Bacillus subtilis spoVIF (yjcC) gene, involved in coat assembly and spore resistance

Ritsuko Kuwana1, Satoko Yamamura1, Hiromi Ikejiri1, Kazuo Kobayashi2, Naotake Ogasawara2, Kei Asai3, Yoshito Sadaie3, Hiromu Takamatsu1 and Kazuhito Watabe1

1 Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan
2 Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
3 Saitama University, Urawa, Saitama 338-8570, Japan

Correspondence
Kazuhito Watabe
watabe{at}pharm.setsunan.ac.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 (t2) after the onset of sporulation, and yjcB, yjcZ and yjcC were transcribed by the SigK RNA polymerase beginning at t4 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.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Some bacteria, such as Bacillus subtilis, change from vegetative cells to spores following an alteration in environmental conditions, for example nutritional starvation. The mature spores can remain in the dormant stage for long periods of time and are resistant to heat, toxic chemicals, lytic enzymes and other factors that cause cell damage (Aronson & Fitz-James, 1976; Goldman & Tipper, 1978; Gould, 1983; Driks, 1999). Spore formation is the result of a complex process of macromolecular assembly that is controlled at different stages. Genes involved in sporulation are mostly transcribed during sporulation by RNA polymerase containing developmentally specific sigma factors. These sigma factors, SigF, SigE, SigG and SigK, are temporally and spatially activated and regulate gene expression in a compartment-specific fashion. SpoIIID, SpoVT and GerE also regulate transcription from some SigE-, SigG- and SigK-dependent promoters, respectively (Piggot & Losick, 2001; Stragier & Losick, 1996). The mature spores of B. subtilis have three distinct structures, the core, cortex and coat, which can be observed using transmission electron microscopy (Aronson & Fitz-James, 1976). The core is the central part of the spore and is surrounded by a membrane and a thick peptidoglycan layer called the cortex (Driks, 2003). The coat is the outermost layer and is composed of many proteins arranged in an electron-dense, thick outer layer and a thinner, lamellar inner layer (Driks, 1999). The coat layers provide a protective barrier against the outside environment (Gould, 1983). Dramatic morphological events occur during sporulation (Errington, 1993). Assembly of the coat is initiated by expression of a group of morphogenetic proteins, SpoIVA, SpoVID, YrbA (SafA) and CotE, in the mother cell compartment (Roels et al., 1992; Stevens et al., 1992; Driks et al., 1994; Webb et al., 1995; Lewis & Errington, 1996; Price & Losick, 1999; Takamatsu et al., 1999a; Ozin et al., 2000). They control the assembly of the coat structural components in the middle or late stage of sporulation and their absence has a profound impact on coat structure and function (Piggot, 1973; Roels et al., 1992; Beall et al., 1993; Takamatsu et al., 1999a; Driks et al., 1994). The final period of spore development, termed maturation (stage VI), occurs with little overt change in morphology, but during this period the characteristic properties of resistance, dormancy and germinability appear in rapid sequence (Errington, 1993).

The Bacillus subtilis genome-sequencing project revealed about 4100 protein-encoding genes, half of which have unknown functions (Kunst et al., 1997). The systematic disruption of the remaining genes has already been carried out by the Japanese and European Consortia for Functional Analysis of the B. subtilis Genome (Kobayashi et al., 2003). We have previously found unique genes involved in sporulation or germination as part of the B. subtilis genome project (Asai et al., 2001; Kodama et al., 1999, 2000; Takamatsu et al., 1998, 1999a, b, 2000). In this study, we tried to identify sporulation-specific genes in the upstream region of the cotVWXYZ gene cluster using the LacZ colony assay method described by Kuwana et al. (2002). We found that three genes, yjcA, yjcB and yjcC, were transcribed after the onset of sporulation. An additional ORF, which was also expressed during sporulation, was found between yjcB and yjcC. We named it yjcZ. In this report, we describe the characterization of these four genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, media and general techniques.
The B. subtilis strains used in this study are listed in Table 1. B. subtilis 168 (trpC2) (BGSC 1A1), obtained from the Bacillus Genetic Stock Center (Ohio State University, OH, USA), was used in this study as a wild-type strain. B. subtilis YJCAd, YJCBd, YJCCd, BFS74, YJCEd, YJCFd, BFS3245, BFS3246, YJCId, YJCJd, BFS3247, BFS3248 and YJCMd were obtained from the Japanese and European Consortia for Functional Analysis of the B. subtilis Genome [JAFAN (http://bacillus.genome.ad.jp/) and SubtiList (http://genolist.pasteur.fr/)]. B. subtilis SGF602C, SGE603C, SGG604C and SGK605C were derivatives of 168 (Kuwana et al., 2002). B. subtilis TF100 and TF35 carried mutations in spoIIID and gerE, respectively, due to the insertion of the chloramphenicol resistance gene (cat) from pCBB31 into the coding region of spoIIID and gerE (Sato et al., 1996; K. Kobayashi, unpublished).


