIIB BSU36750 spoIID 0,751 BSU00640 spoIIE BSU24420 BSU25770 spoIVCA 0,84 BSU23440 spoVAA -1,79 BSU16980 spoVS -0,77 BSU10700 gerPC BSU10690 gerPD BSU10680 gerPE BSU10670 gerPF BSU30920 cotI BSU30910 cotSA BSU24600 sinI BSU24620 tasA BSU24640 yqxM -1,38 BSU31080 bslA -1,133 BSU24630 sipW BSU34370 epsA BSU34360 epsB BSU34350 epsC BSU34340 epsD BSU34330 epsE BSU34320 epsF BSU34310 epsG BSU34300 epsH others -1,963 1,75E-05 BSU37240 ywiE -3,15 1,74E-04 -1,824 7,01E-07 BSU07880 yfkJ -1,242 4,21E-02 -1,476 8,54E-05 BSU15760 prpC -0,564 9,70E-03 -1,462 3,70E-04 BSU15770 prkC -0,592 3,44E-02 -1,325 1,13E-03 BSU00650 yabS -2,051 1,51E-20 -0,986 7,32E-04 BSU00660 yabT -1,806 1,08E-11 -1,392 1,28E-05 Kinases and phosphatases -1,511 2,08E-05 BSU35250 ftsX 0,915 4,47E-05 -1,194 2,06E-03 BSU35260 ftsE 1,179 3,76E-05 0,00073 Cell division 9,05E+09 BSU38080 ywcI -1,866 4,54E-11 -1,879 2,12E-08 BSU38090 vpr -1,168 4,29E-09 -0,808 7,50E-03 BSU28390 racE 0,411 1,92E-02 Matrix genes BSU34430 racX -1,588 2,92E-12 -1,094 2,12E-02 BSU28390 racE 0,411 1,92E-02 -1,691 8,00E-06 BSU37030 racA -1,662 9,29E-12 -1,655 8,21E-10 BSU40970 parA 0,577 1,88E-02 -1,335 9,39E-07 BSU17850 lexA 1,151 6,18E-08 -0,906 1,59E-02 BSU00190 dnaX 0,492 7,93E-03 -0,838 1,07E-04 BSU29230 dnaE 0,369 3,65E-02 8,60E-03 BSU40440 dnaC 0,467 4,01E-02 6,60E-04 BSU00010 dnaA 0,736 9,85E-05 1,14E-06 BSU00330 yabA -2,361 2,27E-66 spoIIIAB -1,904 2,61E-02 Replication BSU22910 ypfA 0,744 3,06E-03 BSU14090 ykuI 0,557 7,30E-06 -2,137 1,52E-16 BSU13420 ykoW 0,704 1,93E-03 3,54E-02 C-di-GMP -1,817 3,64E-17 BSU25580 comEB 0,584 2,76E-02 spoIIAB -1,675 2,38E-05 competence spoIIAA -1,499 5,32E-04 BSU35620 lytC 0,606 2,65E-02 9,62E-03 BSU35640 lytA 1,085 9,61E-04 -0,412 2,80E-02 BSU35630 lytB 1,089 3,36E-05 Sporulation genes autolytic genes 4,14E-02 BSU16360 fliQ 0,699 4,97E-02 2,79E-02 BSU16340 fliZ 0,632 4,14E-02 -0,765 1,32E-02 BSU16210 fliF 0,636 2,89E-02 -1,955 4,14E-18 BSU16190 flgC 0,748 2,80E-02 -1,716 3,01E-11 BSU16180 flgB 0,781 3,44E-02 BSU39810 csbC -2,792 2,94E-03

Table Annex 5

 Annex . List of genes differentially expressed in yabAT71D (left) and ∆yabA (right) during sporulation stage T3. Padj represents p-value adjusted.

