Pierre Genevaux
email: pierre.genevaux@ibcg.biotoul.fr

Roland Barriot

Justine Latour

Marie-Pierre Castanié-Cornet

Gwennaele Fichant
email: gwennaele.fichant@ibcg.biotoul.fr

J-domain proteins in bacteria and their viruses

Keywords: HscA/HscC, NERD, CheY, PilZ, SNARE, HCP

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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The DnaK/DnaJ/GrpE chaperone machine Universally conserved molecular chaperones maintain cellular protein homeostasis by acting at virtually every step of protein biogenesis pathways. Among these chaperones the Heat Shock Protein 70 (HSP70) has been associated with almost every known essential chaperone functions [START_REF] Balchin | In vivo aspects of protein folding and quality control[END_REF][START_REF] Rosenzweig | The Hsp70 chaperone network[END_REF][START_REF] Mayer | Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones[END_REF]. HSP70 assists the folding of newly synthesized polypeptides, remodels native protein complexes to control their activities, facilitates protein targeting to membranes and protein translocation, reactivates aggregated proteins and participates to protein disaggregation and degradation in cooperation with major disaggregases and proteases [START_REF] Rosenzweig | The Hsp70 chaperone network[END_REF][START_REF] Dahiya | Chapter One -Functional principles and regulation of molecular chaperones[END_REF][START_REF] Żwirowski | Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding[END_REF]. In these cases, HSP70 functions rely on its ability to interact in an ATP-dependent manner with short extended hydrophobic stretches of polypeptide segments within its protein clients [START_REF] Mayer | Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones[END_REF][START_REF] Rudiger | Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries[END_REF].

In bacteria, multifunctional HSP70 chaperones are named DnaK [START_REF] Georgopoulos | Toothpicks, serendipity and the emergence of the Escherichia coli DnaK (Hsp70) and GroEL (Hsp60) chaperone machines[END_REF]. Among these, the Escherichia coli DnaK chaperone is from far the best characterized representative. DnaK is one of the most abundant chaperones in E. coli (about 10 000 copies per cell) that is expressed constitutively at 37°C and further induced following certain stresses, including heat shock or protein aggregation [START_REF] Genevaux | The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions[END_REF]. DnaK has been associated with all the HSP70 chaperone functions described above and shown to interact co-and/or post-translationally with over six hundred protein clients, including cytosolic proteins (~80%), inner membrane proteins (~11 %), outer membrane proteins (~3%) and periplasmic proteins (~3%), highlighting the key role played by DnaK in almost all E. coli key cellular processes [9]. Remarkably, DnaK often cooperates with other generic chaperones that are part of the E. coli proteostasis network, either during severe protein denaturing stresses, including ClpB, HSP33, IbpA/B, GroESL and HtpG, or during de novo protein synthesis with Trigger Factor [START_REF] Bruel | Hsp33 controls elongation factor-Tu stability and allows Escherichia coli growth in the absence of the major DnaK and trigger factor chaperones[END_REF][START_REF] Castanié-Cornet | Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane[END_REF][START_REF] Agashe | Function of bacterial chaperones trigger factor and DnaK in multi-domain protein folding: Increase in yield at the expense of folding speed[END_REF][START_REF] Genevaux | In vivo analysis of the overlapping functions of DnaK and trigger factor[END_REF][START_REF] Deuerling | Trigger factor and DnaK cooperate in folding of newly synthesized proteins[END_REF][START_REF] Mogk | Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation[END_REF][START_REF] Genest | Hsp90 and Hsp70 chaperones: Collaborators in protein remodeling[END_REF][START_REF] Obuchowski | Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates[END_REF]. In addition to its direct involvement as a proteostasis network chaperone, DnaK was shown to control the E. coli heat-shock response by modulating intracellular levels of the heat-shock sigma factor σ 32 , in cooperation with the essential membrane protease FtsH [START_REF] Rodriguez | Molecular Basis for Regulation of the Heat Shock Transcription Factor sigma(32) by the DnaK and DnaJ Chaperones[END_REF]. Mutation in dnaK is both cold and high temperature [START_REF] Genevaux | The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions[END_REF][START_REF] Bertelsen | Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate[END_REF][START_REF] Kityk | Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones[END_REF]). The open/closed conformation of the SBD of DnaK and interaction with substrates is regulated by the ATP/ADP occupancy in the NBD [START_REF] Mayer | Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones[END_REF][START_REF] Bertelsen | Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate[END_REF][START_REF] Kityk | Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones[END_REF][START_REF] Palleros | ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis[END_REF]. Indeed, it has been shown that the ATP-bound DnaK is characterized by a low affinity and fast exchange rate for its substrates with the lid open, whereas the ADP-state shows a high affinity and low exchange rates for substrates with the lid covering the substrate. Under this form, the DnaK-ADP bound peptide client is transiently protected from misfolding and unproductive interaction [START_REF] Goloubinoff | Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins[END_REF][START_REF] Imamoglu | Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein[END_REF][START_REF] De Los Rios | Hsp70 chaperones use ATP to remodel native protein oligomers and stable aggregates by entropic pulling[END_REF]. To efficiently interact with its substrate, DnaK necessitates the assistance of its J-domain protein (JDP) co-chaperone partner (also known as DnaJ, Hsp40 or J-proteins), which stimulate DnaK's intrinsically weak ATPase activity and specifically transfers preselected substrate to DnaK (Fig. 1A; [START_REF] Kampinga | The HSP70 chaperone machinery: J proteins as drivers of functional specificity[END_REF][START_REF] Kampinga | Function, evolution, and structure of J-domain proteins[END_REF][START_REF] Kelley | The J-domain family and the recruitment of chaperone power[END_REF][START_REF] Pukszta | Co-evolution-driven switch of Jprotein specificity towards an Hsp70 partner[END_REF]). In addition to JDP co-chaperones, most DnaK/HSP70 chaperones also require a nucleotide exchange factor (NEF), named GrpE in E. coli, which mediates the dissociation of ADP, thus facilitating binding of a new ATP and substrate release from DnaK, which eventually leads to folding of the substrate [START_REF] Brehmer | Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange[END_REF][START_REF] Ang | Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature[END_REF][START_REF] Harrison | Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK[END_REF][START_REF] Siegenthaler | The importance of having thermosensor control in the DnaK chaperone system[END_REF].

The JDP co-chaperone family of proteins is defined by the presence of a compact domain of approximately 70 amino acid residues, named the J-domain, which is essential for a functional interaction with DnaK (Fig. 1B). The J-domain of the E. coli DnaJ protein is composed of four helices, with helices II and III forming an antiparallel coiled-coil fold anchored by helix I.

Helices II and III are connected by a solvent-exposed loop that contains the highly conserved His-Pro-Asp (33HPD35) tripeptide signature of JDPs [START_REF] Kelley | The J-domain family and the recruitment of chaperone power[END_REF][START_REF] Szyperski | NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain[END_REF]. The 33HPD35 tripeptide of the interconnecting loop as well as several exposed basic residues of helix II and III were shown to be critical for the J-domain activity in vivo and in vitro (Fig. 1B; [START_REF] Greene | Role of the J-domain in the cooperation of Hsp40 with Hsp70[END_REF][START_REF] Suh | Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ[END_REF][START_REF] Wall | The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication[END_REF][START_REF] Genevaux | Scanning mutagenesis identifies amino acid residues essential for the in vivo activity of the Escherichia coli DnaJ (Hsp40) J-domain[END_REF]). The recently solved structure of the J-domain in complex with a DnaK-ATP mutant locked in its open conformation showed that the J-domain simultaneously interacts with both the NBD and the SBD of DnaK [START_REF] Kityk | Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones[END_REF]. In this case, the HPD tripeptide is projected toward the NBD docked inter-domain linker of DnaK. Such a remarkable structure pin points a significant network of interactions involving the J-domain and DnaK, and explains how a J-domain can indeed couple two critical steps in the DnaK chaperone cycle, i.e., the stimulation of ATP hydrolysis and the concomitant trapping of the substrate by DnaK (Fig. 1A). For a detailed recent review about the mechanism of DnaK-Jdomain interaction see Mayer and Gierasch [START_REF] Mayer | Recent advances in the structural and mechanistic aspects of Hsp70 molecular chaperones[END_REF].