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Table 1. Bacterial strains and plasmid used in this study

 
To construct the yjcZ mutant, a synthetic DNA fragment was generated by a two-step PCR method. Oligonucleotide primers PUC-F (5'-GTTTTCCCAGTCACGACG-3') and PUC-R (5'-GAATTGTGAGCGGATAAC-3') were used to amplify a 1·1 kbp segment carrying the chloramphenicol resistance gene (cat) from pCBB31. Oligonucleotide primers YJCBA-F1 (5'- GGATGCGGACGGGAACATTG-3') and YJCBB-R1-2 (5'-CGTCGTGACTGGGAAAACCCCATAGATAAGCACCTCCT-3') were used to amplify an 806 bp segment upstream of yjcZ from the B. subtilis 168 chromosome. Oligonucleotide primers YJCBB-F2 (5'-GTTATCCGCTCACAATTCCTTTGGATACGGATTCGGCG-3') and YJCBA-R3 (5'-TGTCTTGCCCTGCTAACAGG-3') were used to amplify a 699 bp segment downstream of yjcZ from the B. subtilis 168 chromosome. Since the 5' sequences of primers YJCBB-R1-2 and YJCBB-F2 were complementary to sequences of PUC-F and PUC-R, respectively, the PCR products of 806 and 699 bp each contained an overlap region with the 1·1 kb cat DNA fragment. One microlitre of each PCR product was added to the 100 µl PCR reaction mix and then a second round of PCR was carried out with primers YJCBA-F1 and YJCBA-R3. The product of the second round of PCR was a 2·6 kbp synthetic DNA fragment generated by the PCR-mediated recombination. The PCR product was introduced into strain 168 by transformation, selecting for chloramphenicol resistance (5 µg chloramphenicol ml-1) to produce the transformant YJCZdd. Transformants were tested by PCR to confirm that insertion into chromosomal DNA was by the expected double crossover.

B. subtilis strains were grown in Difco sporulation (DS) medium (Schaeffer et al., 1965). The conditions for sporulation of B. subtilis have been described previously (Takamatsu et al., 2000). Recombinant DNA techniques were carried out by standard protocols (Sambrook et al., 1989). Methods for the preparation of competent cells, for transformation and for preparation of chromosomal DNA of B. subtilis have been described previously (Cutting & Vander Horn, 1990).

LacZ assay for evaluation of gene expression.
The Japanese and European Consortia for Functional Analysis of the B. subtilis Genome constructed the pMutin strains that we used in this study. Each strain contains a lacZ transcriptional fusion protein to monitor gene expression (Vagner et al., 1998). Chromosomal DNA from the strains was extracted and introduced into the spoIIAC (sigF) mutant by competent cell transformation. Resultant cells were grown on DS agar medium containing X-Gal for 48 h at 37 °C and the colony colour was monitored to detect expression of each gene as described previously (Kuwana et al., 2002).

