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The conserved WhiA protein family is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motive and has been identified as a transcription factor in actinomycetes. InBacillus subtilis, the absence of WhiA influences cell division and chromosome segregation, however, it is still unclear how WhiA influences these processes, but the protein does not seem to function as transcription factor in this organism. To further investigate the function of WhiA inB. subtilis, we performed a yeast two-hybrid screen to find interaction partners, and a Hi-C experiment to reveal possible changes in chromosome conformation. The latter experiment indicated a reduction in short range chromosome interactions, but how this would affect either cell division or chromosome segregation is unclear. Based on adjacent genes, a role in carbon metabolism was put forward. To study this, we measured exometabolome fluxes during growth on different carbon sources. This revealed that in ΔwhiAcells the pool of branched-chain fatty acid precursors is lower. However, the effect on the membrane fatty acid composition was minimal. Transcriptome data could not link the metabolome effects to gene regulatory differences.IMPORTANCEWhiA is a conserved DNA binding protein that influences cell division and chromosome segregation in the Gram-positive model bacteriumB. subtilis. The molecular function of WhiA is still unclear, but a previous study has suggested that the protein does not function as a transcription factor. In this study, we used yeast two-hybrid screening, chromosome conformation capture analysis, metabolomics, transcriptomics and fatty acid analysis to obtain more information about the workings of this enigmatic protein.

International audience; Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ∆stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase-substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.

Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The serine/threonine kinases (STKs) of the Hanks family control cell division and envelope homeostasis, but only a few kinase substrates and associated molecular mechanisms have been identified. In this work, we identified CwlA as the first STK-PrkC substrate in the human pathogen Clostridiodes difficile and showed that CwlA is an endopeptidase involved in daughter cell separation. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, therefore controlling the hydrolytic activity in the cell wall. High level of CwlA at the cell surface led to cell elongation, whereas low level caused cell separation defects. We thus provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division.

International audience; Clostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is a STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. When compared with the wild-type strain, a ΔprkC mutant was able to sporulate and germinate but was less motile and formed more biofilm. Moreover, a ΔprkC mutant was more sensitive to antimicrobial compounds that target the cell envelope such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the ΔprkC mutant compared to the wild-type strain. Finally, in a hamster model of infection the ΔprkC mutant had a colonization delay that did not significantly affect overall virulence.

International audience; YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.

International audience; In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA replication during the cell cycle, as well as in the mechanisms that cope with stress-induced replication blocks. Similar to eukaryotes, bacteria use Hanks-type kinases and phosphatases for signal transduction, and protein phosphorylation is involved in numerous cellular processes. However, it remains unclear whether protein phosphorylation in bacteria can also regulate the activity of proteins involved in DNA-mediated processes such as DNA replication or repair. Accumulating evidence supported by functional and biochemical studies suggests that phospho-regulatory mechanisms also take place during the bacterial cell cycle. Recent phosphoproteomics and interactomics studies identified numerous phosphoproteins involved in various aspect of DNA metabolism strongly supporting the existence of such level of regulation in bacteria. Similar to eukaryotes, bacterial scaffolding-like proteins emerged as platforms for kinase activation and signaling. This review reports the current knowledge on the phosphorylation of proteins involved in the maintenance of genome integrity and the regulation of cell cycle in bacteria that reveals surprising similarities to eukaryotes.