Daniella M Scalice
email: daniella.m.scalice@nasa.gov

M A Chan

France Lagroix

Fieldwork D Scalice

J Ryan- Davis

CO-CREATING ETHICAL PRACTICES AND APPROACHES FOR FIELDWORK

published or not. The documents may come

Introduction:

After highly publicized incidents of unethical field sampling in summer 2021, geoscientists gathered for conversations about the importance of ethics in fieldwork at a Town Hall at the 2021 American Geophysical Union (AGU) Fall Meeting. The Town Hall was convened by the AGU Geomagnetism, Paleomagnetism and Electromagnetism Section and the Geological Society of America (GSA). A working group of Town Hall organizers and panelists was formed afterward, and has worked to disseminate the discussions and recommendations from the Town Hall, as well as develop and implement new activities as resources for considering ethics in our science.

Supporting Culture Change in the Geosciences: The Town Hall discussions highlighted expanding the discussion of ethics in geoscience to ensure Indigenous perspectives, knowledges, and relationship to the land are centered. Follow on conversations illuminated examples and resources for best practices in building relationship and collaboration between researchers and Indigenous communities. The dialogues also included how to manage sample collections-finding new ways to share samples rather then re-sampling-as well as devising new systems for proposing and reviewing studies to increase accountability. The development of new sampling protocols and techniques and incorporating those into foundational training in the geosciences was also a central theme. The major recommendation from the Town Hall centers on "equally valuing Indigenous and Western perspectives and knowledges, with actions that include building relationships with Indigenous communities, teaching ethical sampling protocols in geoscience curricula, and broadly implementing sharable sample archives" [1].

Developing Resources and Recommendations: It is clear that geologic sampling is a complex topic, relevant across multiple Earth science and planetary communities. In order to make appropriate recommendations, a GSA-administered survey was broadly distributed in fall 2022 to assess the culture and attitudes of sampling. The survey was co-sponsored by eight different geoscience related societies/committees, including GSA, AGU, and others [2]. The survey was followed by a GSA meeting forum to solicit community input, with a summary of highlights to be shared broadly across multiple groups [3].

Overall, there is consensus that the communities could use more resources on ethics, and advice on how to navigate appropriate permissions and interaction with stakeholders for both national and international field campaigns. In many cases, sampling can affect important geoheritage sites. Some formal statements already established by professional societies can provide guidance to amplify and evolve existing codes of conduct and possible statements on ethics of sampling for journal publications. Geologic materials ultimately need appropriate curation and archiving with metadata, in order to facilitate future sharing and exchange of information or samples.

It is also clear that unethical geological sampling is a long-standing issue. A workshop held at the 2022 AGU meeting developed educational resources from which best practices and considerations around respecting geological and cultural heritages could be discussed. Cases dominantly explored issues related to sampling on continental land, but also turned towards our oceans and remote sampling from satellites. The material will be packaged in order to start and support conversations of ethical sampling more broadly across communities globally.

Acknowledgments: We appreciate AGU Town Hall attendees, GSA forum attendees, and many survey respondents, who all contributed input that is incorporated into the ideas presented here. Billy M. Williams led the Town Hall session and continued conversations, and Eric A. Davidson moderated and contributed to organizing the Town Hall. We thank Sapóoq'is Wíit'as Ciarra Greene, Brooks Hanson, Vicki McConnell, the AGU Geomagnetism, Paleomagnetism and Electromagnetism Section, and the Geological Society of America for their roles in organizing the Town Hall. We acknowledge Melissa Kirven Brooks and Aaron Gronstal of NASA Ames Research Center, and the Geoethics Reading Group of Caltech for foundational discussions. We sincerely appreciate input from the AGU Indigenous Action Committee, and David Mogk's help with the GSA survey. Finally, we are grateful to Tom Heldal of the Norwegian Geological Survey and Eric Blinman, Mary Weahkee and Shelby Jones from the New Mexico Office of Archeological Studies (OAS) for their insightful talks at the AGU 2022 Workshop, to Suzanne McEnroe for co-organizing, and to AGU Ethics and Equity Center for sponsoring the AGU 2022 workshop.


Magali Ventroux

Marie-Francoise Noirot-Gros
email: marie-francoise.noirot-gros@inrae.fr

Marie-Francoise Noirot

Prophage-encoded small protein YqaH counteracts the activities of the replication initiator DnaA in Bacillus subtilis

Keywords: small ORF, SEP, skin element, DnaA, Spo0A, Bacillus subtilis

A small ORF peptide encoded by the gene-intervening element skin of Bacillus subtilis interacts with two major regulators counteracting their activities during vegetative and developmental cell cycles.

INTRODUCTION

small ORF (smORF)-encoded peptides (SEP) have emerged as a new class of small proteins widespread in both eukaryotic and prokaryotic genomes [START_REF] Albuquerque | small ORFs: a new class of essential genes for development[END_REF][START_REF] Couso | Classification and function of small open reading frames[END_REF][START_REF] Hellens | The emerging world of small ORFs[END_REF][4][START_REF] Storz | Small proteins can no longer be ignored[END_REF]. Due to their small size (for the smallest <30 aa or microproteins up to 100 aa) SEPs have often been overlooked during genome annotation. However, with advanced computation and ribosome profiling-based biochemical methods, small proteins are now more widely identified and some have been functionally characterized [4,[START_REF] Chu | Identification and characterization of sORF-encoded polypeptides[END_REF][START_REF] He | Enrichment-based proteogenomics identifies microproteins, missing proteins, and novel smORFs in Saccharomyces cerevisiae[END_REF][START_REF] Makarewich | Mining for Micropeptides[END_REF][START_REF] Straub | Cross-species genome-wide identification of evolutionary conserved microproteins[END_REF][START_REF] Vanorsdel | Identifying new small proteins in Escherichia coli[END_REF]. Growing evidence indicates that smORFs often encode bioactive peptides [START_REF] Chu | Identification and characterization of sORF-encoded polypeptides[END_REF][START_REF] Saghatelian | Discovery and characterization of smORF-encoded bioactive polypeptides[END_REF]. However, their contribution to cellular functions remains largely unexplored.

Small proteins act as regulators of diverse cellular processes in eukaryotes [START_REF] Staudt | Regulation of protein function by "micro-Proteins[END_REF]. In plants, characterized small proteins regulate transcription factors by sequestering them into non-functional states, preventing DNA binding or transcriptional activation [START_REF] Dolde | Synthetic microproteins: versatile tools for posttranslational regulation of target proteins[END_REF][START_REF] Graeff | Regulation of protein function by interfering protein species[END_REF]. In Drosophila melanogaster, smORFs represent about 5 % of the transcriptome and play an important role in controlling Drosophila development by triggering post-translational processing of transcriptional regulators [START_REF] Albuquerque | small ORFs: a new class of essential genes for development[END_REF][START_REF] Zanet | Pri sORF peptides induce selective proteasome-mediated protein processing[END_REF]. In humans, SEPs have been discovered with specific subcellular localization, suggesting they can fulfil biological functions [START_REF] Slavoff | Peptidomic discovery of short open reading frame-encoded peptides in human cells[END_REF]. For instance, a 69 aa long peptide, MRI-2, has been described to play a role in stimulating DNA repair through binding to the DNA end-binding protein complex Ku [START_REF] Slavoff | A human short open reading frame (sORF)-encoded polypeptide that stimulates DNA end joining[END_REF]. smORFs represent about 2 % of the genome of Saccharomyces cerevisiae [START_REF] Erpf | The long history of the diverse roles of short ORFs: sPEPs in fungi[END_REF]. Their function remains largely elusive but few have been identified to play regulatory roles in diverse physiological processes such as iron homeostasis [START_REF] An | The lateannotated small ORF LSO1 is a target gene of the iron regulon of Saccharomyces cerevisiae[END_REF] or DNA synthesis [START_REF] Erpf | The long history of the diverse roles of short ORFs: sPEPs in fungi[END_REF][START_REF] Chabes | Yeast Sml1, a protein inhibitor of ribonucleotide reductase[END_REF][START_REF] Lee | Dif1 is a DNA-damageregulated facilitator of nuclear import for ribonucleotide reductase[END_REF]. Genomic analysis of S. cerevisiae revealed that a substantial fraction of smORFs are conserved in other eukaryotes even as phylogenetically distantly related as humans, thus emphasizing their biological significance [START_REF] Kastenmayer | Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae[END_REF].