RNA preparation and Northern analysis.
B. subtilis cells were grown in DS medium and 20 ml samples were harvested every hour throughout sporulation. RNA for Northern blots was then prepared by a modification of the procedure described by Igo & Losick, (1986). Aliquots (10 µg) of the RNA preparation were analysed by size fractionation through a 1 % (w/v) agarose gel containing 2·2 M formaldehyde and transferred to a positively charged nylon membrane (Roche). The membrane was stained with 0·04 % Ethylene Blue to measure the concentrations of 16S and 23S RNAs in the preparations (Herrin & Schmidt, 1988) (data not shown). The RNAs on the membrane were hybridized to specific probes for yjcA, yjcB, yjcZ and spoVIF (yjcC). The 0·2 kb probe for yjcA, corresponding to nt 41–220 downstream of the putative translation initiation codon of yjcA, was prepared by PCR with primers YJCA41 (5'-CGGTCTTTCGGCTCGCC-3') and YJCA220RT7 (5'-TAATACGACTCACTATAGGGCGAGCTGTGCACCAATAGCAGCT-3'). The 0·2 kb probe for yjcB, corresponding to nt 21–200 downstream of the putative translation initiation codon of yjcB, was prepared by PCR with primers YJCB21 (5'-CATGAAGACACGATGGC-3') and YJCB200RT7 (5'-TAATACGACTCACTATAGGGCGAGGATCGGCTTGGATATTCGC-3'). The 0·1 kb probe for yjcZ, corresponding to nt 10–140 downstream of the putative translation initiation codon of yjcZ, was prepared by PCR with primers YJCZ10 (5'- GGATACGGATTCGGCGG-3') and YJCZ140RT7 (5'-TAATACGACTCACTATAGGGCGAGGATGCACCTACGATGATGA-3'). The 0·2 kb probe for spoVIF (yjcC), corresponding to nt 51–250 downstream of the putative translation initiation codon of spoVIF (yjcC), was prepared by PCR with primers YJCC51 (5'-ATAATCAATTTTTTAAGAACATCG-3') and YJCC250RT7 (5'-TAATACGACTCACTATAGGGCGAGCGCTTGTAATAGATTCCACAA-3'). The underlined regions in the primers represent the T7 promoter sequence. Each RNA probe was prepared using the Roche digoxigenin labelling system and hybridization was performed with the DIG Northern Starter Kit (Roche).

Mapping of the 5' terminus of yjcA, yjcB, yjcZ and spoVIF (yjcC) mRNA during sporulation.
Cells were grown in DS medium and 20 ml samples were harvested at appropriate times during sporulation. RNAs for primer extension analysis were prepared by a modification of a procedure described by Igo & Losick (1986). Primer extension was performed with 5'-digoxigenin-labelled primers, YJCA80RD (5'-ATCGTGTCAAACACCAGCAGGCGGG-3'), YJCB70RD (5'-CATCCAAGTCTTTTCTGATACTGCCC-3'), YJCZ60RD (5'-AGCATAACCGCCATAACAG-3') and YJCC95RD (5'-AAATTAGCGTTTTGGAGTGATCCGGC-3'). They were complementary to nt 80 downstream of the translational start point of yjcA, to nt 70 downstream of the translational start point of yjcB, to nt 60 downstream of the translational start point of yjcZ and to nt 95 downstream of the translational start point of spoVIF (yjcC), respectively. The RNAs (20 µg) to be tested and the oligonucleotide primer were hybridized at 60 °C for 1 h. SuperScript II reverse transcriptase (Invitrogen) was added and the mixture was incubated at 42 °C for 1 h. DNA ladders for size markers were created using the same 5'-digoxigenin-labelled primers using the dideoxy chain-termination method (Takara). The products of primer extension were resolved on DNA sequencing gels and detected as recommended by Roche.

Spore resistance.
Cells were grown in DS medium at 37 °C for 18 h after the end of exponential growth and spore resistance was assayed as described previously (Takamatsu et al., 1999a). The cultures were either heated at 80 °C for 30 min or treated with lysozyme (250 µg ml-1 final concn) at 37 °C for 10 min. After the cultures were serially diluted 100-fold in distilled water, appropriate volumes of the dilutions were spread on Luria–Bertani agar plates, which were incubated overnight at 37 °C. The proportion of survivors was determined by counting the colonies.