	BSU27980 BSU34280 BSU25570 BSU04820	spoIVFA epsJ comEC immR	1,08 -9,685 4,02E+01 1,48E-04 -1,012 4,73E-04 -11,015 9,28E-04	BSU23401 BSU35220	spoVAEA -1,321 3,11E-05 minJ 0,606 5,92E-03
	BSU27670 BSU34265 BSU35460 BSU12560	spoVB epsK comFB xpf	1,110 4,48E+07 -9,65 3,85E+01 -0,546 3,33E-02 -6,017 1,03E+09	BSU17420 Matrix genes Kinases and phosphatases spoVK	-2,098 2,19E-15
	BSU15170 BSU25860 BSU24730 BSU05010	spoVD yqcG comGA rapI	1,117 1,22E+08 -13,196 2,20E-11 -0,779 3,89E-02 -9,373 2,78E+08	BSU15810 BSU34380 BSU00660	spoVM slrR yabT	-2,168 1,14E-14 -1,486 2,07E-06 -1,298 1,09E-06
	BSU15810 BSU25870 BSU24720 BSU05020	spoVM yqcF comGB phrI	0,85 -12,927 1,09E-11 2,65E-03 -0,604 4,20E-02 -10,494 6,19E+03	BSU00560 BSU34360 BSU00650	spoVT epsB yabS	-1,467 8,06E-13 -0,73 3,51E-02 -1,027 8,03E-06
	T71D vs WT T3 spoVFA 0,674 epsM 1,23 comQ 0,825 mgsR 3,887 9,93E+07 1,30E-02 6,50E-03 7,34E-03 Name log2FC padj spoVFA 0,674 1,30E-02 motility/flagelle BSU16730 BSU34240 BSU31710 BSU24770 Id BSU16730 BSU37460 rapF 0,619 2,42E-02 BSU03050 ldh 7,865 4,34E-14 Sigma factors BSU16740 spoVFB 0,831 8,47E-04 BSU16470 sigD -1,195 1,05E+09 BSU10300 aprE 0,693 1,07E-02 BSU03060 lctP 7,532 6,87E-08 BSU00980 sigH -1,17 1,87E-06 BSU15210 spoVE 1,25 1,36E-03 BSU35360 hag -1,711 2,20E-11 BSU14240 rok -0,711 4,81E-03 BSU33950 cggR 1,823 5,49E+07 BSU23450 sigF 0,898 1,94E-02 BSU15810 spoVM 0,85 2,65E-03 BSU16180 flgB -1,379 3,13E+09 C-di-GMP BSU03981 mtlA 5,190 2,77E+06 BSU16470 sigD -1,195 1,05E+09 BSU17420 spoVK 0,967 5,69E-04 BSU16190 flgC -1,678 2,56E+06 0 BSU03990 mtlD 3,350 4,29E-04 BSU04730 sigB 2,435 2,38E+09 BSU09400 spoVR 1,11 1,74E+06 BSU16280 flgD -2,218 5,84E+02 Replication BSU03982 mtlF 4,519 2,83E+08 BSU23100 sigX -0,455 6,51E-03 BSU16980 spoVS 0,943 3,89E-04 BSU16290 flgE -1,971 5,62E+04 BSU00330 yabA -1,160 7,81E-03 BSU38760 cydA 3,895 2,79E+08 Sporulation genes BSU12440 phrA -12,631 1,78E-62 BSU12430 rapA -12,639 7,35E-45 BSU25840 phrE -14,316 8,94E-15 BSU25830 rapE -13,223 2,44E-11 BSU36690 rapB -0,76 8,49E-03 BSU13530 kinE -0,611 3,69E-02 BSU13660 kinD -0,426 2,74E-03 BSU37130 spo0F -0,852 1,58E-02 BSU27930 spo0B -0,494 1,58E-02 BSU36750 spoIID 0,782 1,50E-02 BSU00640 spoIIE 0,897 1,28E-03 BSU23530 spoIIM 0,879 3,63E+09 BSU25530 spoIIP 1,124 1,11E-04 BSU36550 spoIIQ 1,648 1,87E+06 BSU36970 spoIIR 3,881 9,64E-04 BSU24430 spoIIIAA 1,238 2,52E+09 BSU24420 spoIIIAB 1,327 5,83E+06 BSU24410 spoIIIAC 1,198 1,89E+09 BSU24400 spoIIIAD 1,178 8,55E-04 BSU24390 spoIIIAE 1,104 3,54E+09 BSU22800 spoIVA 1,055 5,77E+08 BSU24360 spoIIIAH 1,124 1,18E+09 BSU24410 spoIIIAC 1,198 1,89E+09 