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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]

Proteins SpoIIQ and SpoIIIAH interact through two membranes to connect the forespore and the mother cell during endospore development in the bacterium Bacillus subtilis. SpoIIIAH consists of a transmembrane segment and an extracellular domain with similarity to YscJ proteins. YscJ proteins form large multimeric rings that are the structural scaffolds for the assembly of type III secretion systems in gram-negative bacteria. The predicted ring-forming motif of SpoIIIAH and other evidence led to the model that SpoIIQ and SpoIIIAH form the core components of a channel or transporter through which the mother cell nurtures forespore development. Therefore, to understand the roles of SpoIIIAH and SpoIIQ in channel formation, it is critical to determine whether SpoIIIAH adopts a ring-forming structural motif, and whether interaction of SpoIIIAH with SpoIIQ would preclude ring formation. We report a 2.8-Å resolution structure of a complex of SpoIIQ and SpoIIIAH. SpoIIIAH folds into the ring-building structural motif, and modeling shows that the structure of the SpoIIQ-SpoIIIAH complex is compatible with forming a symmetrical oligomer that is similar to those in type III systems. The inner diameters of the two most likely ring models are large enough to accommodate several copies of other integral membrane proteins. SpoIIQ contains a LytM domain, which is found in metalloendopeptidases, but lacks residues important for metalloprotease activity. Other LytM domains appear to be involved in protein-protein interactions. We found that the LytM domain of SpoIIQ contains an accessory region that interacts with SpoIIIAH.

The sigma subunit of bacterial RNA polymerase is required for specific binding of the enzyme to promoters. This specificity is probably directed by two regions of most sigma factors that make sequence-specific contacts at two regions of promoters, the -10 and -35 regions. We found that a single amino acid substitution in the -10 recognition region of sigma E from Bacillus subtilis trapped RNA polymerase in a stable complex with promoter DNA in which it was unable to initiate transcription. Our results are consistent with the view that promoter utilization by RNA polymerase proceeds through several intermediate steps and suggest that the -10 recognition region of sigma factors may participate in a step that follows initial promoter binding.

Examination of the effects of 56 single-base-pair substitutions in the spoIIG promoter and studies of the interaction of the spo0A product (Spo0A) with this promoter in vitro demonstrated that Spo0A acts directly to enable this promoter to be used by sigma A-associated RNA polymerase (EC 2.7.7.6). The spoIIG operon from Bacillus subtilis is transcribed during sporulation by a form o RNA polymerase containing sigma A, the primary sigma factor in vegetative cells. The spoIIG promoter is unusual in that it contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We found that single-base-pair substitutions in the around the -35-like sequence, and substitutions in a region upstream from this position, around position -87, reduced promoter activity. DNase I protection and electrophoretic gel mobility shift assays were used to demonstrate that Spo0A binds specifically to these regions in vitro. Evidently, the -35-like sequence is part of a Spo0A binding site and therefore is possibly not a sigma A-recognition sequence. These results support a model in which Spo0A activates the spoIIG promoter after the onset of endospore formation.

The sporulation operon spoIIA of Bacillus subtilis consists of three cistrons called spoIIAA, spoIIAB, and spoIIAC. Little is known about the function of spoIIAA and spoIIAB, but spoIIAC encodes a sigma factor called sigma F, which is capable of directing the transcription in vitro of genes that are expressed in the forespore chamber of the developing sporangium. We now report that the products of the spoIIA operon constitute a regulatory system in which SpoIIAA is an antagonist of SpoIIAB (or otherwise counteracts the effect of SpoIIAB) and SpoIIAB is, in turn, an antagonist of SpoIIAC (sigma F). This conclusion is based on the observations that (i) overexpression of spoIIAB inhibits sigma F-directed gene expression, (ii) a mutation in spoIIAB stimulates sigma F-directed gene expression, (iii) a mutation in spoIIAA blocks sigma F-directed gene expression, and (iv) a mutation in spoIIAB relieves the block in sigma F-directed gene expression caused by a mutation in spoIIAA. The SpoIIAA/SpoIIAB/SpoIIAC regulatory system could play a role in controlling the timing of sigma F-directed gene expression and/or could be responsible for restricting sigma F-directed gene expression to the forespore chamber of the sporangium.

The construction of allele-specific suppressor mutations has enabled us to demonstrate that a sporulation-specific transcription unit in Bacillus subtilis, the spoIIG operon, is transcribed by a form of RNA polymerase associated with sigma A, the principal sigma factor in vegetative cells. The spoIIG operon encodes sporulation-specific factor sigma E, and its transcription is directed from a promoter that is activated about 1 hr after the onset of endospore formation. This promoter contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We have found that substitution of an arginyl residue for the glutamyl residue at position 196 of sigma A (Glu-196----Arg) suppresses the deleterious effect of a thymidine-to-cytidine base substitution at position -11 in the spoIIG promoter. This suppression was allele-specific, since it did not suppress the effects of base substitutions in other positions in the spoIIG promoter or the effects of a thymidine-to-guanosine change at -11. These results support a model in which a form of RNA polymerase containing sigma A is utilized in an unusual manner to activate the transcription of the spoIIG operon well after the onset of endospore formation.

In Bacillus subtilis transcription of spoVG is activated within minutes after the initiation of sporulation. Mutations in several spo0 genes prevent the activation of spoVG transcription. We have found a sigma-like protein that is capable of directing core RNA polymerase to use the spoVG promoter in an in vitro run-off transcription assay. This sigma-like protein was not found to be associated with RNA polymerase in a spo0A or spo0B mutant but was present in a spo0H mutant. We suggest that one role of the spo0A gene product in transcription of spoVG is the modulation of RNA polymerase activity by this sigma-like protein.