Preparation of spores.
The B. subtilis strains were grown in DS medium at 37 °C as described previously and mature spores were harvested 18 h after the cessation of exponential growth (t18) and washed once with 10 mM sodium phosphate buffer (pH 7·2) (Takamatsu et al., 2000). To remove cell debris and vegetative cells, the pellets were suspended in 0·1 ml lysozyme buffer [10 mM sodium phosphate (pH 7·2), 1 % (w/v) lysozyme, complete protease inhibitor cocktail (Roche)] and incubated at room temperature for 10 min. They were then washed repeatedly with buffer (10 mM sodium phosphate, pH 7·2, 0·5 M NaCl) at room temperature (Takamatsu et al., 2000). After these treatments, more than 99 % of the wild-type, yjcA, yjcB and yjcZ spores were refractile and almost no dark or grey spores were visible under phase-contrast microscopy. We could not prepare the spoVIF (yjcC) mutant spores because this mutant was sensitive to lysozyme (data not shown).

Spore germination.
Purified spores were heat-activated at 80 °C for 15 min, cooled and then suspended in 10 mM Tris/HCl (pH 7·5) buffer to an OD660 of 0·5. Either L-alanine (10 mm) or agfk (10 mm L-asparagine, 10 mM D-glucose, 10 mM D-fructose and 10 mM potassium chloride) was added. Germination was monitored by measuring the decrease in OD660 of the spore suspension at 37 °C for up to 90 min (Kodama et al., 1999).

Solubilization of proteins from mature spores for SDS-PAGE.
Spore proteins were solubilized in 0·1 ml loading buffer [62·5 mM Tris/HCl (pH 6·8), 10 % (w/v) SDS, 10 % (v/v) 2-mercaptoethanol, 10 % (v/v) glycerol, 0·05 % (w/v) Bromophenol Blue] and boiled for 5 min (Kuwana et al., 2002). The proteins were separated by 14 % SDS-PAGE and visualized by Coomassie brilliant blue R-250 staining (Takamatsu et al., 2000) (data not shown).

Transmission electron microscopy.
Purified spores and sporulating cells were fixed with 2·5 % glutaraldehyde, then with 2 % OsO4 and embedded in Quetol 653. Thin sections of spores and sporulating cells stained with 3 % (w/v) uranyl acetate were observed with a JEM-1200EX electron microscope operating at 80 kV (Takamatsu et al., 1999a).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Selection of sporulation-specific genes
To characterize B. subtilis genes of unknown function, a series of strains has been constructed by integration of derivatives of the plasmid pMutin (Vagner et al., 1998). In each pMutin strain, lacZ is located downstream of the 5' portion of a target gene, allowing the pattern of target gene expression to be monitored. In this study, we screened for sporulation-specific genes in the region of the B. subtilis genome between yjcA and yjcM.

Sporulation-specific genes are transcribed during sporulation by an RNA polymerase containing developmentally specific sigma factors, namely SigF, SigE, SigG and SigK. These factors, forming a sigma cascade, are temporally and spatially activated and regulate gene expression in a compartment-specific fashion (Piggot & Losick, 2001). In the sigma cascade, SigF is essential for the activation of the other sigma factors, SigE, SigG and SigK, during sporulation. The expression of the 13 functionally unknown genes, yjcA, yjcB, yjcC, yjcD, yjcE, yjcF, yjcG, yjcH, yjcI, yjcJ, yjcK, yjcL and yjcM, was analysed by monitoring LacZ activity in the wild-type and spoIIAC (sigF) defective cells. The expression of three genes, yjcA, yjcB and spoVIF (yjcC), was significantly reduced in spoIIAC (sigF)-deficient cells (data not shown). Based on these results, we decided to characterize the region between yjcA and spoVIF (yjcC) in detail.

