INTRODUCTION

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Endospore formation in Bacillus subtilis involves a series of temporally and spatially ordered changes in cell morphology and gene expression (16). In response to starvation, B. subtilis initiates a developmental process by the formation of an asymmetric septation that divides the bacterium into two compartments, the mother cell and forespore. As development proceeds, the mother cell engulfs the forespore and eventually lyses, releasing the mature spore. Mature spores are resistant to long periods of starvation, heat, toxic chemicals, lytic enzymes, and other factors that could damage a cell (10). Spores germinate and start growing when surrounding nutrients become available. Genes involved in sporulation have been identified, and their biological functions have been analyzed (30, 31). These genes are mostly transcribed during sporulation by RNA polymerase containing developmentally specific sigma factors; these sigma factors, including SigF, SigE, SigG, and SigK, are temporally and spatially activated and regulate gene expression in a compartment-specific fashion (11, 29, 31).

The outermost portion of Bacillus spores consists of cortex, spore coat layer, and, in some cases, exosporium. The cortex, a thick layer of peptidoglycan, is responsible for maintenance of the highly dehydrated state of the core, contributing to the extreme dormancy and heat resistance of spores (9, 18). The coat is composed of dozens of proteins (27), arranged in an electron-dense thick outer layer (the outer coat) and a thinner, lamellar inner layer (the inner coat) (7). These layers provide a protective barrier against bactericidal enzymes and chemicals, such as lysozyme and organic solvents (10). For example, some proteins have been shown to be required for proper spore coat formation in B. subtilis spores. SpoIVA is synthesized 2 h after cessation of exponential growth (T₂) in the mother cell compartment and plays a central role in the proper formation of both cortex and coat. Sporulating cells of a spoIVA mutant fail to synthesize a cortex, and they produce a mislocalized coat (23). The SpoIVA protein is assembled into a spherical shell around the outer surface of the forespore (22) and is thought to be required for the formation of a basement layer on which spore coat proteins assemble (23, 28). YrbA is also synthesized from T₂ of sporulation in the mother cell compartment and is required for the assembly of some coat proteins in B. subtilis spores (34). One of the coat protein components, CotE, also plays a central role in morphogenesis of spore coat and is required for the assembly of the outer coat (37). cotE mutant spores are resistant to heat and chemicals but are lysozyme sensitive and germinate slower and less efficiently than wild-type spores (37). The CotT protein of B. subtilis is synthesized as a 10.1-kDa precursor, which is processed to a coat polypeptide of 7.8 kDa, and inactivation of the cotT gene resulted in spores with an altered appearance of the inner coat layers and slow germination in response to a germination solution containing fructose, glucose, and asparagine (4). These observations indicate that some specific machinery is required for coat assembly and suggest that the coat proteins are tightly fixed to form the strong protective layers which are covering spores.

The B. subtilis genome sequencing project revealed about 4,100 protein-encoding genes, of which half have unknown functions (14). The identification of these genes will contribute useful information to the study of sporulation, germination, and spore dormancy of Bacillus at the gene level. We have previously reported the characterization of the yaaH gene, which is involved in germination of spores, located in the region between dnaA and abrB on the B. subtilis chromosome (12). In the region between abrB and spoVC in the B. subtilis chromosome, six open reading frames (ORFs) (yabD, yabE, yabF, yabG, yabH, and yabJ) were newly identified by the B. subtilis genome sequencing project (21). We systematically inactivated these genes and examined the resulting phenotypes and periods of expression of these genes. In this report, we describe the function of a gene, yabG, which was revealed to be expressed only during sporulation.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, media, and general techniques. The B. subtilis and Escherichia coli strains used in this study are listed in Table 1. ASK202, ASK203, ASK204, and ASK205 are derivatives of 168 (12). Oligonucleotide primers 8141F (5'-AGATCTATAGGGGATATGGTAGCC-3') and 8141R (5'-AGATCTCTCGAGCGAGCAATACTTCAATAG-3') were used to amplify a 448-bp segment internal to yabG from the B. subtilis 168 chromosome. The PCR product was restricted at the BglII sites introduced by the primers and inserted into BamHI-restricted pMutin1 to create plasmid pMU141. Oligonucleotide primers 8141RTF (5'-CGAGGCAGATCTTGCGACACCGATTAGAAC-3') and 8141RTR (5'-GGAGGATCCCCTATTTGAAATTGCAC-3') were used to amplify a 1,042-bp segment internal to yabG from the B. subtilis 168 chromosome. The PCR product was restricted at the BamHI and BglII sites introduced by the primers and inserted into BamHI-restricted pMutinT3 to create plasmid pMU141RT. pMutinT3 was pMutin1 into which the t1t2 terminator from the rrnB operon of E. coli had been introduced between the erythromycin resistance gene and the spac promoter (19, 35). pMU141 and pMU141RTR were introduced into strain 168 by transformation, a single crossover with selection for erythromycin resistance (0.5 µg/ml) yielding strains NIS8141 and NIS8141RT, respectively. The recombination of DNA was confirmed by Southern blotting and PCR.

