Marie-Françoise Noirot-Gros

An expanded view of bacterial DNA replication

A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.

Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB

Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non‐SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non‐SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram‐positive) bacteria, which we denote ‘segregation and condensation proteins’ (Scps). ScpA genes are localized next to smc genes in nearly all SMC‐ containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature‐ sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so‐called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two‐hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non‐SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.

Multiple Interactions between the Transmembrane Division Proteins of Bacillus subtilis and the Role of FtsL Instability in Divisome Assembly

About 11 essential proteins assemble into a ring structure at the surface of the cell to bring about cytokinesis in bacteria. Several of these proteins have their major domains located outside the membrane, forming an assembly that we call the outer ring (OR). Previous work on division in Bacillus subtilis has shown that four of the OR proteins-FtsL, DivIC, DivIB, and PBP 2B-are interdependent for assembly. This contrasts with the mainly linear pathway for the equivalent proteins in Escherichia coli. Here we show that the interdependent nature of the B. subtilis pathway could be due to effects on FtsL and DivIC stability and that DivIB is an important player in regulating this turnover. Two-hybrid approaches suggest that a multiplicity of protein-protein interactions contribute to the assembly of the OR. DivIC is unusual in interacting strongly only with FtsL. We propose a model for the formation of the OR through the mutual association of the membrane proteins directed by the cytosolic inner-ring proteins.

Cell-Cycle-Dependent Spatial Sequestration of the DnaA Replication Initiator Protein in Bacillus subtilis

Initiation of DNA replication must be restricted to occur only once per cell cycle. In most bacteria, DnaA protein binds replication origins and promotes the initiation of DNA replication. We have found that in Bacillus subtilis, DnaA only colocalizes with origin regions at early or late stages of the cell cycle, when the replication machinery is assembling or disassembling, respectively. In contrast, DnaA colocalizes with the DNA replication machinery during most of the cell cycle. Indeed, we present evidence that a primary function of YabA, a negative regulator of replication initiation, is to tether DnaA to the polymerase-clamp protein DnaN. Thus, YabA ensures that once the origin is duplicated, it moves away from the replisome and from DnaA. We propose that DnaA colocalization with origins is specific to the time of initiation, and that replisome/YabA-mediated spatial sequestration of DnaA prevents inappropriate reinitiation of DNA replication.

The bacterial condensin/cohesin‐like protein complex acts in DNA repair and regulation of gene expression

Structural maintenance of chromosome (SMC) and the SMC‐interacting kleisin protein families have key functions in the chromosome organization of most organisms. Here, we report that the Bacillus subtilis kleisin, ScpA, can form a ternary complex with the SMC and ScpB proteins in a yeast tri‐hybrid assay, supporting the notion of a bacterial cohesin/condensin‐like complex. Furthermore, ScpA interacts in two‐hybrid assays with the AddAB complex, essential for recombinational repair, with DegS, a two‐component sensor kinase, as well as with other potential transcription regulators. Point mutations in scpA allowing growth under conditions not permissive for the spcA null and not affecting chromosome condensation were isolated. Among these mutations, some affected DNA repair and gene regulation, thus separating ScpA functions in these two pathways from its functions in chromosome condensation and segregation. Some separation‐of‐function mutations in scpA caused a deficiency in the repair of mitomycin C DNA lesions that was suppressed by increasing the intracellular dosage of the interacting AddAB complex. Another mutation in scpA deregulated the expression of genes encoding degradative enzymes that are known to be controlled by the interacting DegS kinase. We propose that the SMC–ScpA–ScpB complex could: (i) recruit the AddAB helicase/nuclease to act in post‐replicative repair; and (ii) form a complex with the DegS sensor kinase that inhibits its kinase activity. Moreover, our results indicate that the role of cohesin and condensin complexes in DNA repair and gene regulation is evolutionary conserved.

Distinctive genetic features exhibited by the Y‐family DNA polymerases in Bacillus subtilis

Translesional DNA polymerases form a large family of structurally related proteins, known as the Y‐polymerases. Bacillus subtilis encodes two Y‐polymerases, referred herewith as Pol Y1 and Pol Y2. Pol Y1 was expressed constitutively and did not mediate UV mutagenesis. Pol Y1 overexpression increased spontaneous mutagenesis. This effect depended on Pol Y1 polymerase activity, Pol Y1 interaction with the β‐clamp, and did not require the presence of the RecA protein. In addition, Pol Y1 overexpression delayed cell growth at low temperature. The growth delay was mediated by Pol Y1 interaction with the β‐clamp but not by its polymerase activity, suggesting that an excess of Pol Y1 in the cell could sequester the β‐clamp. In contrast, Pol Y2 was expressed during the SOS response, and, in its absence, UV‐induced mutagenesis was abolished. Upon Pol Y2 overproduction, both UV‐induced and spontaneous mutagenesis were stimulated, and both depended on the Pol Y2 polymerase activity. However, UV mutagenesis did not appear to require the interaction of Pol Y2 with the β‐clamp whereas spontaneous mutagenesis did. In addition, Pol Y2‐mediated spontaneous mutagenesis required the presence of RecA. Together, these results show that the regulation and the genetic requirements of the two B. subtilis Y‐polymerases are different, indicating that they fulfil distinct biological roles. Remarkably, Pol Y1 appears to exhibit a mutator activity similar to that of Escherichia coli Pol IV, as well as an E. coli UmuD‐related function in growth delay. Pol Y2 exhibits an E. coli Pol V‐like mutator activity, but probably acts as a single polypeptide to bypass UV lesions. Thus, B. subtilis Pol Y1 and Pol Y2 exhibit distinctive features from the E. coli Y‐polymerases, indicating that different bacteria have adapted different solutions to deal with the lesions in their genetic materiel.

ComE/ComE∼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∼P of the early competence (com) genes, including comX which encodes the competence‐specific σX. 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, ComED58E, constructed because of the failure to detect transphosphorylation of purified ComE in vitro, displays full spontaneous competence in ΔcomD cells, an that in vitro ComED58E exhibits significantly improved binding affinity for PcomCDE. We also provide evidence for a differential transcriptional activation and repression of PcomCDE and PcomX. Altogether, these data support the model of ComE∼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 PcomCDE observed even in cells lacking σX is due to the progressive accumulation of ComE, which outcompetes ComE∼P.

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.

Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

DNA polymerase I acts in translesion synthesis mediated by the Y-polymerases in Bacillus subtilis

Translesion synthesis (TLS) across damaged DNA bases is most often carried out by the ubiquitous error‐prone DNA polymerases of the Y‐family. Bacillus subtilis encodes two Y‐polymerases, Pol Y1 and Pol Y2, that mediate TLS resulting in spontaneous and ultraviolet light (UV)‐induced mutagenesis respectively. Here we show that TLS is a bipartite dual polymerase process in B. subtilis, involving not only the Y‐polymerases but also the A‐family polymerase, DNA polymerase I (Pol I). Both the spontaneous and the UV‐induced mutagenesis are abolished in Pol I mutants affected solely in the polymerase catalytic site. Physical interactions between Pol I and either of the Pol Y polymerases, as well as formation of a ternary complex between Pol Y1, Pol I and the β‐clamp, were detected by yeast two‐ and three‐hybrid assays, supporting the model of a functional coupling between the A‐ and Y‐family polymerases in TLS. We suggest that the Pol Y carries the synthesis across the lesion, and Pol I takes over to extend the synthesis until the functional replisome resumes replication. This key role of Pol I in TLS uncovers a new function of the A‐family DNA polymerases.