Deficiency of the initiation events of sporulation in Bacillus subtilis clpP mutant can be suppressed by a lack of the Spo0E protein phosphatase.

Previous results have shown that the Bacillus subtilis clpP gene is required for developmental processes such as sporulation and competence development. Little is known about its function during the initiation of sporulation. We studied the effect of clpP mutation on the early events of sporulation. The expression of the spo0A and spoIIG genes, whose active transcription requires the phosphorylated Spo0A protein (Spo0A approximately P) as the transcription activator, was significantly decreased in the clpP mutant at the onset of sporulation. The expression of spo0H gene encoding sigma(H) protein was also greatly reduced. As expected from these results, the sigma(H) and Spo0A protein levels in the clpP mutant were also decreased during the initiation of sporulation, indicating that the accumulation of Spo0A approximately P was inhibited in the clpP mutant. We, therefore, introduced the mutation of the spo0E gene, which codes for the Spo0A approximately P-specific phosphatase, into the clpP mutant and found that this double mutant restored the expression of the spo0A as well as spoIIG genes. These results suggest that ClpP had an indirect influence on the intracellular concentration of Spo0A approximately P by regulating the activity of the Spo0E phosphatase during the initiation of sporulation.

Suppression of temperature-sensitive sporulation mutation in the Bacillus subtilis sigA gene by rpoB mutation.

We isolated a temperature-sensitive sporulation defective mutant of the sigA gene, encoding a major sigma factor, sigma(A) protein, in Bacillus subtilis, and designated it as sigA21. The sigA21 mutation caused a single-amino acid substitution, E314K, in region 4 of the sigma(A) protein. In this mutant, expression of the spoIIG gene, whose transcription depends on both sigma(A) and the phosphorylated Spo0A protein, Spo0A approximately P, a major transcription factor during early stages of sporulation, was greatly reduced at 43 degrees C. To obtain further information on the mechanism of sigma(A) function during the early spore development, we isolated a spontaneous sporulation-proficient suppressor mutant at 43 degrees C. This extragenic suppressor mutation was mapped within the rpoB gene, encoding the beta subunit of RNA polymerase, and was found to have a single-amino acid substitution, A863G. In this mutant, the expression of the spoIIG is partially restored at 43 degrees C.

Natural genetic competence in Bacillus subtilis natto OK2.

We isolated a Bacillus subtilis natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using a comG-lacZ fusion at the amyE locus. Although transcription of the late competence genes was not detected in the B. subtilis natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the mecA or the clpC mutation derived from B. subtilis 168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for B. subtilis 168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the mecA and clpC mutants of strain OK2 (ComK(OK2)) could activate the transcription of the whole set of late competence genes and suggested that ComK(OK2) was not activated in strain OK2 during competence development. We therefore sequenced the comS gene of OK2 and compared it with that of 168. The comS(OK2) had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.

Genetic analysis of SecA-SecY interaction required for spore development in Bacillus subtilis.

All spontaneous suppressor mutations obtained from a secA12 sporulation-defective mutant in Bacillus subtilis were localized in highly conserved membrane-spanning regions of SecY. The expression of early sporulation genes, kinA and spo0A encoding a histidine kinase and a transcription regulator for several sporulation genes, respectively, was restored in these suppressor mutants. These results indicate that the secretion function of translocase combined with Sec proteins is required for sporulation in B. subtilis.

Thermo-labile stability of sigmaH (Spo0H) in temperature-sensitive spo0H mutants of Bacillus subtilis can be suppressed by mutations in RNA polymerase beta subunit.

We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the sigmaH protein, respectively, and located in the highly conserved region, "2", among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43 degrees C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the beta subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the sigmaH-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43 degrees C. Western blot analysis showed that the level of sigmaH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of sigmaH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigmaH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of sigmaH protein showed that the short life of sigmaH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-sigmaH formation may be involved in stabilization of sigmaH at the onset of sporulation.

The lethal effect of a benzamide derivative, 3-methoxybenzamide, can be suppressed by mutations within a cell division gene, ftsZ, in Bacillus subtilis.

3-Methoxybenzamide (3-MBA), which is known to be an inhibitor of ADP-ribosyltransferase, inhibits cell division in Bacillus subtilis, leading to filamentation and eventually lysis of cells. Our genetic analysis of 3-MBA-resistant mutants indicated that the primary target of the drug is the cell division system involving FtsZ function during both vegetative growth and sporulation.

ClpC regulates the fate of a sporulation initiation sigma factor, sigmaH protein, in Bacillus subtilis at elevated temperatures.

Using a strain carrying a clpC-bgaB transcriptional fusion at the amyE locus, we found that the expression of a clpC operon was induced at the end of exponential growth in a sigmaB-independent manner and ceased around T3.5 in the wild type but not in a spo0H mutant. This suggests that some gene product(s) whose expression is dependent on sigmaH function is required for the turn-off of clpC transcription during an early stage of sporulation. A clpC deletion mutant showed a temperature-sensitive sporulation phenotype and exhibited an abnormally large accumulation of sigmaH in the cell at 45 degrees C after T2, at which time the sigmaH level in the wild type had begun to decrease. These results, together with the fact that spo0H transcription in the clpC deletion mutant was similar to that of the wild type, suggested that ClpC may be responsible for the degradation of sigmaH after the accomplishment of its role in sporulation. Moreover, as expected from these results, overproduction of Spo0A was also observed after the initiation of sporulation in the clpC deletion mutant at 45 degrees C.