Northern analysis. The cells were grown in DS medium at 37°C, and an aliquot was harvested by centrifugation. Total RNA was extracted from the cells as described previously (32). Aliquots containing 5 µg of total RNA were electrophoresed and blotted on a positively charged nylon membrane (Hybond-N⁺; Amersham). Hybridizations were performed with digoxigenin-labeled RNA probes (10 ng) according to the manufacturer's recommended procedure (Boehringer Mannheim Biochemicals). Hybridizations specific for yabG mRNA were conducted with digoxigenin-labeled RNA probes synthesized in vitro with T7 RNA polymerase using as templates PCR products amplified from pMU141. The primers used to introduce a promoter for T7 RNA polymerase for this amplification were 8141F and T7R (5'-TAATACGACTCACTATAGGGCGAAGTGTATCAACAAGCTGG-3').

Primer extension analysis. Total RNA was extracted from strain NIS8141RT growing in DS medium at 4 h after the onset of sporulation. In strain NIS8141RT, the yabG promoter region is fused downstream of the BamHI cloning site to the promoterless lacZ gene of pMutinT3. The RNA sample was subjected to primer extension assays with digoxigenin-end-labeled primers specific for the sequences around the BamHI site and the lacZ gene of pMutinT3 (RT1 [5'-TGTATCAACAAGCTGGGGATC] and RT2 [5'-CCAGGGTTTTCCCGGTCGACC]). Therefore, these primers can detect yabG-specific transcription. The RNA sample (20 µg) was incubated with the primers (1 pmol) for 60 min at 60°C and gradually cooled down to the ambient temperature over 90 min. After the addition of deoxynucleoside triphosphates (2.5 mM each) and reverse transcriptase (GIBCO BRL), the reaction mixtures were incubated for 60 min at 37°C. The cDNA products were electrophoresed through an 8% polyacrylamide-urea gel, blotted to a positively charged nylon membrane, and detected according to the directions of the manufacturer (Boehringer Mannheim Biochemicals). DNA ladders for size markers were created with the same digoxigenin-end-labeled primers by use of a digoxigenin Taq DNA sequencing kit (Boehringer Mannheim Biochemicals).

Preparation of spores. Mature spores were prepared by culturing the bacteria in DS medium at 37°C for 18 h after the end of exponential growth. The spores were harvested by centrifugation and purified by being washed in cold deionized water two times, by lysozyme treatment (0.1 mg/ml) at 37°C for 10 min, and by sonication (NISSEI; Ultrasonic Generator US-300) six times at 4°C for 15 s. The resultant cells were washed with cold deionized water by repeated centrifugation until all cell debris and vegetative cells had been removed.

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 follows. The cultures were heated at 80°C for 30 min; treated with lysozyme (final concentration, 0.25 mg/ml) at 37°C for 10 min or treated with 10% (vol/vol) chloroform at room temperature for 10 min as described by Nicholson and Setlow (20); and then diluted in distilled water, plated on Luria-Bertani agar, and incubated overnight at 37°C. The numbers of survivors were determined by counting colonies.

Spore germination. The purified spores were heat activated at 65°C for 15 min, cooled, and then suspended in 50 mM Tris-HCl (pH 7.5) buffer to an optical density of 0.5 at 660 nm. Either L-alanine (10 mM) or AGFK (3.3 mM L-asparagine, 5.6 mM D-glucose, 5.6 mM D-fructose, and 10 mM potassium chloride) was added. Germination was monitored by measurement of the decrease in the optical density at 660 nm of the spore suspension at 37°C for up to 90 min.

Solubilization of proteins from mature spores. For preparation of proteins from mature spores, spores were harvested at T₁₈ of sporulation and washed once with 10 mM sodium phosphate buffer (pH 7.2). The pellets were suspended in 0.1 ml of lysozyme buffer (10 mM sodium phosphate [pH 7.2], 1% lysozyme), incubated at room temperature for 10 min, and washed with wash buffer (10 mM sodium phosphate [pH 7.2], 0.5 M NaCl). Spore proteins were solubilized in 0.1 ml of loading buffer (62.5 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol, 10% glycerol, 0.05% bromophenol blue) and boiled for 5 min. The resulting samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (15% acrylamide) as described previously (1). The majority of spore coat proteins, including some core proteins, will be solubilized by this procedure, but not all spore proteins will be extracted.

Visualization of YabG-GFP fusion protein. Aliquots of the culture of strains harboring a yabG-gfp fusion on the chromosome sporulated in DS medium were transferred to a microscope slide coated with poly-L-lysine. Fluorescence obtained from the GFP fusion protein was observed under an Olympus fluorescence microscope (AX70) with a U-MNIBA mirror cube unit. The images were captured with a cooled charge-coupled device camera (PXL-1400; Photometrics) and obtained with the image processing software IPLAB SPECTRUM (Signal Analytics Corporation).

NH₂-terminal sequence analysis. Amino acid sequences of the samples were determined as described previously (17). The samples were subjected to SDS-PAGE, electroblotted onto a polyvinylidene difluoride (PVDF) membrane as described above, and briefly stained with Coomassie brilliant blue. After extensive washing, the protein bands of interest were excised and applied to a Procise 492 gas-phase sequencer (Applied Biosystems Division, Perkin-Elmer), and sequences of NH₂-terminal amino acids were determined.