In prokaryotes, small proteins are encoded by 10-20 % of sRNA on average, and are often species-specific [START_REF] Vanorsdel | Identifying new small proteins in Escherichia coli[END_REF][START_REF] Friedman | Common and phylogenetically widespread coding for peptides by bacterial small RNAs[END_REF][START_REF] Miravet-Verde | Unraveling the hidden universe of small proteins in bacterial genomes[END_REF][START_REF] Yang | Identification and validation of novel small proteins in Pseudomonas putida[END_REF][START_REF] Zuber | A peptide profile of the Bacillus subtilis genome[END_REF]. SEPs with characterized functions are involved in various cellular processes [START_REF] Storz | Small proteins can no longer be ignored[END_REF]. In Pseudomonas aeruginosa, PtrA (63 aa long) and PtrB (59 aa long) repress the type III secretion system in response to DNA stress [START_REF] Ha | An in vivo inducible gene of Pseudomonas aeruginosa encodes an anti-ExsA to suppress the type III secretion system[END_REF][START_REF] Wu | PtrB of Pseudomonas aeruginosa suppresses the type III secretion system under the stress of DNA damage[END_REF]. In Escherichia coli, the 43 aa long peptide SgrT interferes with the PTS glucose transport system, allowing cells to utilize alternative non-PTS carbon sources to rapidly adapt to environmental changes in nutrient availability [START_REF] Lloyd | The small protein SgrT controls transport activity of the glucose-specific phosphotransferase system[END_REF]. In Bacillus subtilis, SEPs participate in regulating cell division and stress responses [START_REF] Ebmeier | Small proteins link coat and cortex assembly during sporulation in Bacillus subtilis[END_REF][START_REF] Handler | Peptide inhibitor of cytokinesis during sporulation in Bacillus subtilis[END_REF][START_REF] Schmalisch | Small genes under sporulation control in the Bacillus subtilis genome[END_REF]. A compelling example is the recently characterized developmental regulator MciZ (mother cell inhibitor of FtsZ), a 40 aa long peptide which prevents cytokinesis in the mother cell during sporulation [START_REF] Araújo-Bazán | Synthetic developmental regulator MciZ targets FtsZ across Bacillus species and inhibits bacterial division[END_REF][START_REF] Bisson-Filho | FtsZ filament capping by MciZ, a developmental regulator of bacterial division[END_REF]. About 20 % of the total core protein of the mature spores of B. subtilis is composed of the small acid-soluble spore proteins (SASPs) playing an important role in protecting DNA in the dormant spores [START_REF] Moeller | Roles of the major, small, acid-soluble spore proteins and sporespecific and universal DNA repair mechanisms in resistance of Bacillus subtilis spores to ionizing radiation from X rays and high-energy charged-particle bombardment[END_REF][START_REF] Setlow | I will survive: DNA protection in bacterial spores[END_REF]. Notably, several smORFs identified in intergenic regions were reported to be expressed during sporulation [START_REF] Schmalisch | Small genes under sporulation control in the Bacillus subtilis genome[END_REF]. The sporulation inhibitor sda encodes a 52 aa long protein, which acts as a checkpoint system coordinating DNA replication with sporulation initiation [START_REF] Burkholder | Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis[END_REF][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][START_REF] Rowland | Structure and mechanism of action of Sda, an inhibitor of the Histidine Kinases that regulate initiation of sporulation in Bacillus subtilis[END_REF]. Sda binds to the primary sporulation kinase KinA, preventing its activation as well as the subsequent phosphorelay-mediated activation of the master sporulation regulator Spo0A refer to growth or absence of growth on selective media, reflecting an interacting or loss-of-interaction phenotype, respectively. [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][START_REF] Veening | A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis[END_REF]. By linking DNA replication to a phosphorylation-dependent signalling cascade that triggers cellular development, this system illustrates an important biological role played by an SEP in blocking sporulation in response to DNA stress in Bacillus [START_REF] Veening | A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis[END_REF].

Small proteins encoded by phage or by prophage-like regions of bacterial genomes can hijack the host cellular machineries, as part of a strategy to shift host resources toward the production of viral progeny [START_REF] Duval | Small bacterial and phagic proteins: an updated view on a rapidly moving field[END_REF][START_REF] Liu | A bacteriophage transcription regulator inhibits bacterial transcription initiation by σ-factor displacement[END_REF][START_REF] Liu | The sabotage of the bacterial transcription machinery by a small bacteriophage protein[END_REF]. The bacteriophage T7 gene 2 encodes the 64 aa long gp2 protein essential for infecting E. coli. Studies of its biological role revealed that gp2 inhibits bacterial transcription by binding to RNA polymerase (RNAP), promoting a host-to-viral RNAP switch [START_REF] Nechaev | Purification and characterization of bacteriophage-encoded inhibitors of host RNA polymerase: T-odd phage gp2-like proteins[END_REF][START_REF] Savalia | The role of the T7 Gp2 inhibitor of host RNA polymerase in phage development[END_REF]. Another example is the 52 aa long protein ORF104 of phage 77 infecting Staphylococcus aureus. ORF104 is able to interfere with the host chromosome replication by binding to the ATPase domain of the helicase loader protein DnaI, thus preventing loading of the DNA helicase DnaC [START_REF] Hood | Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication[END_REF][START_REF] Liu | Antimicrobial drug discovery through bacteriophage genomics[END_REF].

In bacteria, DNA replication is initiated by the conserved initiator protein DnaA that assembles to the chromosomal replication origin to elicit local DNA strand opening [START_REF] Hwang | Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF[END_REF][START_REF] Leonard | Regulation of DnaA assembly and activity: taking directions from the genome[END_REF][START_REF] Mott | DNA replication initiation: mechanisms and regulation in bacteria[END_REF][START_REF] Ozaki | DnaA structure, function, and dynamics in the initiation at the chromosomal origin[END_REF]. This step triggers the coordinated assembly of the proteins that will further build a functional replication fork, from the DNA helicase, unwinding the DNA duplex, to the many components of the replisome that form the replication machinery [START_REF] Messer | The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication[END_REF]. In addition to its initiator activity, DnaA acts as a transcription factor repressing or activating genes [START_REF] Goranov | A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA[END_REF][START_REF] Messer | DnaA as a transcription regulator[END_REF][START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF]. The activity of the initiator DnaA is tightly controlled to coordinate chromosomal replication initiation with other cellular processes during the bacterial cell cycle [START_REF] Katayama | Regulation of the replication cycle: conserved and diverse regulatory systems for DnaA and oriC[END_REF][START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF]. Part of this control is mediated by protein-protein interactions and involves various protein regulators that bind DnaA and affect its activity [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF][START_REF] Jameson | Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli[END_REF][START_REF] Katayama | The DnaA Cycle in Escherichia coli: activation, function and inactivation of the initiator protein[END_REF][START_REF] Riber | Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli[END_REF][START_REF] Skarstad | Regulating DNA replication in bacteria[END_REF]. In B. subtilis, four proteins, SirA, Soj, DnaD and YabA, have been identified to regulate DnaA activity or its assembly at oriC through direct interaction [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF][START_REF] Bonilla | The primosomal protein DnaD inhibits cooperative DNA binding by the replication initiator DnaA in Bacillus subtilis[END_REF][START_REF] Martin | DNA replication initiation in Bacillus subtilis: structural and functional characterization of the essential DnaA-DnaD interaction[END_REF][START_REF] Murray | Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA[END_REF].

Phage SEPs targeting essential functions for bacterial survival are regarded as promising antimicrobial peptides, and have ignited a strong interest in their identification and characterization [START_REF] Hood | Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication[END_REF][START_REF] Liu | Antimicrobial drug discovery through bacteriophage genomics[END_REF]. The phage-like element skin of B. subtilis encodes about 60 proteins. The skin element is repressed under most physiological conditions [START_REF] Nicolas | Conditiondependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF], silenced by the skin repressor SknR (Fig. 1a) [START_REF] Kimura | Repression of sigK intervening (skin) element gene expression by the CI-like protein SknR and effect of SknR depletion on growth of Bacillus subtilis cells[END_REF]. The excision of skin from the genome is mediated by the self-encoded site-specific recombinase CisA and restores the integrity of the sigK gene encoding the late sporulation factor σ K [START_REF] Kunkel | The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene[END_REF]. Among the skin ORFs, yqaH encodes the 85 aa long polypeptide YqaH previously identified to bind to the replication initiator protein DnaA in a yeast two-hybrid genomic screen [START_REF] Marchadier | An expanded protein-protein interaction network in Bacillus subtilis reveals a group of hubs: exploration by an integrative approach[END_REF][START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF]. When yqaH is overexpressed, Bacillus cells exhibit aberrant nucleoid morphological defects suggestive of replication deficiency [START_REF] Kimura | Repression of sigK intervening (skin) element gene expression by the CI-like protein SknR and effect of SknR depletion on growth of Bacillus subtilis cells[END_REF]. In this study, we further characterized the function of YqaH in antagonizing DnaA activities. In addition, we found that YqaH also interacts with the master regulator Spo0A involved in developmental transitions to sporulation and biofilm formation [START_REF] Dubnau | A protein complex supports the production of Spo0A-P and plays additional roles for biofilms and the K-state in Bacillus subtilis[END_REF]. As with DnaA, Spo0A is a DNA-binding protein, which, in its activated phosphorylated form (Spo0A-P), controls the expression of numerous genes during the early stages of sporulation [START_REF] Molle | The Spo0A regulon of Bacillus subtilis[END_REF]. To better understand the biological role of this smORF in B. subtilis we performed a functional dissection of YqaH. Using a reverse yeast two-hybrid system, we selected yqaH alleles that selectively disrupted the YqaH/DnaA complex. This approach allowed us to link specific DnaA loss-of-interaction with loss-of-function phenotypes related to replicative stress. Interestingly, it also highlighted an intricate role of both DnaA and Spo0A in YqaHmediated sporulation phenotypes.

METHODS

Strains, plasmids and primers

Experiments were performed in B. subtilis strains 168 or BSBA1 (168 trpC2). S. cerevisiae PJ69-4a or α strains were used for yeast two-hybrid experiments [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF]. All strains are listed in Table S1A (available in the onlive version of this article). E. coli strain DH10B [START_REF] Durfee | The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse[END_REF] was used as a cloning host. Plasmid constructs are listed in Table S1B. Primers are listed in Table S2. Sequences of interest cloned or mutated in this study were verified by DNA sequencing.

Bacteria growth

Experiments were conducted at 37 °C in LB medium containing the necessary antibiotics such as ampicillin, 100 µg ml -1 (in E. coli); spectinomycin, 60 µg ml -1 ; kanamycin, 5 µg ml -1 ; chloramphenicol, 5 µg ml -1 or erythromycin, 1 µg ml -1 associated with lincomycin, 25 µg ml -1 (for B. subtilis). B. subtilis strains containing pDG148 and pDG148-yqaH plasmids were grown overnight in LB containing kanamycin. To investigate the effect of yqaH expression during exponential phase growth, ON cultures were diluted at OD 600 of 0.01 onto fresh LB (+Kn) media and grown to mid-exponential phase (OD 600 0.3-0.4). The cultures were then further diluted in LB (+Kn) media containing 0.5 mM IPTG at OD 600 of 0.01 and 200 µl of cultures were then transferred to a 96-well microplate reader to monitor OD 600 at 37 °C for 18 h.