Sporulation-specific expression of the yjcA, yjcB, yjcZ and spoVIF (yjcC) genes
Three successive ORFs, yjcA, yjcB and spoVIF (yjcC), had been predicted in the B. subtilis chromosome (Kunst et al., 1997). We found an additional ORF encoding 49 aa in the region between yjcB and spoVIF (yjcC), and named it yjcZ in this study (Fig. 1). A homology search using BLAST revealed significant similarities of YjcZ to several unknown proteins: bacteriophage SPBc2 protein, YosA in B. subtilis, OB1389 in Oceanobacillus iheyensis, BH1397 in Bacillus halodurans and BA5513 in Bacillus anthracis (data not shown). To determine the expression pattern and the transcription unit, total RNAs were analysed by Northern hybridization (Fig. 2). A 0·6 kb transcript was first detected in cells 2 h (t2) after the onset of sporulation by a probe specific for yjcA (Fig. 2a). A 0·5 kb mRNA was detected in the cells at t4 and the following stages by a probe specific for yjcB (Fig. 2b). Two sizes of transcripts were detected, beginning in t4 cells, by probes specific for yjcZ (Fig. 2c) or spoVIF (yjcC) (Fig. 2d).



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Fig. 1. Nucleotide and deduced amino acid sequences of the yjcZ and spoVIF (yjcC) chromosomal region. The deduced amino acid sequences of each ORF (yjcZ and spoVIF (yjcC) are marked below the nucleotide sequences. Asterisks indicate stop codons. Potential ribosome-binding sites (SD) are double underlined. Inverted arrows are potential stem–loop structures of transcriptional terminators. The nucleotide sequence of each gene promoter is shown, indicating the putative -35 and -10 regions and the transcription start site (+1). Boxed sequences and dotted arrows are putative GerE-binding sites.

 


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Fig. 2. Northern blot analysis of yjcA, yjcB, yjcZ and spoVIF (yjcC) mRNA. Total RNA was prepared from sporulating cells and each mRNA was detected by Northern hybridization using probes specific for yjcA (a), yjcB (b), yjcZ (c) and spoVIF (yjcC) (d). The arrowheads indicate the position of each mRNA hybridizing to a digoxigenin-labelled RNA probe. Lanes 1–9, total RNA (10 µg) isolated from strain 168. The number of hours after the end of the exponential phase of growth is shown at the top. Transcription of yjcA in spoIIAC (SigF-) (lane 10), spoIIGAB (SigE-) (lane 11), spoIIIG (SigG-) (lane 12), spoIVCB (SigK-) (lane 13) and spoIIID (SpoIIID-) mutants (lane 14) at t4 was analysed by Northern hybridization (a). Transcription of yjcB, yjcZ and spoVIF (yjcC) in spoIIAC (SigF-) (lanes 10), spoIIGAB (SigE-) (lanes 11), spoIIIG (SigG-) (lanes 12), spoIVCB (SigK-) (lanes 13) and gerE (GerE-) (lanes 14) mutants at t6 was also analysed (b–d).

 
We then examined the dependency of yjcA, yjcB, yjcZ and spoVIF (yjcC) expression on the sigma factors SigF, SigE, SigG and SigK. We performed Northern analysis with mRNAs prepared from cells deficient in these sigma factors and transcriptional regulators, SpoIIID and GerE. As shown in the right half of the panel in Fig. 2(a), yjcA mRNA was not detectable in sigF or sigE mutant cells, whereas it was detectable in sigG, sigK and spoIIID mutant cells and in wild-type cells analysed at t4 of sporulation. The yjcB mRNA was not found in sigF, sigE, sigG or sigK mutant cells, while it was present in gerE mutant cells analysed at t6 of sporulation (Fig. 2b). No yjcZ mRNA was detected in any of the sigma factor mutants or in gerE-deficient cells analysed at t6 of sporulation (Fig. 2c). Using a specific probe for spoVIF (yjcC), the 0·6 kb mRNA was not detected in any of the sigma factor or gerE-deficient cells analysed at t6 of sporulation, while 0·3 kb mRNA was detected in gerE-deficient cells (Fig. 2d). The 0·6 kb mRNA shown in Fig. 2(d) was probably derived from the yjcZ promoter (P3), because the size of the transcript was consistent with the mRNA that hybridized with a probe specific for yjcZ (Fig. 3e). The results shown in Fig. 2 suggest that the expression of yjcA is dependent on SigE RNA polymerase in the mother cell compartment, but not on SpoIIID. Transcripts from the yjcB and spoVIF (yjcC) promoters (P2 and P4) are both dependent on SigK RNA polymerase, but not on GerE in the mother cell compartment. Expression from the yjcZ promoter P3 is dependent on both SigK RNA polymerase and GerE in the mother cell compartment.



