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RNA polymerase sigma factor σF initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation, σF governs transcription of spoIIIG, encoding the late prespore-specific regulator σG. However, transcription of spoIIIG is delayed relative to other genes under the control of σF, and after synthesis, erg is initially kept in an inactive form. Activation of erg requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of σG. We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with σG. Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with σG. The σGE156K isoform restored σG-directed gene expression to spoIIIA mutant cells. However, expression of sspE lacZ in the spollIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, β-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of σGE156K is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidentally with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of era in the prespore. [ABSTRACT FROM AUTHOR]

RNA polymerase sigma factor sigma(F) initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation. sigma(F) governs transcription of spoIIIG, encoding the late prespore-specific regulator sigma(G). However, transcription of spoIIIG is delayed relative to other genes under the control of sigma(F), and after synthesis, sigma(G) is initially kept in an inactive form. Activation of sigma(G) requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of sigma(G). We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with sigma(G). Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with sigma(G). The sigma(GE156K) isoform restored sigma(G)-directed gene expression to spoIIIA mutant cells. However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, beta-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of sigma(GE156K) is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of sigma(G) in the prespore.

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.