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During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σG coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation. [ABSTRACT FROM AUTHOR]

During sporulation in Bacillus subtilis, the prespore-specific developmental program is initiated soon after asymmetric division of the sporangium by the compartment-specific activation of RNA polymerase sigma factor sigma(F). sigma(F) directs transcription of spoIIIG, encoding the late forespore-specific regulator sigma(G). Following synthesis, sigma(G) is initially kept in an inactive form, presumably because it is bound to the SpoIIAB anti-sigma factor. Activation of sigma(G) occurs only after the complete engulfment of the prespore by the mother cell. Mutations in spoIIIJ arrest sporulation soon after conclusion of the engulfment process and prevent activation of sigma(G). Here we show that sigma(G) accumulates but is mostly inactive in a spoIIIJ mutant. We also show that expression of the spoIIIGE155K allele, encoding a form of sigma(G) that is not efficiently bound by SpoIIAB in vitro, restores sigma(G)-directed gene expression to a spoIIIJ mutant. Expression of spoIIIJ occurs during vegetative growth. However, we show that expression of spoIIIJ in the prespore is sufficient for sigma(G) activation and for sporulation. Mutations in the mother cell-specific spoIIIA locus are known to arrest sporulation just after completion of the engulfment process. Previous work has also shown that sigma(G) accumulates in an inactive form in spoIIIA mutants and that the need for spoIIIA expression for sigma(G) activation can be circumvented by the spoIIIGE155K allele. However, in contrast to the case for spoIIIJ, we show that expression of spoIIIA in the prespore does not support efficient sporulation. The results suggest that the activation of sigma(G) at the end of the engulfment process involves the action of spoIIIA from the mother cell and of spoIIIJ from the prespore.