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.T he replication of the bacterial chromosome is carried out by a large multiprotein machine, the replisome, in which the activities of individual polypeptides are highly coordinated to achieve efficient and faithful DNA replication. The components of bacterial replisomes have been characterized extensively, revealing the molecular mechanisms at work in a DNAreplication apparatus (1, 2). Localization studies indicated that the replication machinery is preferentially at midcell, suggesting a factory model of replication in which the DNA template moves through a rather stationary polymerase (3). In contrast, the origin regions of the chromosomes are moving toward the cell poles during cell-cycle progression (4, 5). However, other aspects of DNA replication still remain unclear. For instance, it is not known how the replication machinery coordinates its action with other cellular processes in a variety of environmental conditions or what the determinants that specify replisome or origin positions within the cell are. Mutants affected in these biological processes have not been reported yet, possibly because they display weak or inconsistent phenotypes caused by redundant functions.To gain insight into this unexplored area, we used genomewide yeast two-hybrid screens (6) to identify the proteins that physically associate with known replication proteins from the Gram-positive bacterium Bacillus subtilis. To circumvent one of the main limitations of the approach, the false-positive interactions, we verified experimentally the specificity of every potential interaction identified in the screens. The resulting protein network is composed of 91 specific interactions connecting 69 proteins. Over one fourth of the interactions were described previously in bacteria or eukaryotes, showing that our approach yields biologically significant interactions. The remaining interactions are previously uncharacterized, and in combination with data from the literature, many of their biological roles can be hypothesized. They link DNA replication with DNA recombination and repair, potential origin-and replisome-anchoring membrane complexes, signaling pathways, and numerous proteins of unkno...

SummaryRibonucleases J1 and J2 are recently discovered enzymes with dual 5Ј-to-3Ј exoribonucleolytic/ endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild-type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5Ј-to-3Ј exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.

Endogenous peptidoglycan (PG)-hydrolyzing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PGs of pathogens and commensal bacteria may also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation were dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase (encoded by oatA) led to bacterial growth arrest, indicating the potential lethality of oatA and a need for its tight regulation. A novel regulatory factor, SpxB (previously denoted as YneH), exerted a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multistep response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two-component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis. Peptidoglycan (PG)7 is the major and essential component of the bacterial cell envelope, the main function of which is to preserve cell integrity by withstanding internal osmotic pressure (1, 2). It is also responsible for cell shape, participates in cell division, and serves as support for attachment of other cell wall molecules such as teichoic acids, proteins, and exopolysaccharides (3). The bacterial PG is a giant multilayer polymer that envelops the cell as a rigid sacculus. It is composed of N-acetylglucosamine-N-acetylmuramic acid disaccharide pentapeptide blocks that are synthesized intracellularly and transported through the cytoplasmic membrane as lipid-disaccharide pentapeptides. These blocks are covalently linked to the pre-existing PG polymers by high molecular weight penicillin-binding proteins (4).To allow cell division and surface expansion, the rigid PG sacculus has to be relaxed. This is achieved by PG ruptures, which could be introduced in several ways. First, not all possible covalent bonds are formed during PG synthesis; for example, only 36% of possible PG cross-links between stem peptides are formed in Lactococcus lactis (5). Bacteria also possess a number of endogenous bacterial enzymes (collectively called autolysins) that disrupt PG and can result in cell lysis. According to their hydrolytic bond specificity and products, autolysins are classified as muramidases, lytic transglycosylases, glucosaminidases, amidases, and peptidases (6). Bacteria often possess several autolysins, e.g. L. lactis encodes a main autolysin (N-acetylglucosaminidase AcmA) (7, 8) and four minor PG hydrolases (9, 10) as well as prophage-encoded bacteriolytic enzymes (11).In the human or animal host, antimicrobial PG lytic enzymes such as lysozyme constitute a first line of defense against infection. Bactericidal propertie...

Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.