BSU24430 spoIIIAA 1,238 2,52E+09 BSU24390 spoIIIAE 1,104 3,54E+09 BSU24380 spoIIIAF 1,136 3,38E+06 BSU24370 spoIIIAG 0,897 6,58E-03 BSU24360 spoIIIAH 1,12 1,18E+09 BSU26390 spoIIIC 1,27 8,58E+06 BSU22800 spoIVA 1,06 5,77E+08 BSU27970 spoIVFB 1,13 5,00E+09 BSU24230 spoIVB 1,04 1,20E-03 BSU25770 spoIVCA 1,57 2,89E-13 BSU25760 spoIVCB 1,02 1,85E+07 BSU34290 epsI -9,499 4,84E+00 BSU28070 comC -1,395 2,93E-04 Others BSU34300 epsH -9,269 5,16E+02 BSU25580 comEB -0,949 1,69E+09 BSU07880 yfkJ 1,791 7,48E-04 BSU34310 epsG -9,627 2,40E+01 BSU25590 comEA -1,347 1,76E-02 BSU37240 ywiE 3,840 1,34E+08 BSU34320 epsF 2,93 2,11E-03 BSU35500 degS -0,738 1,18E-03 BSU04740 rsbX 2,017 6,72E-04 BSU34330 epsE 1,93 6,13E+07 competence BSU04710 rsbV 2,413 1,61E+09 BSU34340 epsD 1,39 3,53E-04 BSU28920 lytT -0,651 8,62E+09 BSU04720 rsbW 2,626 5,57E+08 BSU34350 epsC 0,997 4,02E-04 BSU28930 lytS -0,678 1,34E+08 BSU24140 prpC 1,041 1,66E-03 BSU34360 epsB 1,22 9,89E+09 BSU35650 lytR -0,443 2,39E-02 BSU08970 prkA 1,067 2,80E-03 BSU34370 epsA 1,30 1,94E-04 BSU31120 lytG -1,099 1,02E+09 BSU00650 yabS 1,300 9,11E+05 BSU24640 yqxM 1,572 1,83E+08 BSU35780 lytD -0,792 4,05E+08 BSU00660 yabT 1,211 6,12E+08 BSU31080 bslA 1,168 0,000239 BSU35620 lytC -1,479 3,94E+04 Kinases and phosphatases BSU24630 sipW 1,86 1,15E+07 BSU35630 lytB -2,263 3,98E-06 BSU35220 minJ -0,562 1,13E-02 BSU24620 tasA 2,968 3,64E-06 BSU35640 lytA -2,486 4,49E-01 BSU27990 minD -0,827 1,34E+04 BSU24600 sinI 0,652 2,37E-02 autolytic genes BSU28000 minC -0,978 3,18E+07 Matrix genes BSU13680 motB -0,830 1,20E-02 BSU15290 ftsZ -0,469 1,27E-02 BSU28380 gerM 1,114 9,22E-04 BSU13690 motA -1,639 3,45E+09 BSU35250 ftsX -1,964 3,64E-06 BSU28410 gerE 0,525 3,43E-02 BSU35360 hag -1,711 2,20E-11 BSU14850 ftsW -0,786 6,85E-04 BSU11740 cotZ 1,209 6,88E+08 BSU16340 fliZ -1,601 2,30E+05 BSU35260 ftsE -2,087 5,71E+00 BSU11750 cotY 1,134 7,94E+09 BSU16320 fliY -1,912 6,77E+03 BSU15280 ftsA -0,567 1,38E-02 BSU11770 cotW 0,824 2,16E-02 BSU35320 fliT -1,413 1,21E+05 BSU15420 divIVA -1,239 7,73E-02 BSU11780 cotV 0,986 1,99E-04 BSU35330 fliS -1,371 4,79E+03 Cell division BSU12090 cotT 0,935 1,66E-03 BSU16370 fliR -1,572 1,62E+09 BSU38090 vpr 0,447 4,21E-02 BSU30910 cotSA 0,798 3,29E-02 BSU16360 fliQ -1,806 8,82E+05 BSU38080 ywcI 1,041 2,65E-04 BSU30900 cotS 0,689 3,04E-02 BSU16350 fliP -1,750 8,50E+05 BSU37030 racA 1,031 4,33E+09 BSU11730 cotO 0,955 3,76E-03 BSU16310 fliM -1,873 1,57E+05 BSU34430 racX 0,522 3,78E-02 BSU17970 cotM 0,963 1,66E-03 BSU16300 fliL -1,800 1,43E+08 BSU00210 recR 0,617 8,45E-04 BSU06910 cotJC 1,21 1,02E-02 BSU16270 fliK -2,268 1,06E-01 