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Fig. 3. Transcription analysis of yjcA, yjcB, yjcZ and spoVIF (yjcC) by Northern hybridization. Total RNA (10 µg) was prepared from sporulating cells of strains 168 (wild-type) (lane 1) and mutants in yjcA (lane 2), yjcB (lane 3), yjcZ (lane 4) and spoVIF (lane 5). Each mRNA was detected by Northern hybridization using probes specific for yjcA (a), yjcB (b), yjcZ (c) and spoVIF (yjcC) (d). The arrowheads indicate the position of each mRNA hybridizing with a digoxigenin-labelled RNA probe. The loci and probes used in these experiments, the prospective promoters for yjcA (P1), yjcB (P2), yjcZ (P3) and spoVIF (yjcC) (P4), and the terminators (T1–3) are shown in (e).

 
Transcriptional units of these four mRNAs were confirmed by additional Northern blot analysis (Fig. 3). The 0·6 kb mRNAs that hybridized with a yjcA-specific probe were found in yjcB, yjcZ and spoVIF (yjcC) mutant cells at t4 of sporulation (Fig. 3a), which indicates that the transcript derived from the yjcA promoter P1 ends at the yjcA terminator T1 (nt position 1 252 045–1 252 074 in GenBank) (Fig. 3e). As shown in Fig. 3(b), the 0·5 kb mRNAs corresponding to yjcB were not detected in yjcB and yjcZ mutant cells analysed at t6 of sporulation. This result indicates that the transcriptional unit of the yjcB gene is derived from the yjcB promoter P2 and ends at the terminator T2 (Figs 1 and 3e). mRNA larger than 0·5 kb was detected with a yjcB-specific probe in yjcZ mutant cells (Fig. 3b, lane 4). This transcript would have arisen from the modified size of yjcZ, which was constructed by insertional disruption with the cat gene as described in Methods. Using a probe specific for yjcZ, 0·2 and 0·6 kb mRNAs were detected in yjcA and yjcB mutant cells as well as wild-type cells analysed at t6 of sporulation (Fig. 3c). This result suggested that the 0·2 and 0·6 kb mRNAs were both derived from the yjcZ promoter P3. The smaller transcript ended at terminator T2 and the larger one was a read-through product of the gene, which ended at terminator T3 (Fig. 3c, e). As shown in Fig. 3(c), a 0·2 kb mRNA, but not a 0·6 kb one, was found in spoVIF (yjcC) mutant cells at t6 of sporulation. The amount of the 0·2 kb mRNA was obviously reduced in spoVIF (yjcC) mutant cells compared to wild-type cells (Fig. 3c; lane 5). The 0·5 kb mRNA, which would be derived from the yjcB promoter P2, was so rare that it was not clearly detectable with the yjcZ probe. We conclude that the activity of the yjcB promoter P2 was lower than that of the yjcZ promoter P3. As shown in Fig. 3(d), a probe specific for spoVIF (yjcC) hybridized with the 0·3 and 0·6 kb mRNAs in both yjcA and yjcB mutant cells analysed at t6 of sporulation. The 0·3 kb mRNA, but not the 0·6 kb mRNA, was detected in yjcZ mutant cells (Fig. 3d; lane 4). The 0·6 kb mRNA would correspond to the transcript derived from the region between the yjcZ promoter P3 and the spoVIF (yjcC) terminator T3 (Fig. 1 and 3e). These results indicate that spoVIF (yjcC) has a putative promoter P4 and a terminator T3 after the spoVIF (yjcC) stop codon (Fig. 1 and 3e).

