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Bacterial spores are encased in a multilayered proteinaeeous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar iocalizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring- or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a muitilayered assembly but also that it exhibits uneven spatial distribution of particular proteins. doi: 10.1128/JB.01103-09

The yeeK gene of Bacillus subtilis is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in yabG mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the yeeK gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of yeeK did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in yabG mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.

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 spollG 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 Spophenotype 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.sup.-] 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~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 predicted amino acid sequence of Bacillus subtilis ycsK exhibits similarity to the GDSL family of lipolytie enzymes. Northern blot analysis showed that ycsK mRNA was first detected from 4 h after the onset of sporulation and that transcription of ycsK was dependent on SigK and GerE. The fluorescence of the YcsKgreen fluorescent protein fusion protein produced in sporulating cells was detectable in the mother cell but not in the forespore compartment under fluorescence microscopy, and the fusion protein was localized around the developing spores dependent on CotE, SafA, and SpoVID. Inactivation of the ycsK gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, lysozyme, or chloroform. The germination of ycsK spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride and LB medium was also the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. In addition, zymogram analysis demonstrated that the YcsK protein heterologously expressed in Escherichia coli showed lipolytic activity. We therefore propose that ycsK should be renamed lipC. This is the first study of a bacterial spore germination-related lipase.

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.sub.2]) after the onset of sporulation, and yjcB, yjcZ and yjcC were transcribed by the SigK RNA polymerase beginning at [t.sub.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.

Results reveal that deletion of the yabQ gene from the operon yabP-yabQ-divlC-yabR, leads to a decreased spore production suggesting that it is essential for sporulation. Data further indicate that the yabQ gene is also involved in formation of the spore cortex.

Results show that yabG gene is expressed only during sporulation of Bacillus subtilis. Data further indicate that the gene encodes a sporulation specific protein which has role in the coat protein composition.

The expression of 21 unique genes found in the region from danA to abrB of the Bacillus subtilis chromosome was examined. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Research findings showed that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T2 of sporulation and that it is needed for the L-alanine-stimulated germination pathway.

Insertional inactivation of the yrbA gene of Bacillus subtilis weakened the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical intensity at the same rate as the wild-type spores upon incubation with L-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose and KCl was also extremely poor. In this medium, yrbA spores showed only a small loss in optical density and yielded a mixture of phase-bright, -gray and -dark spores. Results indicated that YrbA is implicated in the assembly of some coat proteins, which are involved in both spore lysozyme resistance and germination.

In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulphhydryl groups exerted significant effects strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibitedGraphical Abstract the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the MEROPS peptidase database. [ABSTRACT FROM AUTHOR]

Western blot and Northern blot analyses were used to investigate elements that control CotS assembly and cotS gene expression into the spore coat of Bacillus subtilis. The results showed little evidence for the transcription of cotS mRNA in spores of sigma(super K). CotS was initially found in solubilized protein samples from wild-type cells, five hours after commencing sporulation. The results of immunoelectron microscopy further showed high concentration of CotS in the inner coat and the outer portion of the cortex of the mature spore.

Amino acid substitution of threonine 128 of SecA by alanine or isoleucine Bacillus subtilis yields four azide-resistant Sec A mutants that exhibit normal protein translocation and cell growth in the presence of 1.5 millimoles of sodium azide, unlike the wild-type strain that is inhibited. Purified mutant SecA proteins have three times higher values of basal and elevated level ATPase activities than the wild-type SecA. The decrease in ATPase activity of the mutants when 1.5 millimoles sodium azide is added is less than that in the wild-type, indicating that protein translocation in B. subtilis requires elevated ATPase activity, which is resistant to sodium azide in the mutants.

The Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, and its product were characterized. The mutational site of div-341 was located at the position Pro-431 in the B. subtilis div+ gene, where Pro was replaced by Leu. The properties of a purified B. subtilis Div protein produced by Escherichia coli were similar to those of E. coli SecA. The Div protein consists of a homodimer with a 94-kDa subunit carrying ATPase activity. However, the Div protein did not function in E. coli cells.