JDPs can be grouped into three major classes (A, B and C) based on the conserved domains that are present in addition to the J-domain [START_REF] Genevaux | The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions[END_REF][START_REF] Kampinga | The HSP70 chaperone machinery: J proteins as drivers of functional specificity[END_REF][START_REF] Kelley | The J-domain family and the recruitment of chaperone power[END_REF]. Adjacent to their N-terminal Jdomain, class A JDP members possess a glycine/phenylalanine (G/F) rich region that connects the J-domain to a zinc-binding domain (ZBD) composed of eight cysteines coordinating two zinc ions (zinc 1 and 2 sites) and a large C-terminal domain (CTD) composed of two beta domains (CTD1 and CTD2) that is followed by a short dimerization domain (DD) (Fig. 1C). Note that class A JDPs were shown to function both as generic DnaK/HSP70 co-chaperones and as bona fide chaperones capable of preventing protein aggregation independently of HSP70. While the CTD appears to be the main substrate binding domain, the role of the G/F region and the ZBD is less well understood. The G/F region, which appeared to be highly unstructured and more variable in sequence [START_REF] Pellecchia | NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone[END_REF], was shown to be involved in substrates binding [START_REF] Perales-Calvo | Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates[END_REF] and to be critical for proper activation of the DnaK chaperone cycle in vivo and in vitro, possibly triggering functional substrate-bound DnaK [START_REF] Wall | The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone[END_REF][START_REF] Cajo | The role of the DIF motif of the DnaJ (Hsp40) co-chaperone in the regulation of the DnaK (Hsp70) chaperone cycle[END_REF]. The ZBD domain was also proposed to exhibit a dual function, with each zinc site contributing differently. The crystal structure of the ZBD together with the CTD of the Saccharomyces cerevisiae Ydj1 (DnaJ) showed that they form an L-shaped structure with the zinc 1 site at the ankle and the zinc 2 site in the middle of the shorter arm of the L-shape [START_REF] Li | The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate[END_REF]. Biochemical analyzes suggest that zinc 1 is most likely involved in substrate binding [START_REF] Li | The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate[END_REF][START_REF] Linke | The roles of the two zinc binding sites in DnaJ[END_REF], while zinc 2 appeared to be important for DnaJ-mediated activation of the DnaK chaperone cycle [START_REF] Linke | The roles of the two zinc binding sites in DnaJ[END_REF][START_REF] Lu | The conserved carboxyl terminus and zinc finger-like domain of the cochaperone Ydj1 assist Hsp70 in protein folding[END_REF][START_REF] Wu | The crystal structure of the C-terminal fragment of yeast Hsp40 Ydj1 reveals novel dimerization motif for Hsp40[END_REF]. It should be noted that a role for the DnaJ ZBD as a potential redoxregulator has also been proposed [START_REF] Mattoo | Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK[END_REF][START_REF] Kröncke | Nitric Oxide Inhibits the Cochaperone Activity of the RING Finger-like Protein DnaJ[END_REF][START_REF] Choi | Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2, Free Radic[END_REF]. Class B JDP members have a similar domain architecture to that of class A, except that they do not have the zinc-binding domain (Fig. 1C), and were also shown to function as generic chaperones/co-chaperones. In contrast, class C JDPs only have the J-domain in common with class A and B JDPs. In this class of JDPs, the Jdomain can be found at the N-or at the C-terminal ends (Fig. 1C), or even in the middle of the JDP architecture [START_REF] Kelley | The J-domain family and the recruitment of chaperone power[END_REF]. It is believed that class C JDPs might deliver specific substrates or confer specific cellular localization to DnaK/HSP70 chaperones, which contrasts with the proposed more generic properties of class A and B JDPs.

The three HSP70/JDP chaperone/co-chaperone machines of E. coli

Most organisms from bacteria to human generally encode multiple HSP70/JDP chaperone/co-chaperone machines, with JDPs being significantly more diverse than their HSP70 partners. The bacterium E. coli was proposed to be a representative model organism to study HSP70/JDP chaperone/co-chaperone functions and pairs' specificity [START_REF] Genevaux | The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions[END_REF][START_REF] Kluck | Structure-function analysis of HscC, the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones[END_REF][START_REF] Vickery | Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation[END_REF][START_REF] Liberek | Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK[END_REF] analyses of the E. coli HSP70/JDP chaperones/co-chaperones have shown that these proteins form three independent machines with specific functions [START_REF] Kluck | Structure-function analysis of HscC, the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones[END_REF][START_REF] Vickery | Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation[END_REF][START_REF] Liberek | Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK[END_REF].

The multitasking and stress-responsive E. coli DnaK chaperone described above has been shown to functionally interact with three JDPs, namely DnaJ, CbpA and DjlA [START_REF] Liberek | Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK[END_REF][START_REF] Genevaux | DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction[END_REF][START_REF] Ueguchi | An analogue of the DnaJ molecular chaperone in Escherichia coli[END_REF]. DnaJ is encoded from the same operon as DnaK and is considered as the main DnaK co-chaperone, assisting DnaK in almost all of its known generic activities, including de novo protein folding and translocation, protein disaggregation, control of the heat-shock response, bacterial motility, viral replication or plasmid maintenance (phenotypes review in [START_REF] Genevaux | The Hsp70 chaperone machines of Escherichia coli: a paradigm for the repartition of chaperone functions[END_REF]). CbpA can also assist DnaK for protein folding, for the prevention of protein aggregation and the remodeling of protein complexes, and can replace DnaJ function in vivo when overexpressed [START_REF] Ueguchi | An analogue of the DnaJ molecular chaperone in Escherichia coli[END_REF][START_REF] Bird | Functional analysis of CbpA, a DnaJ homolog and nucleoid-associated DNA-binding protein[END_REF]. In addition, CbpA was shown to be nucleoid-associated in stationary phase and this inhibited all tested CbpA co-chaperone functions. Finally, the DjlA co-chaperone of DnaK is a membrane anchored class C JDP with a cytoplasmic C-terminal J-domain. DjlA was shown to interact with DnaK both in vivo and in vitro, and a soluble variant of DjlA lacking its transmembrane region was shown to efficiently stimulate DnaK ATPase activity and to assist DnaK in the reactivation of denatured substrates in vitro [START_REF] Genevaux | DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction[END_REF][START_REF] Clarke | Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway[END_REF][START_REF] Genevaux | The djlA gene acts synergistically with dnaJ in promoting Escherichia coli growth[END_REF][START_REF] Gur | All three J-domain proteins of the Escherichia coli DnaK chaperone machinery are DNA binding proteins[END_REF]. In contrast to DnaJ and CbpA, no generic chaperone function has been found for DjlA, suggesting that DjlA might recruit DnaK at the inner membrane for specific function related to protein translocation or assembly of membrane protein complexes.