BSU25470 dnaK 0,595 1,71E-03 BSU06900 cotJB 1,188 1,24E-02 BSU16250 fliJ -2,388 2,61E+08 BSU40970 parA -0,556 1,82E-02 BSU06890 cotJA 1,21 9,01E+09 BSU16240 fliI -2,458 5,89E-05 BSU17850 lexA -0,700 1,68E-03 BSU30920 cotI 0,725 3,40E-02 BSU16230 fliH -2,367 8,05E-02 BSU28390 racE -0,469 3,59E-03 BSU36060 cotH 0,859 1,79E-02 BSU16220 fliG -2,259 6,53E+00 BSU16940 recA -0,335 1,34E-02 BSU17030 cotE 1,095 1,34E+09 BSU16210 fliF -2,143 8,56E-05 BSU23020 recQ -0,996 2,54E+01 BSU05550 cotP 0,718 1,55E-02 BSU16200 fliE -1,986 1,20E+04 BSU28990 dnaB -0,705 1,27E+09 BSU11810 spoVIF 1,228 4,01E-02 BSU35340 fliD -1,949 1,65E-03 BSU22350 dnaD -0,429 2,33E-02 BSU28110 spoVID 1,12 5,31E+08 BSU35410 flgK -1,053 2,99E+08 BSU00020 dnaN -0,844 1,39E+09 BSU00560 spoVT 0,517 2,22E-02 BSU35400 flgL -1,082 8,10E+04 BSU00010 dnaA -1,543 1,56E-03 BSU39810 csbC 4,458 1,77E+07	BSU09400 BSU34290 BSU08970 Id BSU16980 BSU34280 BSU15760 BSU15210 motility/flagelle ∆yabA vs WT T3 spoVR -2,041 6,95E-27 epsI -1,139 3,78E-03 prkA -1,921 9,52E-09 Name log2FC spoVS -1,036 9,05E-05 epsJ -1,138 1,82E-02 prpC -0,571 5,87E-03 padj spoVE -1,328 6,00E-04 BSU04690 rsbT 0,724 1,98E-03 Sigma factors BSU00980 sigH BSU23402 spoVAEB -1,343 1,13E-03 BSU35360 hag 0,571 1,38E-03 BSU04700 rsbU 0,613 2,15E-02 0,536 4,63E-05 BSU15320 sigE BSU16740 spoVFB -1,473 2,60E-07 BSU16180 flgB 0,979 3,92E-03 others -0,91 6,68E-04 BSU15330 sigG BSU16730 spoVFA -1,467 6,60E-07 BSU16190 flgC 1,05 6,64E-04 BSU04820 immR 0,91 6,04E-04 -1,307 9,85E-09 BSU38700 sigY BSU17420 spoVK -2,098 2,19E-15 BSU16200 fliE 0,901 5,29E-03 BSU16170 codY -0,995 2,04E-04 1,058 1,50E-04 BSU01730 sigW BSU15810 spoVM -2,168 1,14E-14 BSU16210 fliF 0,903 6,07E-04 0,916 1,35E-03 BSU23100 sigX 0,43 BSU27120 sigV BSU13660 kinD BSU31450 kinB BSU37130 spo0F BSU28060 spoIIB BSU00640 spoIIE BSU36420 spoIIID BSU23530 spoIIM BSU25530 spoIIP BSU36550 spoIIQ BSU36970 spoIIR BSU24430 BSU24420 BSU24400 BSU24410 BSU24390 BSU24380 BSU24370 BSU24360 BSU26390 spoIIIC BSU36750 spoIID BSU22800 spoIVA BSU24230 spoIVB BSU25770 BSU25760 BSU27980 BSU23440 BSU23430 spoVAB BSU23420 spoVAC BSU23410 BSU23390 spoVAF BSU27670 spoVB BSU15170 spoVD BSU27990 minD 0,5 4,14E-04 -1,289 1,44E-08 BSU35820 gerBC -1,515 4,73E-03 BSU35250 ftsX 0,896 4,01E-05 -1,505 1,20E-11 BSU35810 gerBB -1,647 3,81E-03 BSU35260 ftsE 0,923 1,26E-03 -0,702 2,68E-03 BSU10690 gerPD -0,99 2,75E-03 BSU15280 ftsA 0,476 4,83E-02 spoVAD -0,916 1,48E-02 BSU33050 gerAA -1,295 2,15E-03 BSU15420 divIVA 0,48 3,93E-03 -1,147 2,58E-02 BSU19490 gerT -1,274 1,21E-03 Cell division -1,215 1,73E-02 BSU35800 gerBA -1,772 5,67E-05 