Location of the yjcA, yjcB, yjcZ and spoVIF (yjcC) promoter
To further analyse the dependency of yjcA, yjcB, yjcZ and spoVIF (yjcC) expression on sigma factors, the start points of their transcription were mapped by primer extension analysis (Fig. 4). Fig. 4 shows the results of high-resolution mapping of the 5' terminus of these transcripts. In each case, we performed primer extension analysis using a synthetic oligonucleotide primer and RNA isolated from sporulating wild-type cells (see Methods). The size of the transcript indicated that transcription of yjcA started at a G residue 28 nt upstream of the proposed start codon of yjcA (Fig. 4a). The nucleotide sequence around the yjcA promoter was similar to the consensus sequence of the -35 (DYMTRWW) and -10 (CATAHAWT) promoter region recognized by RNA polymerase containing SigE in B. subtilis (Eichenberger et al., 2003). The transcription of yjcB started at GC residues 48 nt upstream of the proposed start codon of yjcB (Fig. 4b). The transcription of yjcZ and spoVIF (yjcC) started at an A residue 22 nt upstream of the proposed start codon and at an A residue 26 nt upstream of the proposed start codon, respectively (Fig. 4c, d). Here, we propose a more suitable ORF of spoVIF (yjcC) than the predicted ORF in JAFAN and SubtiList based on primer extension analysis (Fig. 1). These results show that the nucleotide sequences around the yjcB, yjcZ, spoVIF (yjcC) promoters are similar to the consensus sequence of the -35 (mACm) and -10 (CATA---Ta) promoter region recognized by B. subtilis RNA polymerase containing SigK (Helmann & Moran, 2001). The dependency of yjcZ expression on GerE suggests that one or more GerE-binding sites are located near the yjcZ promoters. Indeed, two putative GerE-binding sites are present in the region upstream from the yjcZ promoter (Fig. 1) (Ichikawa et al., 1999).



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Fig. 4. Primer extension analysis of yjcA, yjcB, yjcZ and spoVIF (yjcC). RNA prepared from sporulating cells of wild-type at stage t4 for yjcA and at t6 for yjcB, yjcZ and spoVIF (yjcC) was hybridized with digoxigenin-labelled primers. The lanes labelled A, C, G and T contain DNA sequencing reactions with appropriate primers. Primer extension products are marked with arrowheads and the transcription start site at each gene's upstream sequence is marked with an asterisk and a capital letter. Lane 1 on each panel indicates the primer extension products.

 
Properties of mutant spores
The vegetative growth of the mutant cells in DS medium was the same as that of wild-type cells (data not shown). Mature spores of yjcA, yjcB and yjcZ harvested after 24 h cultivation at 37 °C showed resistance to heat and lysozyme, similar to the wild-type spores (Table 2). Mutation of the spoVIF (yjcC) gene reduced spore resistance to heat and lysozyme (Table 2). The sensitivity of spoVIF (yjcC) spores was confirmed by phase-contrast microscopy; lysozyme treatment caused lysis of spoVIF (yjcC) mutant spores (data not shown). Germination of yjcA, yjcB and yjcZ spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose and potassium chloride was also similar to that of wild-type spores (data not shown). We did not test the germination of spoVIF (yjcC) spores because the heat activation treatment prior to incubation with germinants would damage the heat-sensitive mutant spores (see Methods).


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Table 2. Resistance of mutant spores

 
We analysed the spore proteins of yjcA, yjcB and yjcZ mutants by SDS-PAGE. The protein profiles of yjcA, yjcB and yjcZ spores were similar to that of wild-type spores (data not shown).

Morphology of spoVIF (yjcC) mutant spores
We analysed the ultrastructure of spoVIF (yjcC) spores by transmission electron microscopy (Fig. 5). The coat of the wild-type spores has two major layers, a highly electron-dense and thicker outer coat and a fine lamellar inner coat (Fig. 5a) (Driks, 1999). Some changes in coat morphology were observed in spoVIF (yjcC) mutant spores. Almost no coat layers were found around the cortex only a thin dark layer was observed. The core of the spoVIF (yjcC) mutant spore appeared larger than that of wild-type spore (Fig. 5b).