Strain constructions yqaH overexpression

The yqaH wild type (WT) and mutated gene derivatives were PCR-amplified using the yqaH-HindIII-RBS-F/pYqaH-R primer pair and inserted in plasmid pDG148 between HindIII and SalI restriction sites to place yqaH under control of the IPTG-inducible Pspac promoter [START_REF] Stragier | Processing of a sporulation sigma factor in Bacillus subtilis: How morphological structure could control gene expression[END_REF]. The plasmid constructs were then extracted from E. coli and transformed into B. subtilis. To assess expression of YqaH variants, YqaH WT and mutated proteins were expressed as 3Flag-fusion proteins.

Yeast two-hybrid plasmid constructs

Genes encoding full size YqaH, Spo0A, YabA and DnaA were translationally fused to the activating domain (AD) or the binding domain (BD) of the transcriptional factor Gal4 by cloning into pGAD and pGBDU vectors, respectively [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF]. DNA fragments were amplified by PCR using appropriate primer sets (Table S2), double digested by EcoRI and SalI and ligated to the corresponding pGAD or pGBDU linearized vector to generate a translational fusion with Gal4-AD or BD domains. pGAD-and pGBDU-plasmid derivatives were selected onto SD media lacking leucine (SD-L) or uracyl (SD-U), respectively [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF]. Plasmid constructs were first transformed into E. coli prior to be introduced in the haploid yeast strain PJ69-4α (pGAD-derivatives) or PJ69-4a (pGBDU-derivatives). Truncated dnaA fragments (boundaries as illustrated in Fig. 1d) were translationally fused to the AD of Gal4 by gap repair recombination in yeast [START_REF] Weir | PCR mutagenesis and gap repair in yeast[END_REF]. DNA fragments were amplified by PCR using appropriate primer sets (Table S2) to generate 50 bp of flanking homology with the linearized recipient vector pGAD on both sides of the dnaA fragments. PJ69-4α was co-transformed with the PCR fragments containing the truncated dnaA domains and linearized pGAD to give rise to pGAD-truncated dnaA fusions by in vivo recombination.

Sporulation conditions

Sporulation of B. subtilis was induced by nutrient limitation and re-suspension in Sterlini-Mandelstam medium (SM) [START_REF] Sterlini | Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance[END_REF]. The beginning of sporulation (t 0 ) is defined as the moment of re-suspension of the cells in SM medium. To study the effects of yqaH overexpression on B. subtilis sporulation, ON cultures of strains containing the pDG148 constructions were diluted in CH medium at a starting OD 600 of 0.05. When cultures reached an OD 600 of 1, cells were re-suspended in an equivalent volume of SM medium supplemented with 0.5 mM IPTG (t 0 ). To monitor sporulation efficiency, cells were collected at different times after sporulation induction (t 0 ) until t 6 (6 h after induction, defined as a stage of production of mature spores) and t 18 . Asymmetric septa and spores were enumerated (n≥500 cells) by microscopic observations from all samples.

Yeast two-hybrid assay

The yeast two-hybrid assays were performed as described [START_REF] Marchadier | An expanded protein-protein interaction network in Bacillus subtilis reveals a group of hubs: exploration by an integrative approach[END_REF][START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF][START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[END_REF]. PJ69-4a and α haploid yeast strains transformed by pGADand pGBDU-plasmid derivatives were mixed onto YEPD-rich media plates to allow formation of diploids. Diploids containing both pGAD-and pGBDU-type plasmids were then selected on SD-LU and interacting phenotypes were monitored by the ability of diploids to grow on SD-LUHA medium further lacking histidine (H) or adenine (A).

Generation of loss of interaction (LOI) mutation

YqaH DnaA_LOI mutants were identified using a yeast two-hybrid-based assay as described elsewhere [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF][START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[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][START_REF] Quevillon-Cheruel | Structure-function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation[END_REF]. Random mutagenesis of the targeted genes was achieved by PCR amplification under mutagenic conditions that promote fewer than one mis-incorporation per amplification cycle [START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. For yqaH, a library of mutated pGAD-yqaH* was constructed by gap-repair recombination into yeast (PJ69-4α strain). About 1000 individual transformants were organized in 96-well format on plates containing a defined medium lacking leucine (-L) to form an arrayed collection of AD-yqaH* gene fusion mutants. This organized library was then mated with PJ69-4a strains containing either pGBDU-dnaA or pGBDU-spo0A, or an empty pGBDU plasmid as a negative control. Selective pressure for interacting phenotypes was then applied on media lacking -LUH or -LUA. Diploids that failed to grow on interaction-selective media were considered as potentially expressing an LOI mutant of yqaH. Importantly, any particular AD-YqaH*-LOI protein unable to trigger interacting phenotypes in the presence of BD-DnaA while still producing interacting phenotypes when expressed in the presence of BD-Spo0A is defined as a DnaA-specific LOI mutant. The corresponding yqaH-LOI genes were retrieved from the initially organized haploid library and the mutations were identified by DNA sequencing. Only mutations resulting from single substitutions were considered.

Ori/Ter ratio determination

The ratios of origin-proximal and terminus-proximal DNA sequences were determined by quantitative PCR (qPCR). ON cultures of B. subtilis strains containing pDG148 or pDG148-yqaH plasmid derivatives were first diluted to OD 600 of 0.01 in LB supplemented with kanamycin (5 µg ml -1 ) and grown at 37 °C up to mid-exponential phase (OD 600 0.3-0.4). Then the cultures were secondly diluted at OD 600 of 0.02 in LB+kanamycin media supplemented with 0.5 mM IPTG and grown again up to an OD 600 of 0.3-0.4 at 37 °C. Culture samples were collected and mixed with 1/2 volume of sodium azide solution (5 mM final) to stop all metabolic activities prior subjecting them to total lysis. Aliquots of total genomic DNA extracts were kept at -80 °C and were only used once after defrosting. Quantitative real-time PCRs were performed on a Mastercycler ep realplex (Eppendorf) thermocycler device using ABsolute Blue QPCR SYBR Green ROX Mix (ABgene), to amplify specific origin (Ori) or terminus (Ter) proximal sequences. Primers used for sequence amplification were chosen using the Primer3Plus program (http://www.bioinformatics.nl/ cgi-bin/primer3plus/primer3plus.cgi/). Amplification using the ORI pair of primers (oriL3F and oriL3R, Table S2) targeting the 4212889-4211510 region of the B. subtilis chromosome yields a 128 bp size product corresponding to sequence at the left side of the origin. The terminus sequence is a 122 bp long fragment obtained from the Ter pair of primers (terR3F and terR3R, Table S2) amplifying the region 2016845-2017711 at the right side of the terminus. The two primer pairs Ori and Ter exhibited ≥95 % amplification efficiency. Data analysis was performed using the software Realplex (Eppendorf) and quantification was done with the ΔΔCt method.

Gene expression analysis by qPCR

To monitor the expression of the DnaA-regulated gene dnaA, samples of cultures (generated as described for Ori/Ter ratio measurements) were harvested in exponential growth phase at OD 600 of 0.3-0.4, and RNA extractions were performed. To quantify the expression of Spo0A-regulated genes spoIIE and spoIIGA, cells were harvested between stages t 2 and t 3 of sporulation in SM medium. In both cases, a determined volume of culture was mixed with 1/2 volume of sodium azide solution (5 mM final), then collected by centrifugation and subjected to lysis followed by total RNA extraction as described [START_REF] Nicolas | Conditiondependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF]. Total RNA was reverse transcribed and quantitative real-time PCR was performed on cDNA. Primers used for sequence amplification were chosen using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) and are detailed in Table S2. The primer pairs exhibited ≥95 % amplification efficiency. Data analysis was performed using the software Realplex (Eppendorf) and the quantification was done with the ΔΔCt method.

Real-time in vivo monitoring of spoIIG gene expression by luminescence

The promoter of spoIIG fused to the firefly luciferase gene luc [START_REF] Mirouze | Fluctuations in spo0A transcription control rare developmental transitions in Bacillus subtilis[END_REF] was transferred in the BSBA1 background strains by transformation with total genomic DNA. Cell growth was monitored in a 96-well microplate under agitation at 37 °C using a microplate reader (Biotek Synergy2). Luminescence as well as OD 600 were recorded every 5 min. Luminescence signals were expressed as relative luminescence units (RLU) normalized to the bacterial OD. For monitoring of spoIIG expression during sporulation, precultures of strains harbouring plasmid pDG148-yqaH wild-type and mutated derivatives were first performed in LB supplemented with kanamycin (10 µg ml -1 ) over 1 day. Cells from precultures were then inoculated in CH media at very high dilution so they could reach an OD 600 of 1 after 12 h (ON). Then cells were re-suspended in SM medium supplemented with 0.5 mM IPTG and inoculated in 96-well microplates in the presence of luciferin (1.4 mg ml -1 ). The cultures were incubated at 37 °C under agitation in a plate reader equipped with a photomultiplier for luminometry. RLU and OD 600 were measured at 5 min intervals.

Fluorescence microscopy

For observation of nucleoids, cells were grown and harvested as described for Ori/Ter measurements, then rinsed in a minimal transparent media, and stained with FM4-64 dye (to stain the bacterial membranes) and DAPI (to stain the nucleoids) prior to being mounted onto 1.2 % agarose pads. Fluorescence microscopy was performed on a Leica DMR2A (100× UplanAPO objective with an aperture of 1.35) coupled with a CoolSnap HQ camera (Roper Scientific). System control and image processing were achieved using Metamorph software (Molecular Devices). Counts of cells, spores, foci or nucleoids were determined with the ImageJ software, from at least 500 cells.