BSU40960 parB 0,586 1,23E-03 spoVAA -1,272 6,80E-03 BSU10700 gerPC -0,859 2,31E-05 BSU40970 parA 0,77 5,93E-04 spoIVFA -1,436 2,35E-07 BSU10680 gerPE -1,213 2,88E-06 BSU17850 lexA 1,703 6,94E-17 spoIVCB -1,477 1,40E-11 BSU28380 gerM -1,576 1,10E-06 BSU16940 recA 0,61 1,16E-06 spoIVCA -1,78 7,32E-35 BSU03720 gerKB -1,556 8,98E-07 BSU00190 dnaX -0,371 4,14E-02 -1,792 8,44E-09 BSU03700 gerKA -1,947 2,03E-07 BSU25470 dnaK -0,435 3,08E-02 -1,835 1,70E-16 BSU10670 gerPF -1,391 7,82E-09 BSU00020 dnaN 0,603 2,68E-03 -1,331 9,97E-06 BSU03710 gerKC -2,014 2,07E-10 BSU00010 dnaA 0,869 1,43E-06 -1,587 4,01E-10 BSU36070 cotG -2,011 2,27E-02 BSU00330 yabA -2,489 1,91E-69 spoIIIAH -1,38 3,97E-08 BSU36050 cotB -1,589 1,58E-02 Replication spoIIIAG -1,692 4,00E-08 BSU17970 cotM -0,866 1,12E-02 BSU09120 yhcK -0,758 6,10E-03 spoIIIAF -1,564 9,35E-13 BSU11770 cotW -1,344 7,49E-04 BSU14090 ykuI 0,393 1,72E-03 spoIIIAE -1,678 1,04E-10 BSU06910 cotJC -1,638 3,01E-04 C-di-GMP spoIIIAC -1,668 8,30E-09 BSU06900 cotJB -1,722 1,50E-04 BSU02820 rapJ 0,641 5,30E-05 spoIIIAD -1,64 2,39E-06 BSU30910 cotSA -1,6 3,20E-05 BSU36690 rapB 0,628 3,72E-02 spoIIIAB -1,48 3,30E-08 BSU34530 cotR -1,133 1,44E-05 BSU40300 rapG 0,64 2,35E-02 spoIIIAA -1,496 2,62E-07 BSU30920 cotI -1,417 1,27E-05 BSU14240 rok 0,973 5,59E-05 -1,405 5,32E-03 BSU36060 cotH -1,593 6,52E-06 BSU35500 degS 0,585 1,27E-02 -1,939 1,75E-11 BSU05550 cotP -1,437 3,85E-06 BSU03490 srfAB 0,418 1,93E-02 BSU07955 yfkA -0,725 4,83E-03 -1,705 1,30E-09 BSU11760 cotX -1,797 1,73E-06 BSU03480 srfAA 0,449 1,63E-02 BSU07920 yfkE -1,758 1,34E-03 -1,076 4,80E-07 BSU06890 cotJA -1,54 3,57E-07 BSU03510 srfAC 0,451 5,50E-03 BSU07930 yfkD -1,731 1,26E-03 -1,934 3,09E-07 BSU12090 cotT -1,588 2,91E-07 BSU03520 srfAD 0,503 1,64E-03 BSU07830 yfkO -2,956 2,90E-37 -0,709 1,46E-02 BSU11750 cotY -1,569 3,10E-08 BSU31710 comQ -0,792 1,22E-02 BSU07940 yfkC -0,503 4,46E-02 -0,814 3,87E-04 BSU11780 cotV -1,698 1,46E-08 BSU31690 comP -0,387 3,25E-02 BSU07790 yfkQ -1,623 4,19E-02 0,726 4,83E-02 BSU11740 cotZ -1,636 1,11E-09 competence BSU38750 cydB -1,947 2,06E-02 0,465 2,22E-02 BSU11730 cotO -1,959 1,63E-10 BSU35650 lytR 0,916 2,73E-07 BSU38740 cydC -2,068 2,58E-03 0,631 3,06E-06 BSU17030 cotE -2,049 1,53E-17 BSU31120 lytG 0,663 9,28E-03 BSU38730 cydD -1,96 3,10E-04 Sporulation genes BSU28110 spoVID -1,97 5,99E-17 BSU35630 lytB 0,676 1,35E-02 BSU05310 ydeR 9,896 4,47E-137 -1,077 4,79E-02 BSU11810 spoVIF -1,657 7,25E-03 autolytic genes BSU05300 ydeQ 7,999 1,14E-164 1,03E-02 BSU00560 spoVT -1,467 8,06E-13 BSU16220 fliG 0,717 3,49E-02 BSU05290 ydeP 6,859 3,82E-174

