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Several features distinguish laboratory and undomesticated strains of Bacillus subtilis. For example, unlike the laboratory strain 168, the undomesticated strain ATCC 6051 is deficient in sporulation in a rich sporulation medium, 2x SG. ATCC 6051 cannot induce transcription of the spoIIG operon, suggesting that this strain has a defect in initiation of sporulation. To determine the genetic difference between 168 and ATCC 6051, the DNA region responsible for the Spo(-) phenotype was transferred to strain 168. Genetic mapping and DNA sequencing analysis revealed that a stop codon (TAA) for kinA in 168 is replaced with Lys (TAT) in ATCC 6051, making the kinA open reading frame 201 bp longer in the undomesticated strain ATCC 6051. Introduction of kinA from strain 168 restored sporulation in ATCC 6051, indicating that the difference in kinA is responsible for the Spo(-) phenotype of ATCC 6051. A potential rho-independent terminator is located upstream of a stop codon for the extended kinA open reading frame in ATCC 6051. Northern blot analysis showed that transcription of kinA terminated at this terminator, and kinA mRNA is missing a stop codon in ATCC 6051. Moreover, deletion of tmRNA suppresses the sporulation defect in ATCC 6051. These observations indicate that in ATCC 6051 the absence of a stop codon in kinA mRNA affects sporulation, probably by leading to rapid degradation of KinA via the trans-translation process. In ATCC 6051, the kinA mutation affects sporulation but not other Spo0A-dependent phenomena such as biofilm formation, which can be activated by a low level of Spo0A approximately P. This is due to the fact that KinA activity is kept low during the exponential phase via transcriptional and post-translational regulation. Thus, the stop-codon-less kinA probably affects only sporulation. DNA sequencing of 30 B. subtilis strains revealed that another strain also produces stop-codon-less kinA mRNA. This observation suggests that the lack of a stop codon for kinA mRNA may give rise to a selective advantage under certain conditions.

The sporulation-specific SpoVIF (YjcC) protein of Bacillus subtilis is essential for the development of heat-resistant spores. The GerE protein, the smallest member of the LuxR-FixJ family, contains a helix-turn-helix (HTH) motif and is involved in the expression of various sporulation-specific genes. In this study, the gene expression and protein composition of sporulating spoVIF-negative cells were analysed. CgeA, CotG and CotS, which are GerE-dependent coat proteins, were not expressed in the spoVIF-negative cells. Northern blotting showed that SpoVIF regulated the transcription of cgeA, cotG and cotS in a manner similar to that of GerE. In spoVIF-negative cells, gerE mRNA was transcribed normally, but immunoblot analysis using anti-GerE antiserum showed that the quantity of GerE protein was considerably less than that in wild-type controls. Using GFP (green fluorescent protein) fusion proteins, the localization of SpoVIF and GerE was observed by fluorescence microscopy. SpoVIF-GFP was detectable in the mother cell compartment, as was GerE-GFP. These results suggest that SpoVIF directly or indirectly controls the function of the GerE protein, and that SpoVIF is required for gene regulation during the latter stages of sporulation.

In systematic screening four sporulation-specific genes, yjcA, yjcB, yjcZ and yjcC, of unknown function were found in Bacillus subtilis. These genes are located just upstream of the cotVWXYZ gene cluster oriented in the opposite direction. Northern blot analysis showed that yjcA was transcribed by the SigE RNA polymerase beginning 2 h (t(2)) after the onset of sporulation, and yjcB, yjcZ and yjcC were transcribed by the SigK RNA polymerase beginning at t(4) of sporulation. The transcription of yjcZ was dependent on SigK and GerE. The consensus sequences of the appropriate sigma factors were found upstream of each gene. There were putative GerE-binding sites upstream of yjcZ. Insertional inactivation of the yjcC gene resulted in a reduction in resistance of the mutant spores to lysozyme and heat. Transmission electron microscopic examination of yjcC spores revealed a defect of sporulation at stage VI, resulting in loss of spore coats. These results suggest that YjcC is involved in assembly of spore coat proteins that have roles in lysozyme resistance. It is proposed that yjcC should be renamed as spoVIF.

The spores of Bacillus subtilis have characteristic properties and consist of complex structures including various types of proteins. To perform comprehensive analysis of the protein composition of the spores, the proteins extracted from the spore were analysed by a combination of one-dimensional PAGE and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using Turboquest SEQUEST software interfaced with the DNA sequence database of B. subtilis. A total of 154 proteins were identified, and 69 of them were novel. The remaining 85 proteins have been previously reported as sporulation-specific proteins or as proteins that are synthesized in vegetative cells. The expression pattern of each gene deduced to encode novel spore proteins was analysed using a series of strains carrying a lacZ reporter gene. The results revealed that the expression of 26 genes was dependent on sporulation-specific sigma factors, namely sigma(F), sigma(E), sigma(G) and sigma(K). In this study, it is demonstrated that the combination of the techniques of SDS-PAGE and LC-MS/MS, with the mutant library of B. subtilis, is an effective tool for the analysis of complicated cellular structures.

An extensive screening for transcripts with probes specific for the genes in a 108 kb region from rrnO to spo0H of the Bacillus subtilis chromosome led to identification of an operon, yabP--yabQ--divIC--yabR, the expression of which was initiated at the second hour of sporulation and in a sigma(E)-dependent manner. Among three y genes in the operon, deletion of the yabQ gene, which is predicted to encode a protein product of 468 residues with five membrane-spanning domains, resulted in a large decrease in numbers of chloroform-, lysozyme- and heat-resistant spores, compared to findings with the wild-type strain. Electron microscopy revealed that development of the spore cortex was blocked in the yabQ mutant. In addition, although the spore coat was visible, the inner coat layer of the mutant seemed partially detached from the outer coat. In sporangia of the strains harbouring an in-frame fusion of the green fluorescent protein gene to yabQ, fluorescence was detected around the forespore. This localization did not depend on SpoIVA or on CotE functions, both of which determine proper localization of coat proteins and cortex formation. The yabQ deletion did not affect expression of genes involved in cortex synthesis. These results suggest that the YabQ protein localizes in the membrane of the forespore and plays an important role in cortex formation.