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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 41220 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 21200 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 10140 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 51250 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 LuriaBertani 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).
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RESULTS |
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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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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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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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DISCUSSION |
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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 helixturnhelix (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.
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ACKNOWLEDGEMENTS |
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Received 19 April 2003;
revised 18 June 2003;
accepted 3 July 2003.