Overexpression of DjlA was shown to induce the production of a colanic acid capsule in a DnaKdependent manner and more recently, to enhance the production of recombinant membrane proteins for biotechnology purposes [START_REF] Clarke | Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway[END_REF][START_REF] Kelley | Positive control of the two-component RcsC/B signal transduction network by DjlA: a member of the DnaJ family of molecular chaperones in Escherichia coli[END_REF][START_REF] Gialama | Functional Requirements for DjlA-and RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR[END_REF]. Although no detectable phenotype could be observed for the single djlA mutation in E. coli laboratory strains, it was shown that djlA is required for the cytotoxicity of clam pathogen Vibrio tapetis and for Legionella dumoffii and L. pneumophila intracellular growth [START_REF] Lakhal | DjlA, a membrane-anchored DnaJ-like protein, is required for cytotoxicity of clam pathogen Vibrio tapetis to hemocytes[END_REF][START_REF] Ohnishi | Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth[END_REF][START_REF] Ohnishi | Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth[END_REF].

The HscA/HSP70 (39% identity with DnaK) chaperone of E. coli was shown to specifically interact with its HscB class C co-chaperone (encoded from the same operon) to facilitate the assembly of iron sulfur cluster proteins, as described for the mitochondrial HSP70/Jac1 pair [START_REF] Vickery | Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation[END_REF][START_REF] Kim | Nucleotide-dependent interactions within a specialized Hsp70/Hsp40 complex involved in Fe-S cluster biogenesis[END_REF]. In this case, both HscA and HscB bind the IscU protein client known to function as a scaffold protein for the assembly of [Fe-S] clusters. Although the exact mechanism has not been described yet, it has been proposed that HscA/HscB interaction with IscU [Fe-S] facilitates the transfer of the [Fe-S] cluster to acceptor proteins. Note that in contrast with DnaK, the HscA/B machine does not rely on the GrpE nucleotide exchange factor to stimulate ADP-ATP exchange during the chaperone/co-chaperone cycle [START_REF] Brehmer | Tuning of chaperone activity of Hsp70 proteins by modulation of nucleotide exchange[END_REF].

Although less well characterized, HscC (also named Hsc62; 28% identical to DnaK and 29% to HscA) together with the closely related class C JDPs DjlB and DjlC (50% identical) represent the third HSP70/JDP chaperone/co-chaperone machine of E. coli. Note that both hscC and djlC genes are adjacent on the E. coli K-12 chromosome while djlB is located approximately two kb upstream of djlC. Although the hscC gene was induced upon UV radiation, the function of this machine remains unknown. Both DjlB and DjlC possess a putative transmembrane domain at their C-terminal end. Although DjlB has not been investigated, the efficient membrane insertion of DjlC was shown to require SRP, DnaK and YidC [START_REF] Peschke | FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjlC and Flk[END_REF]. In addition, DjlC deleted of its transmembrane domain was shown to specifically stimulate HscC ATPase activity and to assist HscC in the prevention of aggregated luciferase substrate in vitro [START_REF] Kluck | Structure-function analysis of HscC, the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones[END_REF]. Accumulated data obtained with E. coli and many other organisms have revealed that JDP co-chaperones display very diverse architectures and functions related to HSP70, with a central role played by the J-domain in recruiting HSP70 and stimulating both substrate transfer and ATP hydrolysis by HSP70. Remarkably, in combination with other functional domains, the Jdomain containing proteins are thus capable of directing/recruiting the full HSP70 chaperone power to preselected client proteins and at specific cellular localization. In this review, we have used a representative set of well-annotated bacterial and bacteriophage genomes to investigate the reservoir of JDPs in these organisms. The knowledge accumulated in E. coli HSP70/JDP partners was used throughout this work as a guideline for the discovery of new JDP families.

Taxonomic distribution of DnaK/DnaJ/GrpE in bacteria

The distribution of J-domain proteins (JDPs) throughout the bacterial taxonomy was analyzed using 1709 annotated and representative bacterial genomes as a resource. The presence of the canonical DnaJ protein (class A JDP) and the co-occurrence of its two functional partners DnaK and GrpE were first investigated (Fig. 2A; Table S1). Selection of domain profiles for these proteins from functional domain databases were used to identify (i) class A DnaJ (scanned for the presence of a J-domain, a ZBD and CTDs), (ii) DnaK (scanned for the presence of NBD and SBD domains specific to DnaK, and excluding HscA, HscC and other putative DnaK-like proteins) and (iii) GrpE. A detailed procedure is described in supplementary file (SI-Materials and methods). The analysis reveals a high conservation of the class A JDP within bacterial genomes, with only 19 genomes, scattered in different taxonomic groups, having no detectable full length class A JDP. We found that all the class A JDPs contained the canonical HPD tripeptide that is essential for its activity. Among the 19 genomes without class A JDP, 11 have a class B JDP and four have a class C JDP, which leaves four bacterial genomes without any JDP. Interestingly, in the case of JDP-less genomes, both Mycoplasma pullorum B3 and Bacillus kochii BDGP4 strains still possess a dnaK gene, suggesting that these bacterial HSP70 could recruit host JDPs. The two remaining JDP-less genomes (Thermovibrio ammonificans strain DSM 15698 / JCM 12110 / HB-1 ; Desulfurobacterium thermolithotrophum strain DSM 11699 / BSA) do not have any HSP70 and likely rely on other chaperones like GroEL for survival.

Length distribution of the bacterial class A JDPs revealed that these co-chaperone proteins form a compact group with an average length comparable to that of the canonical E. coli DnaJ protein (i.e., 376 amino acids). Accordingly, the architecture of class A JDPs is highly conserved and we only found one class A DnaJ from a plasmid of the metallo-resistant bacterium Cupriavidus metallidurans CH34, which had a grafted C-terminal nucleotidyltransferase domain. Whether such an atypical association is real or due to sequencing error is not known. Remarkably, we found that several bacterial genomes, especially within Actinobacteria, have more than one class A JDP (i.e., 274 genomes among which one genome has 4 copies and five genomes have 3 copies), although the vast majority of the 1709 genomes (~84%) possess one single copy. In addition, the presence of multiple class A JDPs is often accompanied by the presence of several DnaK (Fig. 2A; Table S1). S1). Only 15 genomes potentially encode a DnaK but no GrpE. As observed for class A DnaJ, the presence of multiple GrpE was largely correlated with multiple DnaK, suggesting duplication events involving DnaK-DnaJ-GrpE systems co-localized on the chromosome (see below). While we did not find more than 3 GrpE in a bacterial genome, this number could reach 12 in the case of DnaK. Whether all these DnaK proteins are functional remains unknown. Remarkably, 6 bacterial genomes do not have a DnaK protein, which was confirmed by tblastN searches on whole genomes using as query an orthologous protein from a closely related species (i.e., Mycoplasma crocodyli MP145; Thermovibrio ammonificans strain DSM 15698 / JCM 12110 / HB-1; Desulfurobacterium thermolithotrophum (strain DSM 11699 / BSA); Bacillus methanolicus MGA3; Microterricola viridarii strain ERGS5:02; Tessaracoccus sp. NSG39). Among these genomes, 4 possess GrpE and at least one class A JDP and have dnaK either truncated or annotated as pseudogene, and 2 of them do not have any of the three proteins.

Analysis of the

The bacterial dnaK, dnaJ and grpE genes are frequently found within the same operon.

Thus, in order to investigate this organization at a comprehensive scale, and since operon organizations are not known in the vast majority of these genomes, we have arbitrarily considered the restriction that two consecutive genes are not in the same operon if they are not on the same strand or if their intergenic distance is greater than 150 bp (Fig. 2B). We found 544 tripartite organizations distributed as follows: 202 associations regrouping dnaJ-dnaK-grpE, 174 grpE-dnaK-dnaJ, 87 dnaJ-grpE-dnaK and 81 dnaK-grpE-dnaJ. Remarkably, we did not find any organization having dnaJ flanked by the two others partners. In addition, we identified 929 associations involving a pair of genes, with the most common (434) involving dnaK and grpE (218 cases with dnaK upstream of grpE and 216 cases with dnaK downstream). For the other bipartite organizations the observed numbers are comparable, with 136 dnaK-dnaJ, 120 dnaJ-dnaK, 128 grpE-dnaJ and 111 dnaJ-grpE associations.