Biofilm assay

Production and analysis of air-to-liquid biofilm pellicles were performed as already described [START_REF] García | Phosphorylation of the Bacillus subtilis replication controller YabA plays a role in regulation of sporulation and biofilm formation[END_REF]. Briefly, strains expressing yqaH, wild-type or K17E mutant derivative as well as the control strain were grown in LB to an OD 600 of 1.0 and inoculated in 12-well culture plates containing 3.5 ml of MSgg media at a starting OD 600 of 0.1. Cultures were maintained at 28 °C and 70 % humidity, with no agitation. After 48 h, wells were filled with MSgg media (slowly added at the edge) to lift the biofilm pellicles up to the top of the wells. The pellicles were then peeled off onto a 2.5 cm diameter circular cover slide. The cover slides with intact biofilm pellicles were mounted on an Attofluor Cell Chamber and stained with the Film Tracer 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-Light confocal imager; objectives Nikon CFI Plan Fluor 10×, DIC, 10×/0.3 NA (WD=16 mm); excitation was performed at 470 nm and emission recorded at 505 nm]. Images were processed using IMARIS software (Bitplane). Biofilm images were quantified using the surface function in IMARIS (XTension biofilm) to derive biovolumes [total volume (μm 3 ) per area (μm 2 )], cohesiveness (number of discontinuous components in the area) and mean thickness (µm). Parameters were averaged from eight samples. Pairwise comparisons were performed using the Tukey method (*P≤0.05, **P≤0.01, ***P≤0.001).

Protein extraction and immunodetection

Production of 3Flag-YqaH mutated derivatives was determined from cell lysates of B. subtilis followed by immunodetection using the monoclonal antibody anti-FLAG M2 (Sigma). For cell lysates, cells expressing the 3Flag-YqaH derivatives (OD 600 0.15) were grown in the presence of IPTG for 1 h and collected. Cells were ressuspended in lysis buffer (10 mM Tris pH 7.7, 200 mM NaCl, 1 mM EDTA) and lysed by sonication. Lysates were first centrifuged at 20 000 g for 20 min at 4 °C. The Bradford method was used to estimate the quantity of proteins within soluble fractions prior to loading on a SDS-PAGE 12.5 % polyacrylamide gel for electrophoresis separation. To assess YqaH solubility, the remaining supernatant was further subjected to ultra-centrifugation (100 000 g, 1 h, at 4 °C), and an equivalent amount of proteins (40 ng, assayed by the Bradford method) from the high-speed soluble phase was again loaded onto the SDS-PAGE gel. Proteins were electrophoretically separated and transferred to PVDF membranes. YqaH was detected using a monoclonal anti-FLAG M2 antibody (F2135; Sigma). An IgG goat secondary antibody (A2304; Sigma) conjugated to peroxidase was used at 1:10 000 to detect the anti-FLAG M2. Protein immunodetection was performed using the Clarity Western ECL kit (Bio-Rad) according to the supplier's indications followed by chemiluminescence detection (ChemiDoc imager; Bio-Rad). Images were analysed with the software Image Lab.

Structure predictions

Secondary structure predictions were performed with the computer server Jpred 3 (http://www.compbio.dundee.ac.uk/wwwjpred/) and the 3D structure was predicted using Alphafold [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF] (https://alphafold.ebi.ac.uk).

RESULTS

Skin element smORF-encoded YqaH interacts with DnaA and Spo0A

YqaH was originally identified to interact with the replication initiator DnaA in a yeast two-hybrid screen of a B. subtilis genomic library [START_REF] Noirot-Gros | An expanded view of bacterial DNA replication[END_REF]. Interestingly, in a subsequent screen targeting the sporulation transcriptional factor Spo0A, we also identified YqaH as a binding partner (Fig. 1b). Other DnaA-binding proteins such as SirA, Soj, DnaD and YabA exert regulatory functions by binding to different functional domains of DnaA (Fig. 1b,d). We characterized the domain of DnaA able to elicit the interaction phenotypes with YqaH in a yeast two-hybrid binary assay. After testing several protein fragments spanning various DnaA domains, we delineated the C-terminal domain IV as necessary and sufficient for interaction with YqaH (Fig. 1c,d). This 118 The YqaH protein is predicted to fold into three αhelices (http://www.compbio.dundee.ac.uk./wwwjpred/). Single-substituted amino acids are highlighted in red and blue, and the nature of the substitutions is indicated beneath. Upper case indicates an LOI phenotype monitored on both -His and -Ade selective media. Lower case indicates that the LOI phenotype has been obtained on the most selective -Ade media only but not on -His media, indicative of a partial loss of interaction. The four mutations highlighted in blue are mapped on the cartoon structure shown in (c). (b) YqaHLOI mutational screen. Amino acid substitutions affecting interaction with DnaA and/or Spo0A are indicated with their associated interaction phenotype. (+) indicates unchanged interaction phenotypes compared to YqaH WT, and characterized by a full ability of diploids to grow on both selective -LUH and -LUA media. (-) refers as a total loss of interaction phenotype leading to absence of growth on both -LUH and -LUA media. (+/-) refers as a partial loss of interaction leading to some growth on -LUH but not -LUA; see also Fig. S1 for additional explanation. (c) Top: mapping of four substituted residues (green sticks) on the cartoon representation of an Alphafold-predicted YqaH structure (per-residue confidence score PLDDT>90) [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF], visualized by PyMOL (2.4.1). Bottom: electrostatic surfaces and charge distribution (APBS software package into PyMOL) with ±5 kT e -1 electrostatic potential plotted on the solvent-accessible surface. The charge of substituted amino acids is indicated.

aa long fragment spans residues 328-446, and carries the signature motif for DnaA binding to dsDNA. This result distinguishes YqaH from the other regulators SirA, Soj, YabA and DnaD that inhibit DnaA binding to oriC, through interacting with the central AAA+ATPase domain III (aa 111-328, Fig. 1d). SirA also inhibits DnaA oligomerization by interacting with domain I. The ability of YqaH to interact with two key players of bacterial vegetative growth and transition to sporulation development prompted us to further explore the biological functions of YqaH .

Induction of yqaH expression triggers phenotypes similar to those of a DnaA mutant

The interaction of YqaH with DnaA hints at a potential role in replication initiation control. We first investigated the effect of yqaH overexpression on cell growth and viability. All B. subtilis strains either carrying a plasmid expressing yqaH under control of an IPTG-inducible promoter or carrying a control plasmid without the yqaH gene were treated under identical conditions. Addition of IPTG at an early exponential stage specifically halted cell growth after 2 h in cells expressing yqaH (Fig. 2a). We found that cell viability was affected almost instantly in the presence of yqaH, leading to an about 30-fold decrease after 3 h (Fig. 2a). Overexpression of yqaH also affects cell morphology, causing significant filamentation (Fig. 2b). A closer examination of nucleoids revealed aberrant segregation of chromosomes with both diffused and compacted nucleoids unequally distributed within the filamented cells, as well as a large portion of cells with no DNA. Most importantly, septum-entrapped nucleoids were also observed, evocative of a nucleoid occlusion defect [START_REF] Wu | Nucleoid occlusion and bacterial cell division[END_REF]. These observations highlighted that the synthetic overexpression of yqaH from a plasmid triggered a large panel of chromosomal disorders evocative of a replication and/or segregation stress, as described previously [START_REF] Kimura | Repression of sigK intervening (skin) element gene expression by the CI-like protein SknR and effect of SknR depletion on growth of Bacillus subtilis cells[END_REF]. Using an epitope-tagged YqaH protein, we showed that YqaH was immuno-detected in cells carrying the IPTG-inducible yqaH gene but not in the control, indicating that the chromosomal copy of yqaH within the skin element remained completely silent under our experimental conditions (Fig. S2).

We then investigated the role of YqaH on DnaA-dependent transcriptional regulation. Among the genes of the DnaA-regulon, DnaA negatively regulates its own expression by binding to the dnaA promoter region [START_REF] Goranov | A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA[END_REF][START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF][START_REF] Merrikh | Control of the replication initiator DnaA by an anti-cooperativity factor[END_REF]. We monitored the dnaA mRNA levels in the presence or absence of YqaH and showed that expression of yqaH led to a 2-fold increase in dnaA mRNA, consistent with a regulatory defect (Fig. 2c). Taken together, these results are in agreement with a role of YqaH in counteracting DnaA activity.

Induction of yqaH expression impairs sporulation

We investigated the possible role of YqaH in Spo0A functions, by examining the effect of yqaH overexpression on sporulation. Cells carrying plasmids with or without the yqaH gene were induced into sporulation by the re-suspension method.

To prevent an inhibitory effect of YqaH during vegetative growth, yqaH gene expression was induced only at the onset of sporulation (t 0 ) and spore formation was monitored over time. Appearance of asymmetric septa at an early stage (t 2.5 ) was imaged by fluorescence microscopy after staining by a red-fluorescent membrane dye, while the engulfed forespore (t 6 and t 18 ) was revealed using bright-field microscopy (Fig. 3a). The counts of spore-forming bacteria were drastically reduced in the presence of YqaH (Fig. 3b). We also examined the effect of yqaH overexpression on Spo0A-mediated transcriptional regulation. Among genes under control of Spo0A are spoIIE, encoding the protein serine phosphatase SpoIIE, and spoIIGA, encoding a pro-σ E -processing protease SpoIIGA [START_REF] Molle | The Spo0A regulon of Bacillus subtilis[END_REF]. SpoIIE and SpoIIGA are involved in activation of the alternative sporulation sigma factors σ F and σ E in the forespore and mother cell compartments, respectively [START_REF] Baldus | Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes[END_REF][START_REF] Bradshaw | A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases[END_REF][START_REF] Errington | Cell cycle machinery in Bacillus subtilis[END_REF][START_REF] Fujita | High-and low-threshold genes in the Spo0A regulon of Bacillus subtilis[END_REF]. Examination of their expression levels at the onset of sporulation revealed a significant downregulation (about 7-and 8-fold, respectively) in the presence of yqaH (Fig. 3c). Together, these results pointed to a negative effect of YqaH during sporulation, supporting the notion that YqaH could act by inhibiting Spo0A activity.

