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Two highly similar RNA polymerase sigma subunits, σF and σG, govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σF drives synthesis of σG but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σG while discriminating between σF and σG in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σG (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σG in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σG. CsfB is normally produced in the forespore, under σF control, but sigGN45E mutant cells also expressed csfB and did so in a σG-dependent manner, autonomously from σF. Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σG activity. N45 is invariant in the homologous position of σG orthologues, whereas its functional equivalent in σF proteins, E39, is highly conserved. While CsfB does not bind to wildtype σF, a E39N substitution in σF resulted in efficient binding of CsfB to σF. Moreover, under certain conditions, the E39N alteration strongly restrains the activity of sF in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two foresporespecific sigma factors in B. subtilis. [ABSTRACT FROM AUTHOR]

At an early stage during Bacillus subtilis endospore development the bacterium divides asymmetrically to produce two daughter cells. The smaller cell (forespore) differentiates into the endospore, while the larger cell (mother cell) becomes a terminally differentiated cell that nurtures the developing forespore. During development the mother cell engulfs the forespore to produce a protoplast, surrounded by two bilayer membranes, which separate it from the cytoplasm of the mother cell. The activation of σ[supG], which drives late gene expression in the forespore. follows forespore engulfment and requires expression of the spolllA locus in the mother cell. One of the spolllA-encoded proteins SpolllAH is targeted specifically to the membrane surrounding the forespore, through an interaction of its C-terminal extracellular domain with the C-terminal extracellular domain of the forespore membrane protein SpollQ. We identified a homologous relationship between the C-terminal domain of SpolllAH and the YscJ/FliF protein family, members of which form multimeric rings involved in type Ill secretion systems and flagella. If Sp0IIIAH forms a similar ring structure, it may also form a channel between the mother cell and forespore membranes. To test this hypothesis we developed a compartmentalized biotinylation assay, which we used to show that the C-terminal extracellular domain of Sp0IIIAH is accessible to enzymatic modification from the forespore cytoplasm. These and other results lead us to suggest that Sp0IIIAH forms part of a channel between the forespore and mother cell that is required for the activation of σ[supG]. [ABSTRACT FROM AUTHOR]

Endospore formation in Bacillus subtilis is a morphologically complex process in which the bacterium divides into two compartments (forespore and mother cell) that follow different developmental paths. Compartment-specific transcription in the forespore is initiated by RNA polymerase containing σF, and results in the forespore-speciflc production of σG, which directs most of the subsequent forespore-speciflc transcription. The activity of σF is thought to be restricted to the forespore by the sigma factor antagonist SpollAB. We used antibodies against SpollAB to monitor its accumulation during sporulation. We found that SpollAB accumulates early after the initiation of sporulation, and that it was present in the mother-cell compartment 2h after σF became active in the forespore. SpollAB disappeared preferentially from the forespore during development, and its disappearance from the forespore compartment correlated with the activation of σG in that compartment, raising the possibility that SpollAB may be involved regulating σG activity. We tested whether SpollAB could antagonize σG activity by replacing the σH-dependent promoter that drives expression of spolllG, the structural gene for σG, with a σH-dependent promoter. This resulted in a lytic phenotype that was supressed by the simultaneous expression of a plasmid-borne copy of spollAB. This suggests that SpollAB can suppress this effect of σG Moreover, these cells formed spores efficiently. Since σG the forespore by the σF-dependent transcription of its structural gene that normally occurs in wild-type cells, the forespore-specific activity of σG required for Sporulation appears to have resulted from expression of SpollAB. [ABSTRACT FROM AUTHOR]

The Bacillus subtilis spoIIIA locus encodes eight proteins, SpoIIIAA to SpoIIIAH, which are expressed in the mother cell during endospore formation and which are essential for the activation of σG in the forespore. Complementation studies indicated that this locus may be transcribed from two promoters, one promoter upstream from the first gene and possibly a second unidentified promoter within the locus. Fragments of the spoIIIA locus were expressed at an ectopic site to complement the sporulation-defective phenotype of a spoIIIAH deletion, and we determined that complementation required a fragment of DNA that extended into spoIIIAF. To confirm that there was a promoter located in spoIIIAF, we constructed transcriptional fusions to lacZ and found strong sporulation-induced promoter activity. Primer extension assays were used to determine the transcription start site, and point mutations introduced into the -10 and -35 regions of the promoter reduced its activity. This promoter is transcribed by σE-RNA polymerase and is repressed by SpoIIID. Therefore, we concluded that the spoIIIA locus is transcribed from two promoters, one at the start of the locus (P1spoIIIA) and the other within the locus (P2spoIIIA). Based on Campbell integrations and reverse transcription-PCR analysis of the P2spoIIIA region, we determined that P2spoIIIA is sufficient for transcription of spoIIIAG and spoIIIAH. Inactivation of P2spoIIIA blocked spore formation, indicating that P2spoIIIA is essential for expression of spoIIIAG and spoIIIAH. The P2spoIIIA activity is twice the P1spoIIIA activity; therefore, larger amounts of SpoIIIAG and SpoIIIAH than of proteins encoded at the upstream end of the locus may be required. [ABSTRACT FROM AUTHOR]

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]

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 σ[sup F] · σ[sup F] directs transcription of spoIIIG, encoding the late forespore-specific regulator σ[sup G]. Following synthesis, σ[sup G] is initially kept in an inactive form, presumably because it is bound to the SpoIIIAB anti-sigma factor. Activation of σ[sup 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 σ[sup G]. Here we show that σ[sup G] accumulates but is mostly inactive in a spoIIIJ mutant. We also show that expression of the spoIIIGE155K allele, encoding a form of σ[sup G] that is not efficiently bound by SpoIIAB in vitro, restores σ[sup 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 σ[sup 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 σ[sup G] accumulates in an inactive form in spoIIIA mutants and that the need for spoIIIA expression for σ[sup 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 σ[sup G] at the end of the engulfment process involves the action of spoIIIA from the mother cell and of spoIIIJ from the prespore. [ABSTRACT FROM AUTHOR]

The synthesis of structural components and morphogenetic factors required for the assembly of the Bacillus subtilis spore coat is governed by a mother cell-specific transcriptional cascade. The first two temporal classes of gene expression, which involve RNA polymerase sigma σE factor and the ancillary regulators GerR and SpoIIID, are deployed prior to engulfment of the prespore by the mother cell. The two last classes rely on σK, whose activation follows engulfment completion, and GerE. The cotE gene codes for a morphogenetic protein essential for the assembly of the outer coat layer and spore resistance to lysozyme. cotE is expressed first from a σE-dependent promoter and, in a second stage, from a promoter that additionally requires SpoIIID and that remains active under σK control. CotE localizes prior to engulfment completion close to the surface of the developing spore, but formation of the outer coat is a late, σK-controlled event. We have transplanted cotE to progressively later classes of mother cell gene expression. This created an early class of mutants in which cotE is expressed prior to engulfment completion and a late class in which expression of cotE follows the complete engulfment of the prespore. Mutants of the early class assemble a nearly normal outer coat structure, whereas mutants of the late class do not. Hence, the early expression of CotE is essential for outer coat assembly. Surprisingly, however, all mutants were fully resistant to lysozyme. The results suggest that CotE has genetically separable functions in spore resistance to lysozyme and spore outer coat assembly. [ABSTRACT FROM AUTHOR]