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

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

Dissection fonctionnelle d'un nouveau régulateur de l'initiation de la réplication du chromosome bactérien

National audienc

Bacterial protein interaction networks: puzzle stones from solved complex structures add to a clearer picture

Global scale studies of protein-protein interaction (PPI) networks have considerably expanded our view of how proteins act in the cell. In particular, bacterial "interactome" surveys have revealed that proteins can sometimes interact with a large number of protein partners and connect different cellular processes. More targeted, pathway-orientated PPI studies have also helped to propose functions for unknown proteins based on the "guilty by association" principle. However, given the immense repertoire of PPIs generated and the variability of PPI networks, more studies are required to understand the role(s) of these interactions in the cell. With the availability of bioinformatic analysis tools, transcriptomics and co-expression experiments for a given interaction, interactomes are being deciphered. More recently, functional and structural studies have been derived from these PPI networks. In this review, we will give a number of examples of how combining functional and structural studies into PPI networks has contributed to understanding the functions of some of these interactions. We discuss how interactomes now represent a unique opportunity to determine the structures of bacterial protein complexes on a large scale by the integration of multiple technologies

CRISPR interference to interrogate genes that control biofilm formation in Pseudomonas fluorescens

International audienceAbstract Bacterial biofilm formation involves signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Gene inactivation is a powerful approach for functional studies but it is often labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in diverse strain isolates of P . fluorescens , SBW25, WH6 and Pf0-1. We found that CRISPRi is applicable to study complex phenotypes such as cell morphology, motility and biofilm formation over extended periods of time. In SBW25, CRISPRi-mediated silencing of genes encoding the GacA/S two-component system and regulatory proteins associated with the cylic di-GMP signaling messenger produced swarming and biofilm phenotypes similar to those obtained after gene inactivation. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 operon. We conclude that CRISPRi is a reliable and scalable approach to investigate gene networks in the diverse P . fluorescens group

Dynamics of Aspen Roots Colonization by Pseudomonads Reveals Strain-Specific and Mycorrhizal-Specific Patterns of Biofilm Formation

Rhizosphere-associated Pseudomonas fluorescens are known plant growth promoting (PGP) and mycorrhizal helper bacteria (MHB) of many plants and ectomycorrhizal fungi. We investigated the spatial and temporal dynamics of colonization of mycorrhizal and non-mycorrhizal Aspen seedlings roots by the P. fluorescens strains SBW25, WH6, Pf0-1, and the P. protegens strain Pf-5. Seedlings were grown in laboratory vertical plates systems, inoculated with a fluorescently labeled Pseudomonas strain, and root colonization was monitored over a period of 5 weeks. We observed unexpected diversity of bacterial assemblies on seedling roots that changed over time and were strongly affected by root mycorrhization. P. fluorescens SBW25 and WH6 stains developed highly structured biofilms with internal void spaces forming channels. On mycorrhizal roots bacteria appeared encased in a mucilaginous substance in which they aligned side by side in parallel arrangements. The different phenotypic classes of bacterial assemblies observed for the four Pseudomonas strains were summarized in a single model describing transitions between phenotypic classes. Our findings also reveal that bacterial assembly phenotypes are driven by interactions with mucilaginous materials present at roots

Interaction between Yeast Sgs1 Helicase and DNA Topoisomerase III

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The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

Bacterial DNA replication requires DnaA, an AAA+ ATPase that initiates replication at a specific chromosome region, oriC, and is regulated by species-specific regulators that directly bind DnaA. HobA is a DnaA binding protein, recently identified as an essential regulator of DNA replication in Helicobacter pylori. We report the crystal structure of HobA in complex with domains I and II of DnaA (DnaAI–II) from H. pylori, the first structure of DnaA bound to one of its regulators. Biochemical characterization of the complex formed shows that a tetramer of HobA binds four DnaAI–II molecules, and that DnaAI–II is unable to oligomerize by itself. Mutagenesis and protein–protein interaction studies demonstrate that some of the residues located at the HobA-DnaAI–II interface in the structure are necessary for complex formation. Introduction of selected mutations into H. pylori shows that the disruption of the interaction between HobA and DnaA is lethal for the bacteria. Remarkably, the DnaA binding site of HobA is conserved in DiaA from Escherichia coli, suggesting that the structure of the HobA/DnaA complex represents a model for DnaA regulation in other Gram-negative bacteria. Our data, together with those from other studies, indicate that HobA could play a crucial scaffolding role during the initiation of replication in H. pylori by organizing the first step of DnaA oligomerization and attachment to oriC