Bacterial class B JDP and their association with DnaJ and DnaK

In E. coli, the class B JDP is represented by the DnaK co-chaperone CbpA (Fig. 2A; Table S1).

The J-domain and CTD domain profiles were used to search for class B JDPs in bacterial genomes (SI-Materials and methods). We found that only half of the analyzed genomes (863) possess a class B JDP. In 99% of the cases, genomes without class B JDP have a class A JDP (Fig. 2A; Table S1). The size of class B JDPs is also well conserved, approximately between 260-360 amino acids in length, but with more length variation than for class A JDPs (between 360 and 400 amino acids). Analysis of the less conserved G/F region, which connects the Jdomain to CTDs (including ZBD in class A) revealed that this region is generally longer and its distribution more dispersed in class B (Fig. S1), suggesting that this region could contribute to the greater length variability observed for this class of JDPs. Note that in 3 mycoplasma (M. pneumoniae M129; Mycoplasma gallisepticum str. R; Mycoplasma genitalium G37) the class B JDPs were longer than 500 amino acids and corresponded to the TopJ co-chaperone known to be required for terminal organelle function in M. pneumoniae [START_REF] Cloward | Functional domain analysis of the Mycoplasma pneumoniae co-chaperone TopJ[END_REF]. These specific class B JDPs possess two additional regions found in other terminal organelle proteins that are inserted between the J-domain and the CTD, namely a region enriched in aromatic and glycine residues (EAGR), and an acidic and proline-rich region (APR). Although most of the genomes encode one single class B JDP, some genomes do possess several class B JDPs (9% of the genomes), with a maximal of four copies in four bacterial genomes (Fig. 2A; Table S1; two Cyanobacteria, one Verrucomicrobiota, one α-proteobacteria). Remarkably, 93% of the cyanobacteria genomes encode at least two class B JDPs, and 1/5 of them have three or four copies (Fig. 2A; Table S1). As observed for class A JDPs, the presence of several JDPs was associated with the presence of several copies of dnaK gene. In sharp contrast with class A proteins, only 57 genomes out of 863 have class B JDPs and dnaK genes co-localized, indicating that, as observed for the σ s -regulated CbpA JDP in E. coli, co-regulation of dnaK and its class B JDP partners does not emerge as a main trend in bacteria.

Bacterial genomes as a reservoir for new class C JDP functions -General overview of bacterial class C JDPs

As stated above, E. coli possesses four additional JDPs (HscB, DjlA, DjlB and DjlC) that belong to class C JDPs and that only share their J-domain with class A and B JDPs (Fig. 1C).

Although unexplored yet, we anticipate that bacterial pan-genome might represent a vast reservoir of novel class C JDPs connecting specific cellular functions to a J-domain in order to efficiently recruit the Hsp70 chaperone power. Therefore, all the putative class C JDPs were first collected based on the presence of a J-domain only. Among these class C JDPs, DjlA, DjlB/C and HscB were then identified using their domain profiles from functional domain databases or using specific HMMER profiles (SI-Materials and methods). Using this approach we could identify a total of 3326 class C JDPs distributed into 1227 out of the 1709 bacterial genomes (Table S1). When we take into account all class A, B and C JDPs, the total amount of JDPs per bacterial genome reaches approximately 3.7 (6331 JDPs/1709 genomes), a high ratio that reflects the importance of JDP diversity in bacterial physiology. In contrast, the ratio of HSP70, including DnaK, HscA and HscC is 1.5 per genome. Noticeable exceptions are the taxonomic groups composed mostly of Bacillales [START_REF] Collet | Protein polarization driven by nucleoid exclusion of DnaK(HSP70)-substrate complexes[END_REF], Lactobacillales [START_REF] Ohnishi | Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth[END_REF], Mollicutes [START_REF] Cajo | The role of the DIF motif of the DnaJ (Hsp40) co-chaperone in the regulation of the DnaK (Hsp70) chaperone cycle[END_REF] and Erysipelotrichia (2) that have only one class A JDP. Unlike class A and class B JDPs, the length distribution of class C JDPs is more extensive, ranging from about 54 amino acids to more than 1000 amino acids (Fig. S2; Table S1). Remarkably, in sharp contrast to class A and B JDPs, which have their J-domain at the N-terminal part of the protein and the substrate binding domain at their C-terminal, a significant fraction of class C JDPs have their J-domain positioned at the Cterminal or in the middle of the protein (i.e., 1340 C-terminal and 314 middle versus 1533 Nterminal), likely reflecting the functional diversity and the specific cellular localization generally conferred by class C JDPs [START_REF] Kampinga | Function, evolution, and structure of J-domain proteins[END_REF][START_REF] Kelley | The J-domain family and the recruitment of chaperone power[END_REF].

-E. coli DjlA, HscB and DjlB/C co-chaperone distribution in bacteria

DjlA (452 proteins), followed by HscB (331 proteins), were the most represented class C JDPs in our bacterial genomes (Fig. 2A; Fig. 3A; Table S1). DjlA is present in about 25% of the genomes, mainly in γ-proteobacteria (46% of the genomes) and α-proteobacteria (27%), in Bacteroidota (16%), in ε-proteobacteria (5%), in some δ-proteobacteria (3%) and βproteobacteria (2%) and finally in a few Firmicutes (0.9%). The scattered distribution of these proteins among genomes of diverse taxonomic groups, like Firmicutes, β-proteobacteria and δ-proteobacteria, suggests that it may have been acquired by horizontal gene transfer (Fig. 2A). In nearly all cases, the presence of DjlA was accompanied by a class A JDP and a DnaK chaperone, which is in agreement with the membrane-associated function of DjlA wild type and its inability to efficiently replace DnaJ [START_REF] Genevaux | DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction[END_REF][START_REF] Clarke | Point mutations in the transmembrane domain of DjlA, a membrane-linked DnaJ-like protein, abolish its function in promoting colanic acid production via the Rcs signal transduction pathway[END_REF].

As stated above, the HscB co-chaperone specifically interacts with its Hsp70 partner HscA to assemble Iron/sulfur cluster proteins in bacteria [START_REF] Puglisi | The role of chaperones in iron-sulfur cluster biogenesis[END_REF]. We found that HscB is present in 19% of the selected bacterial genomes analyzed, mostly in γ-proteobacteria and βproteobacteria (80%). HscB is also present in 24 genomes of Bacteroidota, 14 of αproteobacteria, 13 of δ-proteobacteria and in nearly all the genomes from Acidobacteria, Nitrospirota and Acidithiobacillia (respectively 7/10, 6/7 and 3/3). Remarkably, 328 of the 330 genomes containing HscB also possess one HscA partner, strongly supporting the tight coupling between HscB and HscA observed in E. coli (Fig. 3A). Yet, we found that two bacteria with HscB do not have HscA (Chromatiales), thus suggesting that the multifunctional DnaK chaperone might replace HscA, as it is the case for HscB/Jac1 homologs in mitochondria [START_REF] Vickery | Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation[END_REF].

Finally, we found that the hscA and hscB genes are co-localized in 274 out of the 328 genomes, as observed in E. coli, where they are located in the same operon.