Functional dissection of YqaH

To further decipher YqaH's mode of action, we searched for YqaH point mutations able to separate its interactions with DnaA and Spo0A. Using a yeast two-hybrid-based assay, we screened an YqaH mutant library for specific LOI phenotypes (Fig. S1). This approach is used to select single amino acid changes in YqaH that selectively disrupt the interaction with only one partner while maintaining proficiency for interaction with the other partner. Such a screening allows the identification of substitutions of residues preferentially located at the protein-protein interface, preserving the global 3D-structure of YqaH, and leading to an LOI phenotype specific for the targeted partner [START_REF] Noirot-Gros | Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis[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][START_REF] Quevillon-Cheruel | Structure-function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation[END_REF]. We identified three single-residue substitutions, K17E, E38K and K48E, in YqaH that elicited a complete LOI phenotype with DnaA without affecting interaction with Spo0A (Fig. 4a,b). These three replacements were all charge-changing substitutions (Fig. 4c). Six additional substitutions affecting residues R16G, M27T, Y37H, E38V A44V and R56W were only partially abolishing interaction phenotypes with DnaA (Fig. 4a,b). Two substitutions, S25T and A40T, were found to partially affect both interaction with DnaA and Spo0A, and five substitutions, D10G, S20L, L28P, L43P and L57P, were totally abolishing interaction phenotypes with both DnaA and Spo0A (Fig. 4b). In the latter case, these substitutions are likely to affect the overall integrity of the YqaH 3D-structure. The YqaH protein structure is predicted to fold into two successive alpha helices separated by a helix-turn-helix (HTH) structural motif (Fig. 4a,c). The residues important for interaction with DnaA localized within the two main helices, presumably involved in a coiled-coil structure (Fig. 4c). Mapping of the charge distribution on the YqaH model shows that the HTH fold is enriched with negatively charged amino acids (Fig. 4c). It is worthy of note that, although several specific DnaA_LOI mutants have been obtained, no substitution that specifically prevented interaction with Spo0A was identified in our screens.

DnaA_LOI mutants of YqaH restore replication and transcriptional regulation defects caused by yqaH overexpression

We investigated the effect of two DnaA_LOI mutants of YqaH carrying substitutions K17E or R56W and exhibiting total (K17E) or partial (R56W) LOI with DnaA in our yeast two-hybrid assay (Fig. 4b). We first verified production levels of the YqaH mutant proteins in the cells upon overexpression from the plasmid compared to the wild-type (Fig. S2). The cellular amounts of YqaH-K17E and R56W were similar to that of the WT and remained soluble after clarification by ultracentrifugation at 100 000 g indicating that the presence of amino acid substitutions did not notably affect the protein stability. (Fig. S2A,B). Examination of the nucleoid morphology showed that both substitutions abolished the nucleoid segregation and condensation defects resulting from expression of the wild-type yqaH observed in Fig. 2b (Fig. 5a). We further examined the effect of YqaH mutants on replication initiation by quantifying the chromosomal origin-proximal and terminus-proximal sequences (Fig. 5b). The average Ori/ Ter ratio was determined by qPCR on genomic DNA harvested from exponentially growing cells as previously described [START_REF] Soufo | Cell-cycle-dependent spatial sequestration of the DnaA replication initiator protein in Bacillus subtilis[END_REF]. The Ori/Ter ratio was about 4 in control cells that did not express yqaH, indicating that two events of replication initiation have taken place in most cells, in agreement with previous observations under similar experimental conditions [START_REF] Murray | Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis[END_REF]. In the presence of yqaH, the Ori/Ter ratio dropped below 2, indicating that less than one replication initiation event per cell has taken place on average (Fig. 5b). This suggests that the YqaH protein exerts a negative effect on replication initiation by counteracting DnaA activity. In the presence of YqaH-DnaA_LOI variants, the Ori/Ter ratio in the cell population was restored (Fig. 5b). The average Ori/Ter ratio was similar to that in control cells upon expression of yqaH-R56W. However, expression of yqaH-K17E restored about 75 % of the initiation rate, indicating a partial although significant complementation. This difference between YqaH variants may be due to a differential LOI with DnaA in cells. We decided to analyse further the number of chromosomal origins in individual cells using the YqaH-K17E mutant. Cells carrying a lacO repeat array near the replication origin and expressing the GFP-LacI fusion were observed by fluorescence microscopy, in the presence or absence of YqaH (Fig. 5c). In the absence of YqaH, the number of LacI foci per nucleoid was 3.6 on average, in agreement with the Ori/Ter ratio (Fig. 5b). This number dropped to 1 on average upon ovrexpression of yqaH, indicating a strong replication deficit. Cells expressing yqaH also exhibited expected aberrant nucleoids and segregation defects. Expression of the yqaH-K17E mutant partially restored replication defects with an average of GFP-LacI foci of about 2.3 and fully restored nucleoid and cell morphology. Together, these observations indicate that YqaH antagonizes DnaA activity in replication initiation by binding to DnaA.

We also examined the effect of the DnaA_LOI mutants on dnaA mRNA abundance. Similarly to Fig. 2c, expression of yqaH increased the abundance of dnaA mRNA relative to control by 2-fold (Fig. 6). In contrast, dnaA mRNA levels detected in strains expressing the yqaH-K17E and R56W variants were similar to the level detected in cells expressing yqaH-WT (Fig. 6). This indicated that YqaH binding to DnaA also antagonized DnaA activity in transcriptional regulation.

YqaH-mediated inhibition of sporulation and biofilm formation requires interaction with DnaA

Although we did not identify any YqaH mutant that specifically lost interaction with Spo0A (Spo0A_LOI) in our yeast two-hybrid screen, we reasoned that the DnaA_LOI mutant YqaH-K17E, which remains fully proficient for interaction with Spo0A, could still provide a way to investigate a potential effect of YqaH on sporulation phenotypes. Indeed, despite a slightly reduced Ori/Ter ratio, overexpression of yqaH-K17E allows normal vegetative cell growth without the DnaA-related chromosome segregation defects (Fig. 5). We compared the sporulation efficiencies of strains expressing yqaH wild-type or the K17E variant. The percentage of cells with a polar septum or engulfing spore was determined by microscopy at t 6 (i.e stage VI, referred as the spore maturation stage). Surprisingly, we found that YqaH-K17E completely restored the sporulation defect mediated by YqaH WT (Fig. 7a) suggesting that the interaction with DnaA could be also responsible for loss of the sporulation phenotype. To further investigate the potential role of YqaH in Spo0A-related processes, we also examined its effect on biofilm formation (Fig. S3). We observed that the production of biofilm pellicles at the air-liquid medium interface was impaired in strains expressing yqaH, leading to a loss of biomass and cohesion. Damaged biofilm pellicles were not observed in cells expressing the DnaA_LOI mutant YqaH-K17E (Fig. S3A,B). As yqaH expression also affects chromosome segregation and vegetative growth, this finding suggests an indirect role of DnaA during biofilm formation.

The sporulation inhibitor Sda is not responsible for YqaH-mediated defects in sporulation

In B. subtilis, most of the DnaA-mediated transcriptional regulation of sporulation genes is indirect and mediated by the protein Sda, as evidenced by genome-wide expression analysis [START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF]. The Sda protein exerts control over sporulation by inhibiting the phosphorylation activity of KinA, required to activate the sporulation regulator Spo0A [START_REF] Burkholder | Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis[END_REF][START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF]. We investigated whether Sda played a role in the YqaH-dependent defect in sporulation by examining the expression of the Spo0A-driven promoter P spoIIG fused with the firefly luciferase reporter gene (luc), in the presence or absence of sda (Fig. 7b-d). In the absence of YqaH, the expression of P spoIIG-luc increased with time to reach a maximum 3 h after induction of sporulation in both sda+ and Δsda backgrounds. A higher expression level was observed in the Δsda strain, in agreement with increased levels of Spo0A-P [START_REF] Hoover | Changes in DnaAdependent gene expression contribute to the transcriptional and developmental response of Bacillus subtilis to manganese limitation in Luria-Bertani medium[END_REF] (Fig. 7b-d). Expression of yqaH led to a similarly strong reduction of luminescence signal in both strains, illustrative of an spoIIG expression decrease (Fig. 7b-d). By comparison, spoIIG expression was abolished in the absence of spo0A, as already described [START_REF] Fujita | High-and low-threshold genes in the Spo0A regulon of Bacillus subtilis[END_REF][START_REF] Seredick | The Bacillus subtilis response regulator Spo0A stimulates sigmaA-dependent transcription prior to the major energetic barrier[END_REF](Fig. 7b). This indicates that in our experimental condition, the overexpression of yqaH is not sufficient to completely counteract the activity of Spo0A. This observation is also corroborated by the strong reduction of sporulation efficiency in both sda+ and Δsda strains (Fig. 7a ande). These results pointed to an sda-independent inhibition of sporulation mediated by YqaH. However, the decrease of spoIIG expression caused by YqaH WT was only partly compensated for by the DnaA_LOI YqaH-K17E mutant, suggesting that interaction of YqaH with DnaA may have a moderate role in the inhibition of Spo0A activity. Yet, the K17E mutation fully abolished the YqaH-dependent sporulation defect in sda+ and Δsda strains, as measured by the ratio of cells containing asymmetric septa or engulfed forespores 6 h after initiation of sporulation (Fig. 7a ande). Together, these results indicated that Sda was not involved in the DnaA-mediated response to sporulation and suggested a combined role of DnaA and Spo0A in YqaH-mediated sporulation phenotypes.

DISCUSSION

Our study sheds light on some of the mechanisms of action of YqaH, a small protein with growth inhibition activities encoded by the B. subtilis skin element. By physically interacting with two master regulators DnaA and Spo0A, YqaH is a multifunctional small peptide, with the potential to act on two key cellular processes. YqaH is able to counteract DnaA, which is essential during vegetative growth as a replication initiator and transcriptional factor. YqaH also interfers with Spo0A, which is an essential regulator of lifestyle transitions in response to changes in environmental conditions, including the transition from vegetative growth to sporulation.