Frontiers in Microbiology | www.frontiersin.org

February 2016 | Volume 7 | Article 184

PJ69-a, pGBDU-C1 : Ura3 Gal4BD-tlpA Laboratory stock

Acknowledgements/ Remerciements/ Agradecimientos

ACKNOWLEDGMENTS

The authors would like to thank Christophe Grangeasse for his valuable comments on the manuscript.

ACKNOWLEDGEMENTS

We would like to thank the following for advice and/or technical assistance: Eleanor Dodson, Andrew Leech and Johan Turkenberg at York, and Stephen McGovern and Sara Santos at INRA. We acknowledge the use of the UMS3444 Protein Science platform and the access to beamlines SWING of the synchrotron SOLEIL and ID23EH1 at the ESRF.

generosity of a large number of people, including my friends, a ti, a quien me sin ti no lo habría conseguido.

En definitiva, a todas aquellas personas que creyeron en mí.

"This project has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 317338"

FUNDING

This work was supported by the grants from the Agence Nationale de la Recherche (2010( -BLAN-1303-01) -01) to MN and IM and the Vetenskapsrådet (2015-05319) and the Novo Nordisk Foundation to IM. TG is supported by the European Union, Marie Curie ITN AMBER, 317338. FUNDING European integrated project, BaSysBio; Biotechnology and Biological Sciences Research Council, UK Studentship (to K.H.J.); EU-Marie Curie Project AMBER FP7-People 317338 (to T.G.G.); CIBLE program 2011 from the région Rh ônes-alpes (to L.T., A.B.). Funding for open access charge: MICALIS INRA.