An expanded protein-protein interaction network in Bacillus subtilis reveals a group of hubs: Exploration by an integrative approach

We have generated a protein-protein interaction network in Bacillus subtilis focused on several essential cellular processes such as cell division, cell responses to various stresses, the bacterial cytoskeleton, DNA replication and chromosome maintenance by careful application of the yeast two-hybrid approach. This network, composed of 793 interactions linking 287 proteins with an average connectivity of five interactions per protein, represents a valuable resource for future functional analyses. A striking feature of the network is a group of highly connected hubs (GoH) linking many different cellular processes. Most of the proteins of the GoH have unknown functions and are associated to the membrane. By the integration of available knowledge, in particular of transcriptome data sets, the GoH was decomposed into subgroups of party hubs corresponding to protein complexes or regulatory pathways expressed under different conditions. At a global level, the GoH might function as a very robust group of date hubs having partially redundant functions to integrate information from the different cellular pathways. Our analyses also provide a rational way to study the highly redundant functions of the GoH by a genetic approach

ComE/ComE similar to P interplay dictates activation or extinction status of pneumococcal X-state (competence)

Since 1996, induction of competence for genetic transformation of Streptococcus pneumoniae is known to be controlled by the ComD/ComE two-component regulatory system. The mechanism of induction is generally described as involving ComD autophosphorylation, transphosphorylation of ComE and transcriptional activation by ComE similar to P of the early competence (com) genes, including comX which encodes the competence-specific sigma(X). However, none of these features has been experimentally established. Here we document the autokinase activity of ComD proteins in vitro, and provide an estimate of the stoichiometry of ComD and ComE in vivo. We report that a phosphorylmimetic mutant, ComE(D58E), constructed because of the failure to detect transphosphorylation of purified ComE in vitro, displays full spontaneous competence in Delta comD cells, an that in vitro ComE(D58E) exhibits significantly improved binding affinity for P-comCDE. We also provide evidence for a differential transcriptional activation and repression of P-comCDE and P-comX. Altogether, these data support the model of ComE similar to P-dependent activation of transcription. Finally, we establish that ComE antagonizes expression of the early com genes and propose that the rapid deceleration of transcription from P-comCDE observed even in cells lacking sigma(X) is due to the progressive accumulation of ComE, which outcompetes ComE similar to P

Bacillus subtilis serine/threonine protein kinase YabT is involved in spore development via phosphorylation of a bacterial recombinase.

We characterized YabT, a serine/threonine kinase of the Hanks family, from Bacillus subtilis. YabT is a putative transmembrane kinase that lacks the canonical extracellular signal receptor domain. We demonstrate that YabT possesses a DNA-binding motif essential for its activation. In vivo YabT is expressed during sporulation and localizes to the asymmetric septum. Cells devoid of YabT sporulate more slowly and exhibit reduced resistance to DNA damage during sporulation. We established that YabT phosphorylates DNA-recombinase RecA at the residue serine 2. A non-phosphorylatable mutant of RecA exhibits the same phenotype as the ΔyabT mutant, and a phosphomimetic mutant of RecA complements ΔyabT, suggesting that YabT acts via RecA phosphorylation in vivo. During spore development, phosphorylation facilitates the formation of transient and mobile RecA foci that exhibit a scanning-like movement associated to the nucleoid in the mother cell. In some cells these foci persist at the end of spore development. We show that persistent RecA foci, which presumably coincide with irreparable lesions, are mutually exclusive with the completion of spore morphogenesis. Our results highlight similarities between the bacterial serine/threonine kinase YabT and eukaryal kinases C-Abl and Mec1, which are also activated by DNA, and phosphorylate proteins involved in DNA damage repair