In Bacillus, several regulatory proteins inhibit DnaA by targeting different functional domains of the protein. In cells committed to sporulation, the protein SirA prevents DnaA from binding to the replication origin oriC by interacting with its structural domains I and III [START_REF] Jameson | Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA[END_REF]. During vegetative growth, the regulatory protein YabA interacts with DnaA during 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] Soufo | Cell-cycle-dependent spatial sequestration of the DnaA replication initiator protein in Bacillus subtilis[END_REF][START_REF] Felicori | Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners[END_REF]. YabA as well as the primosomal protein DnaD affect DnaA cooperative binding to oriC by interacting with DnaA structural domain III [START_REF] Scholefield | YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA[END_REF][START_REF] Merrikh | Control of the replication initiator DnaA by an anti-cooperativity factor[END_REF]. Finally, the ATPase protein Soj negatively regulates DnaA by also interacting with the structural domain III, preventing oligomerization [START_REF] Scholefield | DnaA and ORC: more than DNA replication initiators[END_REF]. Our results indicate that YqaH controls DnaA activity by a mode distinct from the other known regulators, specifically by contacting the structural domain IV responsible for binding of the DnaA-binding-sites (DnaA-boxes) on the Bacillus genome [START_REF] Fujikawa | Structural basis of replication origin recognition by the DnaA protein[END_REF].

B. subtilis cells overexpressing yqaH exhibited various DnaA-related phenotypes that spanned from a general growth defect, aberrant nucleoid morphologies, impaired replication initiation and loss of transcriptional control. These cells also exhibited Spo0A-related phenotypes such as a dramatic reduction of sporulation efficiency, an inhibition of Spo0A-driven gene expression and a strong decrease in biofilm formation. These observations are in agreement with a role of YqaH in counteracting both DnaA and Spo0A activities during vegetative growth and sporulation. However, it is well documented that initiation of sporulation is closely coupled to the cell cycle and DNA replication to ensure it occurs only in cells containing two fully replicated chromosomes [START_REF] Veening | A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis[END_REF]. The intricate relationship between DNA replication and sporulation makes it difficult to separate the DnaA-related phenotypes from those linked to Spo0A. By using YqaH single-point mutants unable to interact with DnaA but proficient for interaction with Spo0A, we identified a mutational pattern on YqaH and confirmed the direct involvement of YqaH in various DnaA-related phenotypes, validating that LOI caused loss of function in Bacillus. In our yeast two-hybrid screens, we did not obtain YqaH Spo0A_LOI mutants. Yet, by using YqaH DnaA_LOI mutants, we revealed that DnaA also played a part in sporulation and biofilm formation.

In B. subtilis, many genes regulated by DnaA are involved in sporulation and biofilm formation, illustrating the complexity of regulatory circuits that control lifestyle transitions [START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF]. However, most of the effect of DnaA on gene expression is indirect and could be attributed to its transcriptional activation of the sporulation checkpoint gene sda [START_REF] Washington | Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis[END_REF]. We showed that Sda was not involved in the sporulation phenotypes triggered by YqaH, as cells exhibited similar sporulation defects in the presence or absence of Sda. Interestingly, the YqaH-K17E DnaA_LOI mutant separated the observed Spo0A-related phenotypes. Indeed, whereas overexpression of the yqaH-K17E mutant enabled total recovery of sporulation efficiency, it did not fully restore the activity of the Spo0A-dependent promoter P spoIIG . This finding suggests that YqaH may partially or transiently antagonize Spo0A activity. Further investigation is necessary to fully characterize the interaction of YqaH with Spo0A.

Defective prophages and integrated elements account for a large fraction of bacterial genomes and may contribute to gene diversity, bacterial adaptation and fitness [START_REF] Bobay | Pervasive domestication of defective prophages by bacteria[END_REF][START_REF] Casjens | Prophages and bacterial genomics: what have we learned so far?[END_REF]. The integration and excision of prophages and prophage-like elements within genes involved in sporulation are described as a phage regulation switch following the co-evolution between a phage and its sporeforming bacterium [100, 101]. In B. subtilis, the skin element is under tight control by the skin repressor protein SknR. Its excision is restricted to the terminal mother cell at a late stage of sporulation after the completion of engulfment of the pre-spore, leading to reconstitution of the late sigma factor sigK. In Clostridium difficile, a smaller skin-like element skin Cd was found to interrupt the sigK gene. Similarly to B. subtilis, the excision of skin Cd occurs in the mother cell during sporulation and reconstitutes an integral sigK [102]. In C. difficile, a skin Cd -less strain of exhibits a strong sporulation defect [102,103]. Other bacilli prophage elements inserted into sporulation-related genes have been described. The SPβ prophage of B. subtilis disrupts the spsM gene, coding for a polysaccharide synthase. The functional SpsM resulting from expression of spsM after excision of SPβ in the sporulating mother cell was found to participate in the formation of the exosporium-like structure on the mature spores [104].

Here we validated the biological role of a small protein encoded by the skin prophage-like element of B. subtilis in negatively interfering with a broad range of cellular processes by counteracting two master regulators, DnaA and Spo0A. The excision of skin in the mother cell, potentially leading to the expression of YqaH, could be also part of a mechanism to improve the proper orchestration of forespore development. However, the significance of counteracting DnaA activity in the mother cell at a late stage of sporulation remains to be understood. In the non-spore-forming Gram-negative bacterium E. coli, a small protein, YfdR, encoded by a cryptic prophage, CPS-53, was found to to DnaA and to play a role in inhibiting replication initiation in vitro [105]. In B. subtilis, analysis of the occurrence of skin excision in the absence of the site-specific recombinase CisA suggested that such a rearrangement can occur by a RecA-dependent homologous recombination process, although at a very low frequency [106]. Whether the skin element could be excised in vegetative cells of B. subtilis is not documented. However, we postulate that the expression of yqaH could also be part of a mechanism to prevent cell growth and the formation of skin-less spores. YqaH homologues are present in integrated prophages of other Gram-positive bacteria of the genera Bacillus and Staphylococcus, as well as in the Gram-negative bacteria Delftia acidirovans, suggesting that YqaH could confer a beneficial advantage in other hosts under conditions other than sporulation.

Fig. 1 .

 1 Fig. 1. YqaH interacts with DnaA and Spo0A. (a) SknR transcriptionally represses the yqaF-yqaN operon of the Skin element. (b, c) Yeast two-hybrid interaction assay. Haploid yeast strains expressing the yqaH, dnaA and yabA gene or DNA fragments fused to the BD or AD domains of GAL4 are separately introduced into haploid yeast strains. Binary interactions are tested by the ability of diploids to grow onto selective media. (d) DnaA interaction with YqaH and regulators. Schematic representation of the architecture of the four functional domains of DnaA with associated functions as illustrated by colours. Numbers refer to amino acid boundaries. The binding of negative regulators to their targeted DnaA functional domain is illustrated accordingly. The YqaH interacting domains of DnaA with associated yeast two-hybrid interacting phenotypes (IP) are indicated. (+) and (-) refer to growth or absence of growth on selective media, reflecting an interacting or loss-of-interaction phenotype, respectively.

Fig. 2 .

 2 Fig. 2. YqaH triggers dnaA-related phenotypes. (a) Effect of yqaH expression on growth. Cells carrying plasmids pDG148 (control, plain lines) or pDG148-yqaH (dashed lines) were examined in the presence of IPTG (0.5 mM) over 5 h. Growth was monitored either by OD 600 (black) or by cell viability, measured as the number of colony forming units per millilitre, normalized by OD 600 (red). (b) Nucleoid morphological defects. Samples of living cells were examined by bright-field (above) and fluorescence microscopy (below) after staining with FM4-64 (membrane dye, false-coloured in red) and DAPI (DNA dye, false-coloured in blue). White and yellow arrows indicate guillotined chromosomes resulting from septal closing over nucleoids and aberrant nucleoid segregation patterns, respectively, and a typical example of chromosomal segregation defects is magnified. Bars, 5 µm. (c) YqaH affects dnaA expression. Cells harbouring either pDG148 or pDG148-yqaH were grown in LB in the presence of IPTG. RNAs from exponentially grown cells (OD 600 0.3) were extracted and expression levels of dnaA were monitored by qPCR in the presence (+) or absence (-) of YqaH. Data are presented as mean±se; n≥16 from two independent experiments and three biological replicates per experiment. ***P<0.001 (t-test).

Fig. 3 .

 3 Fig. 3. YqaH triggers sporulation and Spo0A-related phenotypes. (a, b) Overexpression of yqaH inhibits sporulation. Cells carrying plasmids pDG148 (control) or pDG148-yqaH were grown in Sterlini-Mandelstam medium in the presence of IPTG (0.5 mM) added at the onset (t 0 ) of sporulation. Sporulating cells were observed at different times after initiation of sporulation. (a) Snapshot captures of light and fluorescence microscopy at indicated times. Cells were stained with the fluorescent membrane dye FM4-64 (left panel). (b) Sporulating cells were quantified by monitoring asymmetric septa, engulfed forespores and free spores, in the presence (+) or absence (-) of YqaH. Ratios were determined from observation of ≥500 cells over two independent experiments and three biological replicates per experiment. (c) YqaH affects expression of Spo0A-regulated genes. Expression levels of spoIIE and spoIIGA genes from the Spo0A regulon were monitored by real-time qPCR in the presence (+) or absence (-) of YqaH and expressed as relative expression ratio compared to control (-). Data are presented as mean±se; n≥8 from two independent experiments. ***P<0.001 (t-test).

Fig. 4 .

