A range of genetical and physiological experiments have established that diverse bacterial cells possess a function called nucleoid occlusion, which acts to prevent cell division in the vicinity of the nucleoid. We have identified a specific effector of nucleoid occlusion in Bacillus subtilis, Noc (YyaA), as an inhibitor of division that is also a nonspecific DNA binding protein. Under various conditions in which the cell cycle is perturbed, Noc prevents the division machinery from assembling in the vicinity of the nucleoid. Unexpectedly, cells lacking both Noc and the Min system (which prevents division close to the cell poles) are blocked for division, apparently because they establish multiple nonproductive accumulations of division proteins. The results help to explain how B. subtilis specifies the division site under a range of conditions and how it avoids catastrophic breakage of the chromosome by division through the nucleoid.

Sporulation in Bacillus subtilis begins with an asymmetric cell division, producing a smaller prespore and a larger mother cell, both of which contain intact copies of the chromosome. The spoIIIE gene is required for chromosome segregation into the prespore compartment. The effects of the spoIIIE36 mutation on sigma F-dependent transcription are an indirect consequence of the failure of certain genes to enter the cellular compartment in which their transcription factor has become active. SpoIIIE may also be required to prevent sigma F from becoming active in the mother cell.

DivIVA is a conserved protein in Gram-positive bacteria and involved in various processes related to cell growth, cell division and spore formation. DivIVA is specifically targeted to cell division sites and cell poles. In Bacillus subtilis , DivIVA helps to localise other proteins, such as the conserved cell division inhibitor proteins, MinC/MinD, and the chromosome segregation protein, RacA. Little is known about the mechanism that localises DivIVA. Here we show that DivIVA binds to liposomes, and that the N terminus harbours the membrane targeting sequence. The purified protein can stimulate binding of RacA to membranes. In mutants with aberrant cell shapes, DivIVA accumulates where the cell membrane is most strongly curved. On the basis of electron microscopic studies and other data, we propose that this is due to molecular bridging of the curvature by DivIVA multimers. This model may explain why DivIVA localises at cell division sites. A Monte-Carlo simulation study showed that molecular bridging can be a general mechanism for binding of proteins to negatively curved membranes.

SummarySporulating cells of Bacillus subtilis undergo a highly polarized cell division and possess a specialized mechanism to move the oriC region of the chromosome close to the cell pole before septation. DivIVA protein, which localizes to the cell pole, and the Soj and Spo0J proteins, which associate with the chromosome, are part of the mechanism that delivers the chromosome to the cell pole. A sporulation-specific protein, RacA, encodes a third DNA-binding protein, which acts in conjunction with Soj and Spo0J to effect efficient polar chromosome segregation. divIVA mutants and soj racA double mutants have an unexpected phenotype in which specific markers to the left and right of oriC can be captured in the prespore compartment but the central oriC region is efficiently excluded. This 'residual' trapping requires Spo0J protein. We suggest that the Soj RacA DivIVA system is required to extract the oriC region from its position determined by the vegetative chromosome segregation machinery and anchor it to the cell pole.

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...

The bacterial cell wall is a highly conserved essential component of most bacterial groups. It is the target for our most frequently used antibiotics and provides important small molecules that trigger powerful innate immune responses. The wall is composed of glycan strands crosslinked by short peptides. For many years, the penicillin-binding proteins were thought to be the key enzymes required for wall synthesis. RodA and possibly other proteins in the wider SEDS (shape, elongation, division and sporulation) family have now emerged as a previously unknown class of essential glycosyltranferase enzymes, which play key morphogenetic roles in bacterial cell wall synthesis. We provide evidence in support of this role and the discovery of small natural product molecules that probably target these enzymes. The SEDS proteins have exceptional potential as targets for new antibacterial therapeutic agents.

Spore formation inthrough the newly formed spore septum. We propose that translocation of the prespore chromosome occurs by a mechanism that is functionally related to the conjugative transfer of plasmid DNA.

The 787 amino acid SpoIIIE protein of Bacillus subtilis is required for chromosome partitioning during sporulation. This process differs from vegetative chromosome partitioning in that it occurs after formation of the septum, apparently by transfer of the chromosome through the nascent septum in a manner reminiscent of plasmid conjugation. Here we show that SpoIIIE is associated with the cell membrane, with its soluble C‐terminal domain located inside the cell. Immunofluorescence microscopy using affinity‐purified anti‐SpoIIIE antibodies shows that SpoIIIE is targeted near the centre of the asymmetric septum, in support of a direct role for SpoIIIE in transport of DNA through the septum. We also report on the isolation of a mutation affecting the N‐terminal hydrophobic domain of SpoIIIE that interferes with targeting to the septum and blocks DNA transfer. This mutation also causes de‐localization of the activity of the normally prespore‐specific sigma factor, σF, consistent with the notion that SpoIIIE can form a seal between the chromosomal DNA and the leading edge of the division septum.