In order to investigate the class C DjlB and DjlC co-chaperones, for which no InterPro specific domain signature is available, and their HscC/Hsp70 chaperone partner, we first built an HMMER profile based on a selected set of proteins that allows the identification of both DjlB and DjlC with confidence but without the possibility to rigorously distinguish between them due to their high similarity (here named DjlB/C; SI-Materials and Methods). Using this approach, we could only find 78 DjlB/C present in 57 genomes (thus 3% of the selected genomes), with 17 genomes having more than one DjlB/C. DjlB/C were spread among different genomes of Proteobacteria, Firmicutes, Planctomycetes and Fusobacteria, thus suggesting gene acquisition through horizontal gene transfers through multiple independent events (Fig. 3B; Table S1). The size of DjlB/C proteins is highly variable, from 153 to 1123 amino acids, with additional grafted domains, including TPR (Tetratricopeptide repeat), DUF805, RDD domain containing two transmembrane domains, or the HCP-like region homologous to type VI secretion system Hcp protein [START_REF] Zoued | Architecture and assembly of the Type VI secretion system[END_REF]. HscC chaperone homologs were detected in only 73 genomes, with 55 of them having at least one DjlB/C (Fig. 3B). When present, the hscC and djlC, or djlB genes often colocalized on the chromosome (in more than 80% of the cases). Such a feature should thus be considered important when exploring a possible partnership between an HscC and DjlB/C proteins, as it is the case in E. coli [START_REF] Kluck | Structure-function analysis of HscC, the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones[END_REF].

-Novel class C JDPs

Apart from DjlA, HscB, DjlB/C, our analysis identifies 2465 novel class C bacterial JDPs distributed into 1019 genomes (60% of the genomes) (Table S1). The number of these class C JDPs varies considerably per genome, from 1 up to 15 representatives found in a strain of Archangium gephyra. The number of class C JDPs is very high in certain δ-proteobacteria and Cyanobacteria [START_REF] Sato | Expression analysis of multiple dnaK genes in the cyanobacterium Synechococcus elongatus PCC 7942[END_REF][START_REF] Düppre | Specific and promiscuous functions of multiple DnaJ proteins in Synechocystis sp[END_REF], and there is no correlation between their number and the size of the genome (r ≃ 0.3). Remarkably, despite the numerous class C JDPs found in Cyanobacteria none of the E. coli ones could be detected (Fig. 2A, Fig. 3). In contrast, Firmicutes and some groups within Actinobacteria have very few or no class C JDPs (Fig. 3C).

In order to investigate whether the new class C J-domains were associated with known protein domains and/or functions, all class C JDP protein sequences were submitted to InterProScan. Remarkably, we found that at least one conserved protein domain was grafted to the J-domain in about one third of the JDP protein sequences (913/2465). The remaining 1552 class C JDPs had no domain that could be assigned (Table S1). Among the JDPs that possess conserved domains (either with known function or with "domain of unknown function" DUF domains) adjacent to their J-domain we could identify at least 66 different associations (Table S2), likely representing new groups of class C JDPs spread in different classes of bacteria.

While most of the new domain pairings only contained one to 10 representatives, some groups contained more than 200 proteins. All the conserved protein domains identified either upstream or downstream of a J-domain with more than 10 protein members are shown in Fig. 4A.

-Putative cellular functions of new class C JDPs

Analysis of the possible functions of the domains associated with the J-domain shows that the majority of the regions are potentially involved in protein-protein interaction and assembly of protein complexes (325/913), including TPR, WD40, LRR, SH3 or Armadillo repeats (Fig. 4; Table S2). TPR domains are also present in DjlB/C [START_REF] Kluck | Structure-function analysis of HscC, the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones[END_REF] and in the previously studied cyanobacterial JDP co-chaperone Sll1384 involved in phototaxis [START_REF] Düppre | Specific and promiscuous functions of multiple DnaJ proteins in Synechocystis sp[END_REF][START_REF] Chen | DnaJ-like protein gene sll1384 is involved in phototaxis in Synechocystis sp. PCC 6803[END_REF]. In addition, TPR domains are found in the human Hop co-chaperone, which is known to use its TPR domains to bind either HSP70 or HSP90 [START_REF] Schmid | The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop[END_REF][START_REF] Batista | Low sequence identity but high structural and functional conservation: The case of Hsp70/Hsp90 organizing protein (Hop/Sti1) of Leishmania braziliensis[END_REF]. This suggests that such bacterial class C JDPs could recruit DnaK or other chaperones in specific protein complexes to orchestrate their assembly or disassembly.

A substantial number of class C JDPs possess domains likely involved in DNA related functions (66/913), including (i) DNA binding with the λ phage repressor-like HTH and the RHH DNA-binding domains, or (ii) DNA processing with the FtsK gamma domain known to orientate DNA translocation in E. coli, the BAH domain (Bromo adjacent homology domain) generally found in chromatin-associated proteins and in protein-protein interaction module specific to transcription regulation, the ERCC4 nuclease domain, and the NERD domain with possible nuclease function as well. In addition, J-domains were also found associated with RNA binding domain or processing (58/913), including the KTSC and the exonuclease domains, and potentially the P-loop ATP binding domain (36/913) often found in helicases. This suggests the selective recruitment of DnaK to DNA replication or repair and for gene regulation [START_REF] Jonas | Proteotoxic stress induces a cell-cycle arrest by stimulating Lon to degrade the replication initiator DnaA[END_REF][START_REF] Goldfless | DNA repeat rearrangements mediated by DnaK-dependent replication fork repair[END_REF]. The fact that a J-domain is potentially associated with nuclease activity additionally suggests that these bacteria could make use of the JDP/HSP70 chaperone machine to specifically degrade foreign DNA during infection or as a defense mechanism against bacteriophages. J-domains were also found associated with metabolism-related (16/913) or proteolysisrelated domains (7/913), which is in line with DnaK's role in the central metabolism as well as in targeting specific proteins for degradation by proteases [START_REF] Anglès | Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism[END_REF][START_REF] Cho | Individual and collective contributions of chaperoning and degradation to protein homeostasis in E. coli[END_REF][START_REF] Sakr | Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones[END_REF]. How a direct coupling of a Jdomain to such activities would represent a more efficient way to utilize DnaK function remains

unknown. Yet, it is also possible that these domains are not functional but merely serve as specific substrate binding regions or localize DnaK to protein complexes.

We also found that novel class C JDP domains might also be related to stress and cell signaling (102/913; e.g., thioredoxin, GUN4, kinase, Pentapeptide Repeat Protein PRP, PilZ or CheY-like domains), as well as to protein trafficking, secretion and host control (115/913; e.g. SNARE, Sec1-like, T2SS-like, HCP-like, GAS, NTF2, CysZ and MFS transporter). Previous studies of cyanobacterial JDPs showed that the Sll1011 protein of Synechocystis sp. PCC6803 harbors a J-domain associated with a PRP domain, which was shown to bind the DNA gyrase and to protect it from certain toxins in other bacteria [START_REF] Collin | The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents[END_REF]. Although non-essential for growth as a single mutation, the sll1011 mutation was lethal when combined with mutations in other JDP genes [START_REF] Düppre | Specific and promiscuous functions of multiple DnaJ proteins in Synechocystis sp[END_REF], as it is the case for djlA and cbpA in E. coli [START_REF] Genevaux | The djlA gene acts synergistically with dnaJ in promoting Escherichia coli growth[END_REF][START_REF] Ueguchi | A study of the double mutation of dnaJ and cbpA, whose gene products function as molecular chaperones in Escherichia coli[END_REF]. To date, nothing is known about the function of this protein. Two other remarkable examples are the PilZ domain, which was shown to be involved in binding to the cyclic di-GMP second messenger known to regulate biofilm formation [START_REF] Hou | Structural insights into the mechanism of c-di-GMP-bound YcgR regulating flagellar motility in Escherichia coli[END_REF] and the CheY receiver domain of the response regulator CheY that is found in two-component signaling systems in bacteria [START_REF] Jenal | Single domain response regulators: molecular switches with emerging roles in cell organization and dynamics[END_REF]. How a HSP70/J-domain pair directly coupled to these domains could contribute to such signaling pathways in bacteria remains unknown. Furthermore, several class C JDPs identified contain a J-domain associated with a kinase domain (Fig. 4). Intriguingly, the molecular target responsible for the fibrolamellar carcinoma (FLC), a rare liver cancer, is known as the DNAJB1-PRKACA chimera, which is obtained after a 400 kb deletion on chromosome 19 in human [START_REF] Honeyman | Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma[END_REF]. The resulting chimeric protein, which retained its enzymatic activity, was shown to be composed of the J-domain grafted to the C-terminal part of protein kinase A [START_REF] Honeyman | Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma[END_REF][START_REF] Turnham | An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma[END_REF]. Remarkably, the kinase domain is similar to the kinase domain found in the bacterial J-kinase identified (namely IPR000719 and IPR011009 in Table S2). In addition, the presence of a J-domain grafted to HCP-like and T2SSlike domains suggests that the J-domain could recruit DnaK chaperone power specifically to secretion apparatus [START_REF] Collet | Protein polarization driven by nucleoid exclusion of DnaK(HSP70)-substrate complexes[END_REF][START_REF] Aschtgen | The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 Type VI secretion system, is inserted by YidC[END_REF]. Although the multifunctional DnaK, assisted by its generic class A JDP DnaJ, was previously shown to be involved in similar pathways (review in [START_REF] Castanié-Cornet | Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane[END_REF]), the specific association of a J-domain with such domains is intriguing and deserves further analysis.