 4 Fig. 4. Mapping of DnaA_LOI mutations in YqaH. (a)The YqaH protein is predicted to fold into three αhelices (http://www.compbio.dundee.ac.uk./wwwjpred/). Single-substituted amino acids are highlighted in red and blue, and the nature of the substitutions is indicated beneath. Upper case indicates an LOI phenotype monitored on both -His and -Ade selective media. Lower case indicates that the LOI phenotype has been obtained on the most selective -Ade media only but not on -His media, indicative of a partial loss of interaction. The four mutations highlighted in blue are mapped on the cartoon structure shown in (c). (b) YqaHLOI mutational screen. Amino acid substitutions affecting interaction with DnaA and/or Spo0A are indicated with their associated interaction phenotype. (+) indicates unchanged interaction phenotypes compared to YqaH WT, and characterized by a full ability of diploids to grow on both selective -LUH and -LUA media. (-) refers as a total loss of interaction phenotype leading to absence of growth on both -LUH and -LUA media. (+/-) refers as a partial loss of interaction leading to some growth on -LUH but not -LUA; see also Fig.S1for additional explanation. (c) Top: mapping of four substituted residues (green sticks) on the cartoon representation of an Alphafold-predicted YqaH structure (per-residue confidence score PLDDT>90)[START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF], visualized by PyMOL (2.4.1). Bottom: electrostatic surfaces and charge distribution (APBS software package into PyMOL) with ±5 kT e -1 electrostatic potential plotted on the solvent-accessible surface. The charge of substituted amino acids is indicated.

Fig. 5 .

 5 Fig. 5. Restoration of replication initiation defects by YqaH-DnaA_LOI mutation. B. subtilis cells carrying either pDG148 (-), pDG148-yqaH (WT), and pDG148-yqaH LOI mutant derivatives K17E or R56W were grown in the presence of IPTG and assessed for various dnaA-related phenotypes. (a) Nucleoid morphological phenotypes. Cells were treated with DAPI to reveal nucleoids (false-coloured blue) and with the membrane dye FM4-64 (false coloured red). White and yellow arrows indicate septum-entrapped nucleoids and aberrant condensation and segregation, respectively. Bars, 5µm. (b-d) Analysis of DnaA-dependent replication initiation phenotypes. (b) Ori/Ter ratio; origin-proximal and terminus proximal DNA sequences were quantified by qPCR. (c) Visualization of origins foci in living cells. Origins are visualized through binding of the GFP-LacI repressor to LacO operator sequences inserted at a proximal location from oriC. (d) Average number of replication origins per cell determined as the number of GFP-LacI foci upon induced conditions in control (-), WT and K17E-mutant derivative of YqaH. Statistical significance is indicated by asterisks (t-test; Ori/Ter: n=6; dnaA mRNA: n=12; *P<0.05, **P<0.01, ***P<0.001.

Fig. 6 .

 6 Fig. 6. Effect of YqaH LOI mutants on dnaA expression. Cells harbouring either the pDG148, pDG148-yqaH WT or K17E and R56W mutated derivatives were grown in LB in the presence of IPTG. RNAs from exponentially grown cells (OD 600 ̴ 0.3) were extracted and expression levels of the dnaA gene were monitored by qPCR in the absence (-) or in the presence of YqaH (WT, K17E, or R56W). Data are presented as mean±se; n=12 from two independent experiments with two biological replicates per experiment. ***P<0.001 (t-test).

Fig. 7 .

 7 Fig. 7. Effect of the YqaH-K17E mutant on Spo0A activity. (a, e) Sporulation efficiency in B. subtilis 168 sda+ and Δsda. Sporulating cells were quantified by monitoring asymmetric septa, engulfed forespores 6 h after sporulation initiation, in the absence (-) or in the presence of YqaH WT and K17E. Ratios were determined from observation of >500 cells over two independent experiments and three biological replicates per experiment. Pairwise comparisons were performed using the Holm-Bonferroni method (*P≤0.05, **P≤0.01, ***P≤0.001). (b-d) Strains carrying a fusion of the spoIIG promoter to the luciferase coding sequence (luc), and harbouring either plasmid pDG148 (black), pDG148-yqaH WT (green) or the K17E mutant derivative (orange), as well as a strain carrying a PspoIIG-luc in a Δspo0A mutant background (grey) were induced to sporulation in SM medium supplemented with 0.5 mM IPTG. Luminescence was recorded in B. subtilis 168 (sda+) (b) and Δsda (c) strain backgrounds. (d) Grouped-violin plot comparing the maximum levels of luciferase at 3 h after sporulation initiation.

Acknowledgements

The authors are grateful to F. Lecointe for providing the FLB78 strain and to O. Delumeau for his help with protein analysis. We also thank P. Noirot and M-A Petit for their critical reading of the manuscript.

Funding Information

This research has been funded by INRAE.

Author contributions Supervision: M.F.N.G; Conceptualization: M.F.N.G., M.V. Investigation and validation: M.V. Formal analysis: M.F.N.G., M.V. Visualization: M.F.N.G., M.V. Writing, review and editing: M.F.N.G., M.V.

Conflicts of interest

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.


G Boisselier

F Maury

F Schuster

Growth of Chromium Carbide in a Hot Wall DLICVD Reactor

Keywords: DLICVD, Hot Wall CVD, Cr, S Doping

Chromium carbide coatings were grown at 748 K in a hot wall CVD reactor fed by sublimation of bis(benzene)chromium, BBC (MOCVD) and by direct liquid injection using a BBC/toluene solution (DLICVD). The two types of coatings exhibit an amorphous structure and the same C content (22 at.%). DLICVD permits delivering higher mass flow rate of precursors and consequently the growth rate is 3 times higher and the thickness uniformity is better than using MOCVD. Chromium metal deposition has also been investigated by DLICVD in this hot wall reactor using BBC/toluene/additive as precursor. The purpose of the additive is to block carbide formation. Two additives have been studied: (i) hexachlorobenzene (C 6 Cl 6 and (ii) thiophenol (C 6 H 5 SH). The ratio additive/BBC required for Cr metal deposition is a few percent. In this process, C 6 Cl 6 is not decomposed and only traces of Cl (0.4 at.%) are found in the coatings. For a ratio C 6 Cl 6 /BBC > 27% the growth of any coating is blocked. The gas phase containing C 6 H 5 SH is more reactive since the onset of deposition occurs approximately 50 K before the temperature of the chlorinated compound. Furthermore, a sulfur contamination of 3 at.% has been analyzed in the coatings revealing a partial decomposition of the additive. The results are detailed and discussed in relation with previous works.

INTRODUCTION

Industrial CVD processes frequently require (i) low deposition temperature and (ii) high flow rate of molecular precursor to maximize the growth rate and the process throughput. This is achieved using for instance (iii) continuous deposition on a conveyor belt set-up (atmospheric pressure is preferred) or (iv) a large scale isothermal reactor operating generally under low pressure. The first two criteria can be met using both metal organic compounds that significantly decrease the deposition temperature and direct liquid injection (DLI) coupled with a flash vaporization chamber which permits to deliver high flow rates of precursor.

Chromium based coatings deposition by CVD has been investigated for years. It has been demonstrated that chromium metal films (Cr) could be deposited using CrCl 2 at 1323 K. 1 Chromium carbide coatings (CrC is used as general acronym) were deposited using MOCVD processes by our group 2 3 and others. 4 On the other hand, DLICVD process has been used to deposit functional oxide films 5 and more rarely metal thin films. 6 7 In previous works, chromium carbide coatings were deposited at low temperature by low pressure CVD using bis(benzene)chromium (BBC) as metal source either in a hot wall 2 or a cold wall CVD reactor. 3 Interestingly, it was demonstrated that addition of hexachlorobenzene (C 6 Cl 6 in the gas phase prevented the C incorporation into the films and, as a result, metal Cr coatings were formed. 8 If the trends were in agreement with thermodynamic simulations using BBC as sole reactant, the effect of C 6 Cl 6 was not predicted by thermodynamic modeling revealing a kinetic control of this process. 9 Deposition under atmospheric pressure of chromium carbide and Cr metal is also possible using a cold wall DLICVD reactor. A solution of BBC in toluene without and with addition of C 6 Cl 6 was used respectively. 10 In spite of the presence of a high amount of toluene in the reactive vapor phase, the inhibition effect of the chlorine derivative was always observed probably because in cold wall CVD reactor the chemical reactivity in homogeneous phase is significantly reduced.

The kinetics still played a major role as shown by the disagreement with thermodynamic simulation. 11 A further step is presented in this paper, chromium carbide and Cr metal coatings were grown by DLICVD under low pressure in a hot wall reactor using respectively BBC/toluene and BBC/toluene/additive, where additives are C 6 Cl 6 and thiophenol (C 6 H 5 SH). Here, gas phase chemistry is expected to be significant and consequently high carbon incorporation could occur according to thermodynamic calculations. 11

EXPERIMENTAL DETAILS

Experimental conditions are reported in Table I. Si(100) wafers and thermally oxidized silicon were used as substrates (10 × 15 mm). They were degreased with ethanol in an ultra-sonic bath and placed along the longitudinal axis of reactor. The system was vacuum purged for 2 h using a turbo pump. The deposition chamber was a horizontal hotwall CVD quartz reactor, 2.6 cm in diameter and 60 cm length for both processes (MOCVD and DLICVD). The reactor was heated by a one-zonal tubular furnace. It produces for instance an isothermal zone of about 20 cm at 748 K ± 10 K. The pressure was automatically monitored at 50 Torr during the experiments.

For the MOCVD process, commercial BBC was introduced into a glass saturator and sublimated at 378 K. The BBC vapour was transported using N 2 as carrier gas and the pipes from saturator to reactor were heated at 393 K to avoid condensation. The BBC mass flow rate and consequently its mole fraction at the entrance of the reactor (X BBC were calculated according to the procedure described elsewhere 12 and the vapour pressure of BBC. 13 Table I

[BBC] in - 3 × 10 -2 3 × 10 -2 3 × 10 -2 toluene (mol/L) Mole fraction of 10 -3 1 1 × 10 -3 1 1 × 10 -3 1 1 × 10 -3 BBC (X BBC ) [C 6 Cl 6 ] in 0 0 0 3 × 10 -3 toluene (mol/L) [S] in 0 0 6 × 10 -3 0 toluene (mol/L) Additive/BBC (%) - - 20 10 Duration (h) 3 2 2 2
For the DLICVD process, BBC was solubilised in toluene. When they are used, additives like C 6 Cl 6 or C 6 H 5 SH were solubilised directly in the BBC/toluene solution. This permits an easy control of the ratio additive/BBC. The container is maintained at room temperature and pressurised under 2 bars of N 2 atmosphere. The solution was injected into a flash vaporisation chamber using a commercial system (Kemstream). The injected amount of solution is controlled by the injector parameters (opening time and frequency), which are monitored by a computer. The flash vaporisation of the pulsed spray occurred at 473 K. The reactive gas phase is transported to the reactor using an N 2 stream through the vaporization chamber. From the total volume of liquid solution consumed in a run (ca. 160 cm 3 , the flow rate of liquid injected into the flash vaporization chamber is derived (1.3 cm 3 /min). Then using the physical properties of the reactives (density, molecular weight) and assuming they behave like an ideal gas, the composition of the reactive gas phase delivered into the reactor has been determined. The total mass flow rate of the gas phase is 769 sccm (N 2 , 500 sccm; toluene, 268 sccm; BBC, 0.85 sccm). Under the conditions of Table I the dwelling time of the species in the CVD reactor was 6 s.

