Identification of the Product of Sporulation Gene spo0B in Bacillus subtilis

A 1.4-megadalton EcoRI restriction fragment carrying Bacillus subtilis sporulation gene spo0B was cloned from the specialized transducing phage, φ 105spo0B, into a unique EcoRI site of plasmid vector pUB110, and four plasmids having a deletion in the 1.4-megadalton EcoRI fragment were constructed. Analysis of the polypeptides synthesized in B. subtilis minicells harboring these plasmids and the sporulation ability of strain UOT0436 (spo0B136 recE4) harboring these plasmids showed that the spo0B gene product is a polypeptide of 24,000 daltons. Two-dimensional polyacrylamide gel analysis showed that the isoelectric point of this protein is almost neutral.     

Purification of the Early Sporulation Gene spo0F Product of Bacillus subtilis

The RsaI fragment (750 base pairs) containing the entire early sporulation gene spo0F of Bacillus subtilis was inserted in the downstream region of the P_R promoter of the expression vector, pEBR-151. The recombinant plasmid thus obtained was introduced into Escherichia coli HB101 and the synthesis of the spo0F gene product was induced by a temperature shift up. After induction for 7 hr, a protein of molecular weight 14,000 (14 K protein) was overproduced to about 9% of the total cellular protein. The 14 K protein was purified to 94% purity by four steps of column chromatography. Deletion analysis and the sequence determination of the NH₂-terminal amino acid residues of the purified 14 K protein confirmed that the 14 K protein is the spo0F gene product.     

Defects in the nutrient-dependent methylation of a membrane-associated protein in spo mutants of Bacillus subtilis

Summary Methylation of a membrane-associated protein with an apparent molecular mass of 40000 daltons has been observed in Bacillus subtilis. The methylation was nutrient dependent and occurred with a doubling time of 4 ± 1 min. In wild-type strains, the half-life of turnover of the methyl group(s) was 17 ± 6 min. Several isogenic strains of B. subtilis containing spo0 mutations (spo0A and spo0H) were found to be normal in glutamate-dependent methylation of the protein and turnover of the methyl group(s). In strains containing spo0B and spo0E mutations, the methyl group(s) were incorporated in response to glutamate addition but turnover was not at a normal rate. The half-life of methyl group turnover was extended to 45 ± 3 min in these strains. In a spo0K mutant and in spoILI and spoIIF mutants, the protein was not significantly methylated. The methylation of a 40000 dalton protein was also found to be dependent on phosphate. This methylation was observed in wild-type and spo0A and spo0H strains with a doubling time of 4 ± 1 min and a half-life of turnover of the methyl group(s) of 11 + 3 min. In strains containing spo0B, spo0E, and spo0F mutations, the phosphate-dependent incorporation of the methyl group(s) was normal (5 ± 1 min) but the turnover half-life was extended to 46 ± 8 min. It is not known whether the nitrogen-dependent and phosphate-dependent systems methylated the same protein. The spo0 mutants are defective in the initial stages of sporulation, and it has been proposed that the spo0 gene products may play a role in nutrient sensing. The discovery of defects in the methylation of the 40 kDa protein in some of these spo0 mutants supports the proposal that the protein methylation may be part of a nutrient sensing system for the control of growth and sporulation in Bacillus species.     

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.     

Isolation and Characterization of a Temperature-sensitive Sporulation Mutant of Bacillus subtilis

Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 of sporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.     

Early-blocked sporulation mutations alter expression of enzymes under carbon control in Bacillus subtilis

Summary The physiological roles of the gene subset defined by early-blocked sporulation mutations (spo0) and their second-site suppressor alleles (rvtA11 and crsA47) remain cryptic for both vegetative and sporulating Bacillus subtilis cells. To test the hypothesis that spo0 gene products affect global regulation, we assayed the levels of carbon- and nitrogen-sensitive enzymes in wild-type and spo0 strains grown in a defined minimal medium containing various carbon and nitrogen sources. All the spo0 mutations (except spo0J) affected both histidase and arabinose isomerase levels in an unexpected way: levels of both carbon-sensitive enzymes were two- to six-fold higher in spo0 strains compared to wild type, when cells were grown on the derepressing carbon sources arabinose or maltose. There was no difference in enzyme levels with glucose-grown cells, nor was there a significant difference in levels of the carbonindependent enzymes glutamine synthetase and glucose-6-phosphate dehydrogenase. This effect was not due to a slower growth rate for the spo0 mutants on the poor carbon and nitrogen sources used. The levels of carbon-sensitive enzymes were not simply correlated with sporulation ability in genetically suppressed spo0 mutants, but the rvtA and crsA suppressors each had such marked effects on wild-type growth and enzyme levels that these results were difficult to interpret. We conclude that directly or indirectly the spo0 mutations, although blocking the sporulation process, increase levels of carbon-sensitive enzymes, possibly at the level of gene expression.     

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.