Overproducing the Bacillus subtilis mother cell sigma factor precursor, Pro-sigma K, uncouples sigma K-dependent gene expression from dependence on intercompartmental communication.

During sporulation of Bacillus subtilis, proteolytic activation of pro-sigma K and ensuing sigma K-dependent gene expression normally require the activity of many sporulation gene products. We report here that overproducing pro-sigma K at the onset of sporulation substantially uncouples sigma K-dependent gene expression from its normal dependency. Overproducing pro-sigma K in strains with a mutation in spoIIIG, spoIIIA, spoIIIE, or spoIVB partially restored sigma K-dependent gene expression in the mother cell and resulted in accumulation of a small amount of polypeptide that comigrated with sigma K, but these mutants still failed to form spores. In contrast, sporulation of spoIVF mutants was greatly enhanced by pro-sigma K overproduction. The products of the spoIVF operon are made in the mother cell and normally govern pro-sigma K processing, but overproduction of pro-sigma K appears to allow accumulation of a small amount of sigma K, which is sufficient to partially restore mother cell gene expression and spore formation. This spoIVF-independent mechanism for processing pro-sigma K depends on sigma E, an earlier-acting mother cell-specific sigma factor. The spoIIID gene, which encodes a mother cell-specific DNA-binding protein that is normally required for pro-sigma K production, was shown to be required for efficient pro-sigma K processing as well. bof (bypass of forespore) mutations bypassed this requirement for spoIIID, suggesting that SpoIIID is less directly involved in pro-sigma K processing than are spoIVF gene products. However, bof spoIIID double mutants overproducing pro-sigma K still failed to sporulate, indicating that SpoIIID serves another essential role(s) in sporulation in addition to its multiple roles in the production of sigma K.     

Sequential activation of dual promoters by different Sigma factors maintains spoVJ expression during successive developmental stages of Bacillus subtilis

The spoVJ gene of Bacillus subtilis encodes a 36 kDa protein and is expressed only in the mother cell. spoVJ has an interesting pattern of regulation during sporulation because it is expressed from sequentially activated promoters. These promoters, designated P1 and P2, are under the control of different sigma factors, sigma E and sigma K, which become active at separate times during sporulation. Removal of promoter P1, leaving promoter P2 active, resulted in about a 30-minute delay in the formation of heat-resistant spores and demonstrated that the expression of spoVJ from both promoters is essential for normal sporulation. A comparison is made between the sequences of the spoVJ promoters and the promoters of other genes dependent upon sigma E and sigma K.     

Genetic suppression analysis of sigma E interaction with three promoters in sporulating Bacillus subtilis.

Genetic evidence suggests that the sigma (sigma) subunit of RNA polymerase determines the specificity of promoter utilization, by making sequence-specific contacts with DNA. We examined the effects of two single amino acid(aa) substitutions in sigma E on the utilization of mutated derivatives of three different promoters in sporulating Bacillus subtilis. We found allele-specific suppression of mutations in all three promoters by each aa substitution in sigma E. These results provide strong evidence that sigma E interacts with each of these promoters in vivo. Moreover, the specificity of suppression of the mutations by the aa substitutions in sigma E lead us to speculate that the Met124 of sigma E closely contacts two adjacent bp in the -10 region of the promoters.