The coating structures were characterized by X-ray diffraction (Cu K . The average crystallite size of the Cr metal coatings was determined using the Scherrer's formula from the FWHM of the (110) Cr peak. The morphology of the films has been investigated by SEM and the chemical composition has been analyzed by EDS, electron probe microanalysis (EPMA) and SIMS.

RESULTS AND DISCUSSION

Chromium Carbide Coatings: Comparison of MOCVD and DLICVD

Figure 1 shows SEM micrographs of samples from runs #1 and #2 (Table I). The surface morphology of the coating grown by MOCVD is heterogeneous (Fig. 1). It reveals a non continuous film and an equiaxed granular growth. Analyses of several samples along the reactor axis permits to determine a maximum growth rate of ∼0 5 m/h. The surface of the coating grown by DLICVD is smoother and the growth tends to be columnar type (Fig. 1). The films are very compact, excepted the samples at the outlet of the reactor which exhibit a low porosity due to the poor coalescence of the columnar grains. A maximum growth rate of 1.5 m/h has been measured for DLICVD.

Figure 2 shows XRD diagrams of coatings grown by both processes. The coating grown by MOCVD exhibits a nanocrystalline structure while DLICVD films clearly leads to amorphous layers. The XRD patterns of both types of coatings show a broad diffraction peak centred on the main peaks of stable chromium carbides (43-44 ). Abroad shoulder around 35 could be due to traces of oxide. The The growth rate of the layers is different in both cases. Despite the same initial mole fraction of BBC in both processes (Table I), the quantity of deposited material is two times more important in DLICVD (239 mg) than in MOCVD (121 mg), leading to a maximum growth rate of 1.5 m/h for DLICVD and 0.5 m/h for MOCVD. In fact the key factor is the mass flow rate of BBC which was estimated to be two times higher in DLICVD compared to MOCVD. This is an interesting result because the BBC mass flow rate is close to its maximum using the sublimation technique (MOCVD) according to the previous models 12 while it can be increased by DLICVD above the values explored in this work for instance by increasing the liquid concentration or changing the injection parameters. This is an important flexibility of the DLICVD process to maximize the growth rate and to improve the thickness uniformity in the reactor axis.

DLICVD of Chromium Metal Coatings

Two routes have been investigated to deposit Cr metal coatings by DLICVD using a BBC/toluene solution as starting material: addition of two different chemical compounds that inhibit the heterogeneous decomposition of both the aromatic ligands of BBC and toluene molecules of the solvent. As reported in previous work, C 6 Cl 6 is known to have this effect and to be sufficiently stable at the deposition temperature to avoid chlorine contamination of the films 8 but it was not tested in a hot wall reactor or in presence of vapour of solvent. Another additive which has been investigated is C 6 H 5 SH. The molecular ratio additive/BBC were 10% and 20% for C 6 Cl 6 and C 6 H 5 SH, respectively (Table I). The use of C 6 Cl 6 leads to the bcc cubic structure of metallic Cr as confirmed by XRD. In the range 20 to 100 2 , only peaks of Cr, with the main one at 44.4 corresponding to the (110) direction, have been observed. No other XRD peaks that could be due to carbides or oxide have been observed. EPMA analyses have confirmed that no contamination of oxygen is present in the films and the C content is typically 6 at.%. Moreover, the coatings did not exhibit preferential orientation of the growth whatever the local conditions, i.e., there is no structural change along the reactor axis.

Films are smooth and exhibit a metallic brightness. The layer morphology shown in Figure 3 reveals a columnar growth with a nodular aspect on the surface. The average grain size estimated by SEM from the surface morphology was found to increase in the reactor axis with the temperature (and BBC depletion) from 300 nm (693 K) to 600 nm (748 K). The maximum growth rate was 1.4 m/h but it decreases after the maximum due to the BBC depletion. The reactor was coated along ∼35 cm, i.e., only slightly more than half the length of reactor. Over this length the temperature increased from 543 to 753 K and the end of deposition was observed while the temperature was maximum. Calculations based on a mass balance showed that the end of the deposition was not due to the consumption of total BBC but to an excess of C 6 Cl 6 additive compared to the BBC. In fact, a critical ratio C 6 Cl 6 /BBC of approximately 27% seems to inhibit the heterogeneous decomposition of BBC and consequently the growth of any coating.

The coatings grown using thiophenol as additive exhibit also the Cr metal structure (Fig. 4). The samples at the entrance of the reactor were slightly textured in the [110] direction. This [110] preferential orientation of the growth changed to [200] direction along the reactor axis.

Figures 3(c) and (d) show a smooth surface morphology and a granular equiaxed growth. A slight porosity is observed on the cross section which could be related to the dark aspect of the film. Grains are clusters of crystallites, the mean size of which is approximately 420 nm and 62 nm, respectively.

The samples were slightly contaminated by sulphur but no change of the S/Cr ratio was observed along the reactor axis (EDS analysis). EPMA analysis has confirmed a S contamination of coatings of 3.3 at.% and a C content of 4 at.%. Moreover, SIMS analysis has shown the sulphur contamination is homogeneously distributed through the film thickness. The maximum growth rate was 1.9 m/h. It reached about a quarter of the reactor then the growth rate decreased progressively due to BBC depletion up to the outlet of the reactor where the temperature sharply decreased so that no deposition occurred. It is noteworthy that the tube reactor as a whole has been covered. Using this sulfur additive, no critical ratio additive/BBC was observed. This gives a higher flexibility to the process to optimize the growth rate and the thickness uniformity in the reactor axis.

CONCLUSION

Deposition of amorphous CrC coatings at 748 K by DLICVD from BBC/toluene was reported. In spite of the solvent vapour in the reactive gas phase, no excess of C incorporation was found in the films grown by DLICVD compared to those grown by MOCVD. Among the advantages of DLICVD, it was shown that DLI technique permits to deliver higher flow rates of precursor than MOCVD and consequently it is more adapted to feed hot wall CVD reactor. DLICVD of CrC coatings is more flexible to optimize the growth rate and the film thickness uniformity along the reactor axis as required for large scale reactor.

The deposition of polycrystalline Cr metal coatings in such hot wall DLICVD reactor has also been demonstrated at temperature as low as 500 K. Two routes have been investigated: the pulsed injection of a solution of (i) BBC/toluene/C 6 Cl 6 and of (ii) BBC/toluene/C 6 H 5 SH as precursors. The gas phase is slightly more reactive using C 6 H 5 SH compared to C 6 Cl 6 because the onset of deposition occurs at 478 K and 543 K respectively. Only traces of Cl (∼0.4 at.%) were found in the coatings using C 6 Cl 6 while the S content is ∼3 at.% when thiophenol was used as additive indicating that this compound partially decomposed. Furthermore, with C 6 H 5 SH, the length of the coated zone in the tubular reactor was longer and the maximum growth rate was approximately 30% higher. A first remark for the chlorinated additive is that it leads to a better homogeneity of the coating structure along the reactor axis but, due to the BBC depletion, the thickness gradient toward the outlet is sharper than using thiophenol. In fact, beyond a critical ratio C 6 Cl 6 /BBC = 27% the growth of any coating is prevented. The second remark is that traces of Cl are redhibitory for corrosion protective coatings. For these two reasons, there is a greater interest to use C 6 H 5 SH instead of C 6 Cl 6 as additive for low temperature DLICVD of Cr metal coatings.

Fig. 1 .

 1 Fig. 1. SEM micrographs of chromium carbides films grown at 748 K by MOCVD (a, b) and DLICVD (c, d).

Fig. 2 .

 2 Fig. 2. Grazing-incidence (4 XRD patterns of CrC films grown by MOCVD and DLICVD at 748 K on (100)Si substrate.

Fig. 3 .

 3 Fig. 3. SEM micrographs of chromium metal films grown by DLICVD using BBC/toluene/C 6 Cl 6 (a, b) and BBC/toluene/C 6 H 5 SH (c, d) at 748 K.

Fig. 4 .

 4 Fig. 4. XRD pattern of Cr metal films grown by DLICVD using BBC/toluene/C 6 H 5 SH at 748 K.

  . Experimental growth conditions of chromium and chromium carbide coatings.

		MOCVD	DLICVD	DLICVD	DLICVD
	Process	CrC (#1)	CrC (#2)	Cr (#3)	Cr (#4)
	Furnace setpoint	748	748	748	748
	temperature (K)
	Total	50	50	50	50
	pressure (Torr)
	N 2 mass	300	500	500	500
	flow rate (sccm)
	Toluene mass	-	268	268	268
	flow rate (sccm)

Table II .

 II Composition of CrC coating deposited by MOCVD and DLICVD at 748 K compared to the stoichiometry of stable chromium carbides (EPMA data).

	Sample	Cr (at.%)	C (at.%)	O (at.%)	C/Cr
	DLICVD	71.4	21.8	6.8	0.31
	MOCVD	70.2	21.7	8.1	0.31
	Cr 7 C 3	70.0	30.0	-	0.43
	Cr 23 C 6	79.3	20.7	-	0.26