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Fig. 5. Electron microscopy of spoVIF (yjcC) spores. Wild-type 168 (a) and spoVIF (yjcC) mutant spores (b) were collected at t18 of sporulation and analysed by electron microscopy. Bars, 0·5 µm.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have identified four additional B. subtilis sporulation genes, yjcA, yjcB, yjcZ and spoVIF (yjcC), in this study. To identify a putative function, we first analysed their predicted amino acid sequences. Homology searches (BLAST) with the complete genome sequences of Bacillus species which have been published revealed that YjcA, YjcZ and SpoVIF (YjcC) were conserved proteins in Bacillus. However, the functions of these conserved proteins have not been characterized. By contrast, YjcB has no similarity to other known proteins based on results of BLAST searches.

The spoVIF (yjcC) mutant spores showed a unique phenotype. During lysozyme treatment, the spoVIF (yjcC) mutant spores lost their refractility, becoming phase dark and grey (data not shown). In general, dormant spores resist lysozyme digestion because of the impermeability of their complex coat structure exterior to the cortex. Western blot analysis using antibodies against individual coat proteins revealed the loss of some spore proteins in spoVIF (yjcC) mutant spores (unpublished data). Transmission electron microscopic observation also showed that the coat layers of spoVIF (yjcC) spores were almost absent (Fig. 5b). The core of spoVIF mutant spore appeared larger than that of wild-type spore, which suggests that the condensation and dehydration of the core is impaired (Fig. 5b). This result suggests that spoVIF (yjcC) mutation causes a defect at stage VI (Errington, 1993). SpoVIF (YjcC) must be an essential protein for the assembly or synthesis of spore coat proteins. Therefore, we propose that yjcC should be renamed spoVIF. The phenotype of spoVIF (yjcC) is reminiscent of that of spoVID and yrbA (safA) mutants, in which the coat is abnormally assembled (Beall et al., 1993; Takamatsu et al., 1999a). CotE is also a structural protein in the outer-coat layer and is required for morphogenesis of the coat layer of B. subtilis (Zheng et al., 1988; Bauer et al., 1999). These morphological proteins, such as SpoIVA, SpoVID, YrbA and CotE, are transcribed by RNA polymerase containing SigE at t2 of sporulation (Stevens et al., 1992; Roels et al., 1992; Beall et al., 1993; Takamatsu et al., 1999a; Zheng & Losick, 1990). On the other hand, SpoVIF (YjcC) is transcribed by RNA polymerase containing SigK at t4 and inactivation of the spoVIF (yjcC) gene results in more significant changes in the morphology of the spores compared to mutation in the spoVID or yrbA (safA) genes (Beall et al., 1993; Takamatsu et al., 1999a). The phenotype of the spoVIF (yjcC) mutant spores also resembles that of a gerE mutant. The gerE gene is also transcribed at t4 of sporulation by RNA polymerase containing SigK (Cutting et al., 1989). GerE is a DNA-binding protein with a helix–turn–helix (HTH) motif, involved in the transcription of some cot genes (Holland et al., 1987; Cutting et al., 1989; Zheng & Losick, 1990; Zhang et al., 1994; Crater & Moran, 2002). The gerE mutant spores also had decreased resistance to heat and lysozyme. Like spoVIF (yjcC) mutant spores, the coat of gerE mutant spores is incomplete and it is difficult to discriminate between inner- and outer-coat layers (Moir, 1981). As shown in Fig. 3(c), the expression of yjcZ was significantly reduced in spoVIF (yjcC) mutant cells. We speculate that the reduction of yjcZ transcription is caused by a polar effect of the spoVIF (yjcC) mutation or is due to regulation by SpoVIF (YjcC). SpoVIF (YjcC) may possibly, like GerE, be involved in the synthesis of some spore proteins, even though it does not possess the consensus sequence of known DNA-binding motifs. To summarize, we have identified an additional B. subtilis sporulation gene, spoVIF (yjcC), which is required for the efficient synthesis or assembly of a normal, fully resistant spore coat.


   ACKNOWLEDGEMENTS
 
We thank the Japanese and European Consortia for Functional Analysis of the B. subtilis Genome for providing the pMutin strains. This work was supported by Grant-in-Aids for Scientific Research on Priority Areas (C) ‘Genome Biology’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 19 April 2003; revised 18 June 2003; accepted 3 July 2003.