Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis

The spoIID gene, which is involved in Bacillus subtilis sporulation, was fused to the beta-galactosidase gene, lacZ, of Escherichia coli so that the expression of beta-galactosidase would be under the control of the spoIID locus. When the fused product was inserted into the B. subtilis chromosome, production of beta-galactosidase indicated that the spoIID gene was expressed 1.5 h after the start of sporulation. When the spoIID::lacZ fusion was inserted into the chromosome of sporulation mutants, all strains carrying spo0 lesions and those with mutations in spoIIA, spoIIE and spoIIG loci failed to make beta-galactosidase. The proposed provisional order of expression of operons governing stage II is spoIIA----[spoIIG, spoIIE]----[spoIID, spoIIB, spoIIF].     

Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression.

The Bacillus subtilis gsiA operon was induced rapidly, but transiently, as cells entered the stationary phase in nutrient broth medium. A mutation at the gsiC locus caused sporulation to be defective and expression of gsiA to be elevated and prolonged. The sporulation defect in this strain was apparently due to persistent expression of gsiA, since a gsiA null mutation restored sporulation to wild-type levels. Detailed mapping experiments revealed that the gsiC82 mutation lies within the kinA gene, which encodes the histidine protein kinase member of a two-component regulatory system. Since mutations in this gene caused a substantial blockage in expression of spoIIA, spoIIG, and spoIID genes, it seems that accumulation of a product of the gsiA operon interferes with sporulation by blocking the completion of stage II. It apparently does so by inhibiting or counteracting the activity of KinA.     

Variety of sporulation phenotypes resulting from mutations in a single regulatory locus, spoIIA, in Bacillus subtilis

Closely linked mutations in either of the two putative genes of the sporulation locus spoIIA can affect, in quite diverse ways, spore incidence, the production of alkaline phosphatase and DNAase, and the stability of the cells in sporulation medium. It is concluded that the locus has a regulatory function affecting the activation or induction of at least two, and possibly more, sporulation-associated operons.     

Synthesis of spoIIA and spoVA mRNA in Bacillus subtilis

The expression of the spoIIA and spoVA sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoVA loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.     

Control of the expression and compartmentalization of (sigma)G activity during sporulation of Bacillus subtilis by regulators of (sigma)F and (sigma)E

During formation of spores by Bacillus subtilis the RNA polymerase factor sigma(G) ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of sigma(G) ordinarily requires prior activity of sigma(F) in the prespore and sigma(E) in the mother cell. Here we report that in spoIIA mutants lacking both sigma(F) and the anti-sigma factor SpoIIAB and in which sigma(E) is not active, sigma(G) nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of sigma(G) activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for sigma(G), from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to sigma(G). When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, sigma(G) became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature sigma(G) activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of sigma(G) activity, and pro-sigma(E) (but not sigma(E)) can counteract this effect of SpoIIGA. We find that in strains lacking sigma(F) and SpoIIAB and engineered to produce active sigma(E) in the mother cell without the need for SpoIIGA, sigma(G) also becomes active in the mother cell.