Temporal regulation of the Bacillus subtilis early sporulation gene spo0F

The initiation of sporulation in Bacillus subtilis depends on seven genes of the spo0 class. One of these, spo0F, codes for a protein of 14,000 daltons. We studied the regulation of spo0F by using spo0F-lacZ translational fusions and also measured Spo0F protein levels by immunoassays. spo0F-lacZ and Spo0F levels increased as the cells entered the stationary phase, and this effect was repressed by glucose and glutamine. Decoyinine, which lowers GTP levels and allows sporulation in the presence of normally repressing levels of glucose, induced spo0F-lacZ expression and raised Spo0F levels. The expression of spo0F-lacZ was dependent on spo0A, -0B, -0E, -0F, and -0H genes, a spo0H deletion causing the strongest effect. In most respects, the spo0F gene was regulated in a manner similar to that of spoVG. However, the presence of an abrB mutation did not relieve the dependence of spo0F gene expression on spo0A, as it does with spoVG (P. Zuber and R. Losick, J. Bacteriol. 169:2223-2230, 1987).     

Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis.

The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins. Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro. Mutants with deletion mutations in the kinA gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein. Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations. The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the Spo0A protein.     

Identification of the transcriptional suppressor sof-1 as an alteration in the spo0A protein

The mutation sof-1 suppresses the sporulation defect of mutations in either the spo0B, spo0E, or spo0F stage 0 sporulation genes. Through the use of integrative plasmids carrying the portion of the chromosome including the spo0A locus and flanking regions, the sof-1 mutation was localized near the spo0A locus. A plasmid carrying a fragment of DNA with sof genetic activity was constructed. Nucleic acid sequence analysis of this fragment revealed a single base change that resulted in a substitution of lysine for asparagine in the 12th codon of the spo0A gene. The results indicate that certain missense mutations in the spo0A gene bypass the necessity for the spo0B, spo0E, and spo0F gene products in sporulation. Several models for the interaction of these gene products may be imagined.     

New suppressor mutation sur0B of spo0B and spo0F mutations in Bacillus subtilis

Two extragenic suppressor mutations, sur0B20 and sur0F1, which restore the sporulation of spo0B or spo0F mutants of Bacillus subtilis to the wild-type level, were obtained. These suppressor mutations were located in the spo0A gene. Their location is close to that of the sof-1 mutation, which suppresses spo0B, spo0E and spo0F mutations. However, spo0 strains bearing the sur0B20 mutation differed in several phenotypic characteristics from spo0 mutants bearing the sof-1 suppressor. Nucleotide sequence analysis revealed that the sur0B20 and sur0F1 mutations resulted in Glu14 to Val and Asn12 to Lys conversion, respectively, in the spo0A gene. This result indicates that sur0B20 is a new suppressor of spo0b and spo0F mutations, whereas sur0F1 is identical to sof-1.     

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.     

The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A.

SinR is a pleiotropic DNA binding protein that is essential for the late-growth processes of competence and motility in Bacillus subtilis and is also a repressor of others, e.g., sporulation and subtilisin synthesis. In this report, we show that SinR, in addition to being an inhibitor of sporulation stage II gene expression, is a repressor of the key early sporulation gene spo0A. The sporulation-specific rise in spo0A expression at time zero is absent in a SinR-overproducing strain and is much higher than normal in strains with a disrupted sinR gene. This effect is direct, since SinR binds specifically to spo0A in vitro, in a region overlapping the -10 region of the sporulation-specific Ps promoter that is recognized by E-sigma H polymerase. Methyl interference and site-directed mutagenesis studies have identified guanine residues that are important for SinR recognition of this DNA sequence. Finally, we present evidence that SinR controls sporulation through several independent genes, i.e., sp0A, spoIIA, and possibly spoIIG and spoIIE.