AUTHOR CONTRIBUTIONS

TG, SP, LS, and AD analyzed the bibliographic data. TG and SP provided the tables. MN wrote the manuscript. IM revised it critically. All authors gave final approval of the version.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

SUPPLEMENTARY DATA

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online. Conflict of interest statement. None declared.

negative charge at Y90 might alter the conformation of YabA in its C-terminal domain.

Furthermore, this conformational change is not expected to drastically affect the overall 3Dstrucutre of the YabA C-terminal domain since it preserve the ability to interact with DnaN.

Therefore, we propose that the phosphorylation of YabA at Y90 might be the base of the differential binding of YabA with DnaA and DnaN.

Unfortunately, we did not continue in this part of the project to focus on T71 phosphorylation and its effect in YabA function. Although the kinase responsible for phosphorylation of YabA at Y90 residue was not identified, this finding hinted at the possibility of a YabA-mediated control of initiation modulated by tyrosine phosphorylation in B. subtilis.

Supplementary materials and methods

Strains and plasmids

The B. subtilis strains used in this study are listed in Table 1.They were grown in Luria Bertani (LB) medium and the appropriate antibiotics were added when needed. E. coli strains were used for cloning experiments. Selection of E. coli transformants was carried out by using 100 g/ml ampicillin or 50 g/ml kanamycin. Saccharomyces cerevisiae strains were used for yeast-twohybrid assays as described below. Plasmids used in this study are given in Table 2 and corresponding oligonucleotides in Table 3. Cloning of DNA fragments at ectopic loci:

Plasmids (from E. coli sub-cloning) were integrated at the amyE loci by homologous recombination, using B. subtilis competent cells. Following the transformation step, selection was performed on antibiotic resistance plus amy minus phenotype tested on starch plate for integration at the amyE locus.

Creation of point-mutations with the pop-in pop-out method:

B. subtilis strains were constructed at the basis of the 168 (trp -) derivative CCBS185 containing a neomycin resistance gene under control of the Lambda Pr promoter (λPr-neo) inserted into the upp gene. The yabAY90F and yabAY90E mutants were constructed at locus by an improved mutation delivery approach (Fabret et al., 2002;Tanaka et al., 2012) as follows (Figure 6). The primers pair yabA fwd + oY90F rev (a) and oY90F fwd (b) + yabA rev were used to amplify the partially overlapping DNA fragments containing parts of the yabAY90F gene and the franking regions. Primers pair yabA fwd + oY90E rev (a) and oY90E fwd (b) + yabA rev were used for yabAY90E (Table 3). The insertion cassette encoding the Lambda CI repressor and the phleomycin-resistance gene was flanked by mutagenized yabA fragments in direct orientation by joining PCR using primers yabA fwd and yabA rev. The amplified fragment was used to transform CCBS185 cells with selection on phleomycin. The obtained clones were controlled for the loss of neomycin resistance, due to CI that inihibits the activation of the λPr promoter.

Homologous regions allow a recombination process that excises the cassette and the cells were selected for neomycin-resistance. (Hayashi et al., 2005 ;Noirot-Gros et al., 2002). De plus, des tests de complémentation entre un mutant de YabA déficient pour l'interaction avec DnaN (YabA-Nim) et un mutant déficient pour l'interaction avec DnaA (YabA-Aim) ont montré que YabA exerçait son activité en formant un complexe ternaire avec DnaA et DnaN et qu'en outre, ce complexe était associé à la machinerie de réplication pendant une grande partie du cycle réplicatif par l'intermédiaire de DnaN (Hayashi et al., 2005 ;Noirot-Gros et al., 2006). Par ailleurs, en absence de YabA, l'origine oriC est enrichie en DnaA. Le complexe DnaN-YabA exercerait ainsi un rôle de régulateur en séquestrant DnaA à la machinerie de réplication, qui s'éloigne de l'origine pendant la phase d'élongation, afin d'éviter les événements de ré-initiations (Katayama et al., 2010 ;Noirot-Gros et al., 2006 ;Soufo et al. , 2008). Afin d'éviter ces ré-initiations, YabA aurait inhiberait également la fixation de DnaA à l'origine. In vitro, YabA altère la capacité de DnaA à