Finally, 173 class C JDPs had their J-domain associated with domains of unknown function (DUF805, 1232, 1266, 1566, 2759, 3099, 3102, 4101, 4157, 4168, and 4388; AF1342 and PF12339; Fig. 4A). Note that the DUF4101 domain, which is found in 66 class C JDPs of Cyanobacteria, is also present in JDPs of Arabidopsis thaliana. The membrane anchored plant JDP protein ARC6 (Uniprot: Q9FIG9) was shown to be involved in chloroplast division by promoting the assembly or the stabilization of the division ring formed by FtsZ [START_REF] Vitha | ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2[END_REF]. In Cyanobacteria, it has been shown that a representative member of this class C JDP subgroup, namely Ftn2, was also involved in cyanobacterial cell division [START_REF] Koksharova | A Novel Gene That Bears a DnaJ Motif Influences Cyanobacterial Cell Division[END_REF].

-Taxonomic distribution of the most represented domains

Analysis of the taxonomic distribution of the most represented domains adjacent to a Jdomain reveals that except for the likely generic domains identified (i.e., TPR/WD40, SH3-like, Kinase and NTF2 domains), the domains that are potentially involved in more specialized cellular functions or functions related to special groups of bacteria are generally well clustered into certain groups of bacteria. As examples, the DUF4101, PRP and P-loop domains appear specific of class C proteins from Cyanobacteria, the KTSC domain is found only in class C protein of Bacteroidota and Aquificota species, the NERD domain has been identified only in some Actinobacteria, the CheY-like and λ repressor-like DNA-binding domains are identified only in certain genomes of δ-proteobacteria (and also Oligoflexia for two of them), and the PF12339 domain is mainly found in proteins from γ-proteobacteria (Fig. 4B). The presence of these functional domains only in a given taxonomic subgroup could reveal an implication in a function related to the lifestyle of these bacterial species.

-J-domain associates with previously undescribed conserved regions Intriguingly, we did not detect any known conserved domain for 1552 novel class C JDPs from all the databases integrated in InterPro, although these proteins represent almost two thirds of the new JDPs identified. We believe that some of these JDPs may not be isolated sequences and instead, could form subfamilies of J-domains flanked by yet unlabeled conserved regions.

To highlight such potential sequence conservation, we performed all-against-all BlastP comparisons on these 1552 proteins whose results were converted into a graph that was then partitioned using community detection. We retained 8 clusters with at least 15 members for which 8 conserved regions (CR1 to CR8) have been identified after manual inspection of the multiple alignments. The conserved regions have been used to build new HMMER profiles that have been tested against the 1552 class C JDP revealing the specificity of these new profiles since they were not identified in sequences other than the ones belonging to the given cluster (SI-Materials and methods). The various architectures of these new groups of proteins are summarized in Table S3 together with the sequence conservation of the regions illustrated by WebLogos. Note that the largest group, CR1 (195 sequences), was found in different taxonomic subgroups of α-proteobacteria, CR2 (96) in α-and γ-proteobacteria, CR3 (83) and CR4 (78) only in Cyanobacteria, CR5 [START_REF] Li | The crystal structure of the yeast Hsp40 Ydj1 complexed with its peptide substrate[END_REF] mainly in β-proteobacteria, γ-proteobacteria, and Bacteroidota, CR6 [START_REF] Wall | The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone[END_REF] mainly in Actinobacteria, CR7 [START_REF] Greene | Role of the J-domain in the cooperation of Hsp40 with Hsp70[END_REF] in Sphingomonadales, an α-proteobacteria taxonomic subgroup, and CR8 [START_REF] Deuerling | Trigger Factor and DnaK possess overlapping substrate pools and binding specificities[END_REF] coming from different bacterial taxonomic phyla. The position of the Jdomain in these subgroups is shown in Fig. 4A.

Finally, analysis of the novel class C JDPs also revealed the presence of a significant number of very short JDPs of 54 to 100 amino acids in length with either no conserved region adjacent to the J-domain (i.e., 105 JDP sequences in 99 genomes widespread among the bacterial genomes analyzed) or with a J-domain flanked by trans-membrane segments (one group of 83 sequences in Cyanobacteria genomes). In E. coli, the minimal requirement for DnaJ to partially support its DnaK co-chaperone functions in vivo is known to be the J-domain flanked by its adjacent G/F rich region: a construct known as DnaJ12 (N-terminal first 108 amino acids of DnaJ [START_REF] Wall | The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication[END_REF]). Further truncated DnaJ constructs were not active and overexpression of the Jdomain alone was toxic in a strictly DnaK-dependent manner [START_REF] Horne | The Hsp40 J-domain Stimulates Hsp70 When Tethered by the Client to the ATPase Domain[END_REF]. This suggests that short JDPs might be functional in recruiting DnaK to specific protein complexes or perhaps to specifically modulate the DnaK-DnaJ-GrpE chaperone cycle. Remarkably, a 94 amino acids long JDP present in the environmental bacterium Shewanella oneidensis has been recently characterized by Maillot and colleagues [START_REF] Maillot | Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network[END_REF]. The authors showed that this very short JDP, named AtcJ, is essential for bacterial growth at low temperature and that both the J-domain and the adjacent Cterminal region are involved. In this case, the approximately 21 amino acids long adjacent sequence was shown to specifically interact with another protein of unknown function, named AtcC, which is located in the same operon. This suggests that the small sequence adjacent to the J-domain could recruit DnaK to multiprotein complexes involved in cold adaptation. Although the mechanism that confers a JDP-dependent cold resistance is not known, this is the first report characterizing such short JDPs in bacteria. Finally, the association of J-domains with transmembrane segments is also intriguing and suggests a direct involvement of the JDP in recruiting Hsp70 to secretion apparatus or translocases, similar to Pam16 and 18 JDPs that are part of the mitochondrial inner membrane import motor [START_REF] Craig | Hsp70 at the membrane: driving protein translocation[END_REF]. Note that the mutation of a representative of this subclass in the cyanobacteria Synechocystis sp. PCC6803, namely sll1666, showed no growth defect unless it was combined with other mutations in JDP encoding genes, as described above for Sll1011. To our knowledge, nothing is known about the function of this protein.

