Control of the expression and compartmentalization of (sigma)G activity during sporulation of Bacillus subtilis by regulators of (sigma)F and (sigma)E

During formation of spores by Bacillus subtilis the RNA polymerase factor sigma(G) ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of sigma(G) ordinarily requires prior activity of sigma(F) in the prespore and sigma(E) in the mother cell. Here we report that in spoIIA mutants lacking both sigma(F) and the anti-sigma factor SpoIIAB and in which sigma(E) is not active, sigma(G) nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of sigma(G) activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for sigma(G), from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to sigma(G). When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, sigma(G) became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature sigma(G) activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of sigma(G) activity, and pro-sigma(E) (but not sigma(E)) can counteract this effect of SpoIIGA. We find that in strains lacking sigma(F) and SpoIIAB and engineered to produce active sigma(E) in the mother cell without the need for SpoIIGA, sigma(G) also becomes active in the mother cell.     

Separation of chromosome termini during sporulation of Bacillus subtilis depends on SpoIIIE

Bacillus subtilis undergoes a highly distinctive division during spore formation. It yields two unequal cells, the mother cell and the prespore, and septum formation is completed before the origin-distal 70% of the chromosome has entered the smaller prespore. The mother cell subsequently engulfs the prespore. Two different probes were used to study the behavior of the terminus (ter) region of the chromosome during spore formation. Only one ter region was observed at the time of sporulation division. A second ter region, indicative of chromosome separation, was not distinguishable until engulfment was nearing completion, when one was in the mother cell and the other in the prespore. Separation of the two ter regions depended on the DNA translocase SpoIIIE. It is concluded that SpoIIIE is required during spore formation for chromosome separation as well as for translocation; SpoIIIE is not required for separation during vegetative growth.     

Use of asymmetric cell division and spoIIIE mutants to probe chromosome orientation and organization in Bacillus subtilis

Soon after the onset of sporulation in Bacillus subtilis , asymmetric cell division occurs to generate the differentiating prespore and mother cell types. Formation of the septum close to the cell pole initially bisects the nucleoid destined for the prespore, trapping only about one-third of the DNA in the small compartment. The remaining part of the chromosome is then transported through the septum. spoIIIE mutant cells fail to transfer the DNA and arrest with only partially segregated prespore chromosomes. Previous work has shown that the orientation of the chromosome at the time of septation is not random. Here, we use both physical and genetic methods to characterize the trapped DNA. The results show that the chromosome has a very specific orientation at the time of septation, consistent with the action of a centromere-like sequence near oriC . They also demonstrate that the chromosome is folded, or otherwise organized, in a highly ordered manner.     

The Bacillus subtilis soj-spo0J locus is required for a centromere-like function involved in prespore chromosome partitioning

During sporulation in Bacillus subtilis a small prespore cell is formed by an asymmetric cell division. Pre-spore chromosome partitioning occurs by a specialised mechanism in which septation precedes chromosome movement. We show that the spo0J gene is needed to specify the orientation of the chromosome at the time of polar division and to impose directionality on the subsequent transport of the remainder of the chromosome through the septum. Both phenotypes may arise by disruption of a centromere-like apparatus that anchors the oriC region of the prespore chromosome in the pole of the cell.     

Differential gene expression in genetically identical sister cells: the initiation of sporulation in Bacillus subtilis

Early in sporulation, the cell divides asymmetrically to give two sister compartments, a smaller prespore and a larger mother cell. Differential gene expression in these compartments depends on the regulation of the first sporulation-specific sigma factor, sigma(F), which is activated only in the prespore. Regulation relies on the interactions of four proteins -sigma(F), its antisigma SpoIIAB (which also has protein kinase activity), the anti-antisigma SpoIIAA and the protein phosphatase SpoIIE. Before asymmetric division, and in the mother cell after division, sigma(F) is held in an inactive complex with SpoIIAB and ATP; SpoIIAA is in its phosphorylated form. To disrupt the complex so as to liberate sigma(F) in the prespore, dephosphorylated SpoIIAA is needed, and this is made available by SpoIIE. Thereafter, SpoIIAB and SpoIIE are active simultaneously in the prespore, cycling SpoIIAA through phosphorylated and non-phosphorylated forms. This cycle detains SpoIIAB in a state in which it cannot inhibit sigma(F). Results from biophysical techniques, mathematical simulations and enzyme kinetics have now helped to elucidate the dynamics of the protein-protein interactions involved. An understanding of these dynamics largely accounts for the regulation of sigma(F). We show that the system is tuned to be highly efficient in its use of components and extremely economical in conserving ATP.