SummaryCell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the division septum (the divisome). Here we describe the identification of a new cell division protein in Bacillus subtilis . This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell division defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell division, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA , encoding another Z-ring interacting protein, had a synthetic lethal division effect. We conclude that SepF is a new member of the Gram positive divisome, required for proper execution of septum synthesis.

Bacteria use the replication origin-to-terminus polarity of their circular chromosomes to control DNA transactions during the cell cycle. Segregation starts by active migration of the region of origin followed by progressive movement of the rest of the chromosomes. The last steps of segregation have been studied extensively in the case of dimeric sister chromosomes and when chromosome organization is impaired by mutations. In these special cases, the divisome-associated DNA translocase FtsK is required. FtsK pumps chromosomes toward the dif chromosome dimer resolution site using polarity of the FtsK-orienting polar sequence (KOPS) DNA motifs. Assays based on monitoring dif recombination have suggested that FtsK acts only in these special cases and does not act on monomeric chromosomes. Using a two-color system to visualize pairs of chromosome loci in living cells, we show that the spatial resolution of sister loci is accurately ordered from the point of origin to the dif site. Furthermore, ordered segregation in a region ∼200 kb long surrounding dif depended on the oriented translocation activity of FtsK but not on the formation of dimers or their resolution. FtsK-mediated segregation required the MatP protein, which delays segregation of the dif-surrounding region until cell division. We conclude that FtsK segregates the terminus region of sister chromosomes whether they are monomeric or dimeric and does so in an accurate and ordered manner. Our data are consistent with a model in which FtsK acts to release the MatP-mediated cohesion and/or interaction with the division apparatus of the terminus region in a KOPS-oriented manner.

In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a 'repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.

Construction and microscopic imaging of protein fusions to green fluorescent protein (GFP) have revolutionised our understanding of bacterial structure and function. We have undertaken a systematic study of the localisation of over 100 Bacillus subtilis proteins, following the development of high-throughput construction and analysis procedures. We focused on proteins linked in various ways to the DNA replication machinery, as well as on proteins exemplifying a range of other cellular functions and structures. The results validate the approach as a way of obtaining systematic protein localisation information. They also provide a range of novel biological insights, particularly through the identification of a number of proteins not previously known to be associated with the DNA replication factory.

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BackgroundThe FtsK DNA-translocase controls the last steps of chromosome segregation in E. coli. It translocates sister chromosomes using the KOPS DNA motifs to orient its activity, and controls the resolution of dimeric forms of sister chromosomes by XerCD-mediated recombination at the dif site and their decatenation by TopoIV.MethodologyWe have used XerCD/dif recombination as a genetic trap to probe the interaction of FtsK with loci located in different regions of the chromosome. This assay revealed that the activity of FtsK is restricted to a ∼400 kb terminal region of the chromosome around the natural position of the dif site. Preferential interaction with this region required the tethering of FtsK to the division septum via its N-terminal domain as well as its translocation activity. However, the KOPS-recognition activity of FtsK was not required. Displacement of replication termination outside the FtsK high activity region had no effect on FtsK activity and deletion of a part of this region was not compensated by its extension to neighbouring regions. By observing the fate of fluorescent-tagged loci of the ter region, we found that segregation of the FtsK high activity region is delayed compared to that of its adjacent regions.SignificanceOur results show that a restricted terminal region of the chromosome is specifically dedicated to the last steps of chromosome segregation and to their coupling with cell division by FtsK.

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