-Co-evolution of class C J-domains and their associated neighboring domains Since different InterPro domains or HMMER profiles were required to identify J-domains (suggesting divergent subfamilies of J-domains), we asked whether class C J-domains have coevolved with their neighboring domain sequences. To answer this, we computed two phylogenetic trees based exclusively on the J-domain sequences. The first one was constructed on J-domain sequences from JDP groups harboring some domain present in InterPro (1111 sequences) (Fig. S3) and the second on class C JDPs with no known InterPro domain detected.

Note that members of the KTSC group were added as reference (1609 sequences) (Fig. 5A).

Remarkably, based solely on the J-domain sequence, the clusters obtained in both reconstructed trees correspond generally to the groups observed when considering the domains associated to the J-domain. Two exceptions are the CR8 domain (Fig. 5A) and the SH3-like associated J-domains (Fig. S3) that are found at dispersed location in their respective tree, suggesting several independent acquisitions of these two domains along evolution. Two clusters, namely CR7 (Fig. 5A) and the SNARE associated J-domains (Fig. S3) have unexpected locations on the tree. The SNARE/Sec1-like cluster is found inside the DjlA group suggesting that their J-domain sequences are related and that the previous DjlA domain has been replaced by the SNARE one in the ancestor genome of today Cyanobacteria. The CR7 domain (sequences from Sphingomonadales) shows some conserved common aligned positions, especially three cysteines, with the CR1 group found in sequences from different taxonomic subgroups of α-proteobacteria (Table S3). Except one genome of Sphingomonadales, the others own a protein belonging to each of the two groups. From those observations, we expected that the CR1 and CR7 J-domains of Sphingomonadales will form two sister groups on the tree inside the CR1 cluster instead of the two distant clusters that are obtained on Fig. 5A. Similar result was obtained on a tree computed on the complete CR1 and CR7 sequences where the CR7 proteins are branching out with a CR1 protein from another αproteobacteria taxonomic subgroup. Therefore two hypotheses may explain the emergence of the CR7 JDPs: in the common ancestor of the today Sphingomonadales, either the gene coding for CR1 JDP has been duplicated or the ancestral gene coding CR7 proteins has been acquired through a horizontal gene transfer of a CR1 JDP gene from another unidentified αproteobacteria species. In any cases, once acquired in the common ancestor, the CR7 proteins should have subsequently undergone a rapid evolution that also affected the J-domain sequences, which would explain the different location of the CR1 and CR7 J-domain clusters on the tree.

In conclusion, except for the CR8 and the SH3-like associated J-domains, once the association of the J-domain and its neighboring domain has been set up, the sequences of both domains appear to co-evolve. Moreover, the fact that class C JDPs containing a specific CR domain have their J-domain sequences clustered on the tree (Fig. 5A) reinforces the hypothesis that they constitute new groups of class C JDPs whose roles and functions have yet to be determined.

JDP diversity in bacteriophages

Proliferation of viruses requires the effective reprogramming of the host and the synthesis and folding of a large number of viral proteins necessary for genome replication, protein synthesis and viral capsid assembly. The chaperone Hsp70 is often recruited by eukaryotic viruses to assist entry, replication, gene expression, folding and assembly of viral proteins, and to control the host cell cycle [START_REF] Knox | Heat shock protein 40 (Hsp40) plays a key role in the virus life cycle[END_REF][START_REF] Zhang | Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway[END_REF][START_REF] Cao | DnaJA1/Hsp40 is co-opted by influenza A virus to enhance its viral RNA polymerase activity[END_REF][START_REF] Mayer | Recruitment of Hsp70 chaperones: a crucial part of viral survival strategies[END_REF][START_REF] Isken | CRISPR/Cas9-Mediated Knockout of DNAJC14 Verifies This Chaperone as a Pivotal Host Factor for RNA Replication of Pestiviruses[END_REF]. Some viruses, including the simian virus 40 (SV40) and the herpes simplex virus 1 (HSV-1), were shown to encode their own JDPs and express them to control the host Hsp70 [START_REF] Adlakha | The Herpes Simplex Virus 1 Immediate Early Protein ICP22 Is a Functional Mimic of a Cellular J Protein[END_REF][START_REF] Sullivan | T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis[END_REF]. The well-characterized SV40 virus possesses class C JDPs T-antigens that use their N-terminal J-domains for (i) transcriptional activation of viral genes, (ii) viral DNA replication, (iii) capsid assembly, and (iv) controlling host signaling pathways to facilitate viral replication [START_REF] Sullivan | T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis[END_REF]. Growth of bacterial viruses (bacteriophages) was also shown in some cases to rely on bacterial host chaperones and several of the main molecular chaperone families studied nowadays were originally identified for their role in bacteriophage growth [START_REF] Georgopoulos | Toothpicks, serendipity and the emergence of the Escherichia coli DnaK (Hsp70) and GroEL (Hsp60) chaperone machines[END_REF]. Previous work showed that putative gene products showing sequence similarities with the J-domain can be found in some mycobacteriophage and enterobacteriophage genomes [START_REF] Perrody | A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli[END_REF][START_REF] Hatfull | Comparative genomic analysis of 60 Mycobacteriophage genomes: genome clustering, gene acquisition, and gene size[END_REF]. In addition, the T4 related phages RB16, RB42 and RB43 were shown to encode a bona fide class C JDP, named Rki, with a fully functional J-domain capable of interacting with DnaK [START_REF] Perrody | A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli[END_REF]. In this case, it has been shown that Rki functions to control the host stress-response during the early stages of bacteriophage infection, specifically by interacting with the host DnaK/Hsp70 chaperone to stabilize the major host heat-shock factor, σ 32 , thus leading to the induction of the host heat shock genes, including the GroEL chaperone that is essential for RB43 growth. This work shows that control of the host Hsp70 by viral JDPs is well conserved. Therefore, we have used the same identification and classification approach as the one we set up for the bacterial genomes to identify JDPs in bacteriophages. Following this approach, the putative phage JDPs identified among the 968342 annotated phage proteins available in the NCBI database (which are found in 8216 phage genomes) were extracted and further analyzed with InterProScan, classified by JDP class and added into our local database. This analysis lead to the identification of 137 JDPs, including 136 class C JDPs and only one class B found in an Agrobacterium tumefaciens Atu_ph07 phage. Of note, we did not detect full length DnaK in these phages. After removing strictly identical amino acid sequences among the class C JDPs, our sample was reduced to 49 unique sequences. The multiple sequence alignment of these 49 unique JDPs followed by tree reconstruction shows that these phage proteins can be grouped into 17 subgroups of homologous sequences (Fig. S4). Among these proteins only one subgroup (JCP1) possesses a known domain adjacent to the J-domain, namely the protein kinase domain, which is also found in some bacterial class C JDP (Table S2, Fig. 4). The phage sequences were also scanned with our local database of HMM profiles built on the conserved regions discovered in the bacterial JDPs and the CR8 was detected in the Cellulophaga phage phi17:2 sequence AGO47657 from JCP14 (Fig. S4). No conserved regions were found in the other subgroups. The E. coli phage Rki JDP described above was found within subgroup JCP7 together with homologs present in a variety of Citrobacter, Cronobacter, Enterobacter, Klebsiella phages (Table S1). JDPs from mycobacteria phages were the most represented in numbers (25 in group JCP1, 2 in JCP2 and 61 in JCP3 over the 136 phage class C JDPs), most likely due to the significant number of phages that have been sequenced so far. Yet, their diversity in sequences is poor (Fig. S4; Table S1).

Although less numerous than in bacterial genomes, this analysis has revealed the presence of very diverse class C JDPs in bacteriophages, suggesting that hijacking the host HSP70 chaperone machine for phage proliferation is a relatively well conserved feature among viruses. The fact that only JDP co-chaperones and no DnaK were found in bacteriophages genomes is in agreement with such a hypothesis.

Concluding remarks and perspectives

This survey of 1709 representative bacterial genomes revealed for the first time that bacteria possess a remarkable reservoir of yet unexplored JDP proteins (Fig. 6; Fig. 3C). As judged by the very diverse putative domains and functions linked to a J-domain, our analysis suggests that such novel JDP-associated functions might significantly expand the repertoire of yet unknown DnaK functions in bacterial physiology, resilience and host control. The next challenge for microbiologists in the field will be to explore the function of these new JDPs, especially the most represented families identified here, which might be highly relevant with respect to their conservation. Many new J-domain associations identified were only represented by one or two members. Although we believe that the set of genomes examined here is relatively well representative of the bacterial taxonomy, analyzing larger sets of genomes might also increase the numbers of hits for certain groups and perhaps even reveal additional new families. Another striking aspect of this analysis is the identification of eight novel conserved regions associated with a J-domain, named here as CR domains, that were not previously identified as domains and that deserve further interest as some of them are found in many genomes analyzed in the review. Similarly, many class C JDPs identified are very short, with no domain or conserved region assigned to them. Whether these proteins act as solitary modulators of HSP70 or whether the short flanking sequences are sufficient to bind HSP70 clients or to give a specific localization to HSP70 is unknown. Finally, our survey of proteins from 8216 bacteriophage genomes further supports the previous observation that some bacteriophages encode their own class C JDPs, although only for about 1% of them in our case. This is far below the number of JDPs found in bacteria where we found an average of 3.7 JDPs per genome. This suggests that evolving a specialized JDP to hijack or modulate the host HSP70 is not a main feature for bacteriophage growth. Our analysis also revealed that except for one kinase domain and the CR8 conserved region, there is no apparent conservation between the domains associated with a J-domain in bacteriophages and bacteria, further supporting the idea that these JDPs likely have specific phage related functions.

ACKNOWLEDGMENTS:

We thank Petra Langendijk-Genevaux and Yves Quentin for their valuable advice. Clément Birbes, Callum Burnard and Cyril Kurylo are also acknowledged for their contribution at the beginning of this project as first year master students from the University Paul Sabatier Toulouse. This work was supported by ANR-2019/ChapCop to PG. The tree was obtained with online iTOL (https://itol.embl.de/) (B) Distribution of dnaK, dnaJ and grpE chromosomal organizations observed in the 1,709 genomes. The dnaK, dnaJ and grpE genes are depicted as K, J and E respectively. Genes are considered co-localized when they are on the same strand and when their intergenic distance is lower than 150bp. Numbers correspond to the number of times the organization is observed in our genome dataset.

  . E. coli encodes three different HSP70 proteins (DnaK, HscA and HscC) and six JDPs, one class A (DnaJ), one class B (CbpA) and 4 class C (DjlA, HscB, DjlB and DjlC) (Fig 1C). Extensive

  DnaK and GrpE taxonomic distributions largely confirmed their overall presence in bacterial genomes (2223 DnaK and 1768 GrpE) and their co-occurrence with the DnaJ class A partners (Fig. 2A; Table

Fig. 1 :

 1 Fig. 1: DnaK/HSP70 and JDP families in E. coli. (A) The DnaK chaperone cycle. The ATPbound state of DnaK receives the substrate from DnaJ. Interaction of DnaK with DnaJ and the substrate triggers ATP hydrolysis by DnaK, generating a stable complex between the substrate and the ADP-bound state of DnaK. GrpE mediates ADP-ATP exchange, stimulates substrate release and regenerates DnaK-ATP. The substrate binding pocket is indicated with the red arrow. The structures of the open state of DnaK-ATP (PDB 4B9Q) and the closed state of DnaK-ADP (PDB 2KHO) are displayed using Chimera. (B) The three-dimensional structure of the Jdomain of DnaJ (PDB 1XBL) is shown in green, with the conserved HPD motif in orange. (C)The three E. coli HSP70 chaperones with their associated JDP and GrpE partners. The aminoacid length of each protein is indicated. For HSP70 proteins, the nucleotide-binding domain (NBD in dark blue) and the substrate-binding domain (SBD in light blue) are shown. For the JDPs, the J-domain (in green), the G/F glycine-phenylalanine-rich region (orange), the zinc binding domain (ZBD, purple), the C-terminal peptide-binding domains (CTD I and II in red), the dimerization domain (DD in dark grey), the transmembrane (TM in fuchsia) or putative transmembrane (PTM in hatched fuchsia), and the domains of unknown function (in light grey)

Fig. 2 :

 2 Fig. 2: Taxonomic distribution of the main JDP class A, B and C partners of DnaK in bacteria. (A) GTDB taxonomy tree of the 1,709 selected bacterial genomes annotated with bar charts indicating the number of DnaK, GrpE and its JDP class A (DnaJ) class B (CbpA) and DjlA found in each genome. The taxonomic groups are colored at the phyla level, except for Proteobacteria for which the class level is used, according to a color palette. For DnaK, the scale of the dashed line corresponds to values of five and ten copies of DnaK. Note that in three genomes (M. pneumoniae M29, Mycoplasma genitalium G37 and Mycoplasma gallisepticum str R) the class B JDPs are known as TopJ, which is longer than classical class B with a region inserted between the G/F region and the CTD domain (TopJ for P75354, P47442 and Q7NBE5).

Fig. 3 :

 3 Fig. 3: Taxonomic distributions of the HscA-HscB and the HscC-DjlBC systems and of all JDPs identified in selected 1,709 bacterial genomes. The bacteria group colors on the GTDB taxonomic tree of the 1,709 studied genomes correspond to those of Figure 2A. The number of HscA and HscB (A), HscC and DjlB/C (B) found in each genome is indicated by bar charts around the trees. (C) The number of JDPs found from each class in each genome is indicated by

Fig. 4 :Fig. 5 :

 45 Fig. 4: The unexplored reservoir of new J-domain associations in bacterial class C JDPs. (A) Name, number, possible function, and position of the protein domains found in association with a J-domain. The J-domain is shown in green and the adjacent domain(s) in grey at the top of the panel. The frames below the arrows present the names and the number (shown as superscript between brackets) of protein domains found adjacent to a J-domain whose position at the N-terminal, C-terminal or in the middle of the protein is shown. The color indicates the possible broad functional class of these domains (orange stands for protein-protein interactions and assembly of multiprotein complexes; dark blue for secretion systems, transporter and host growth control; light blue for signaling, stress and drug resistance; red for DNA binding and processing; brown for metabolism; green for RNA binding and processing; purple for proteolysis; pink for nucleotide binding and hydrolysis; grey, for domains with non-classified function). The previously unidentified conserved regions found associated with a J-domain in bacteria (unknown conserved regions CR1 to CR8) and in bacteriophages (phage groups JCP1 to JCP17) are also shown. Note that only JCP1 shows similarity with the protein kinase domain found in the new class C JDPs (light blue). (B) Taxonomic distribution in bacteria of the most abundant class C JDP groups (≥ 10 proteins). The groups are defined by the protein domains found associated with the J-domain and are abbreviated when needed as follows: TPR for domains TPR and/or WD40; PRP for domain Pentapeptide repeat; P-loop for domain P-loop NTPase; SNARE for domains SNARE and/or Sec1-like; 123399 for domain PF12339; CheY for domain CheY-like; λ for domain λ repressor-like DNA-binding; HCP for domain HCP-like Type VI secretion system; PK for domain Protein kinase domain; SH3 for SH3-like domain. For each group a bar chart is drawn around the tree and a bar indicates the presence of at least one JDP

Fig. 6 :

 6 Fig. 6: Bacteria as a reservoir of novel JDP functions. The distribution of the different classes of JDPs identified using our set of selected bacterial genomes is shown. JDP class A (pink), class B (blue) and class C (green). At least 66 associations with known domains and 8 with previously unidentified conserved domain have been found among bacterial class C JDPs.