	RESULTS

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Identification of genes transcribed only at sporulation. In the region from abrB to spoVC in the B. subtilis chromosome, six ORFs were newly identified by the B. subtilis genome sequencing project (21). The functions of these ORFs, yabD, yabE, yabF, yabG, yabH, and yabJ, have not yet been analyzed. Their transcription was analyzed by use of lacZ fusions constructed with integrational plasmid pMutin1, and a sporulation-specific gene, yabG, was identified (K. Asai et al., unpublished data). To confirm its expression pattern and transcription unit, total RNA was isolated from B. subtilis 168 (wild type) and analyzed by Northern hybridization. The data in Fig. 1B showed that a single mRNA species of approximately 1.0 kb, which hybridized with a probe specific for yabG, was first detected from T₄ of sporulation. From the nucleotide sequence, yabG was predicted to be monocistronically transcribed and the molecular mass of YabG protein was predicted to be 33 kDa (14, 21). The majority of genes induced during sporulation are transcribed by RNA polymerase containing sporulation-specific sigma factors. In order to determine which sigma factor was concerned with the transcription of yabG, we performed Northern analysis with RNA prepared from sigma factor-deficient mutants and from a gerE mutant at T₆ of sporulation (Fig. 1B). The 1.0-kb mRNA detected with the yabG probe was not found in spoIIAC, spoIIGAB, spoIIIG, and spoIVCB mutants, which were deficient in SigF, SigE, SigG, and SigK, respectively. On the other hand, the signal was still detectable in a gerE36 mutant which was deficient in the DNA-binding regulatory protein GerE.

View larger version (65K): [in this window] [in a new window]   FIG. 1.   Analysis of yabG mRNA by Northern hybridization. (A) Genome structure surrounding yabG and the sizes of the ORFs. The arrow indicates the direction of transcription. (B) Northern hybridization detecting the transcript of yabG. Transcription of yabG in strain 168 (spo+) (wild type) (lanes 1 to 7) and in spoIIAC (SigF) (lane 8), spoIIGAB (SigE) (lane 9), spoIIIG (SigG) (lane 10), spoIVCB (SigK) (lane 11), and gerE36 (GerE) (lane 12) cells was analyzed by Northern hybridization. Total RNA was prepared from the cells at T0 to T6 (lanes 1 to 7) and T6 (lanes 8 to 12). T indicates the harvesting times of cells, hours after the end of the exponential phase of growth. The arrowhead indicates the position of mRNA hybridized with digoxigenin-labeled RNA probe.

Localization of the yabG promoter. To localize precisely the yabG promoter, primer extension analysis was carried out with RNA from sporulating cells of strain NIS8141RT. Two different primers were used for this analysis; both primers yielded the same transcription start site (Fig. 2). Transcription of yabG starts 109 nucleotides (nt) upstream of the yabG GUG codon, at an A residue (Fig. 3A). Sequences centered 10 and 35 nt upstream of the transcription start site are very similar to the 10 and 35 consensus sequences recognized by SigK, with appropriate spacing (16 nt) between these consensus sequences (Fig. 3B).

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Determination of the transcription start site of yabG by primer extension analysis. RNA prepared from sporulating cells of strain NIS8141RT was hybridized with primers RT2 (A) and RT1 (B). Lanes labeled A, G, C, and T are DNA sequencing reactions with appropriate primers. Primer extension products are marked with arrowheads, and the transcription start site on the yaaH upstream sequence is marked with an asterisk and a capital letter.

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Comparison of the 5' upstream region of yabG and a consensus sequence of the 10 and 35 regions recognized by SigK. (A) 5' upstream sequence of the yabG gene. The bases which match the consensus sequence are shaded. (B) Comparison of the promoter sequence of yabG and those of the genes dependent on SigK. The nucleotide sequences of promoter regions are aligned with respect to conserved nucleotides (capital letters) in the 35 and 10 regions relative to transcriptional start sites (underlined). m indicates A or C. References for the sequences of these promoters are as follows: cotA (25), cotB and cotD (38), cotC (36, 38), cotF (7), cotG (23a), cotS (32), gerE (5), and sigK (13).

Detection of a YabG-GFP fusion protein. To explain the subcellular localization of YabG protein, this yabG-gfp fusion was introduced into the chromosome of strain 168 (wild type) and spoIVA178, cotE, and gerE36 strains. The resultant transformants were grown in DS medium and analyzed at 8 h after the cessation of logarithmic growth (Fig. 4). Spore formation was confirmed by observation under phase-contrast microscopy (Fig. 4A, C, E, and G). Expression of this fusion protein did not affect spore resistance or coat protein extraction profile (data not shown). In spoIVA178 cells, the forespores were not distinguishable from the mother cells because this mutant has defects in the formation of cortex and coat (23, 28). In the three transformants of wild-type, cotE, and gerE36 strains, the green fluorescence of YabG-GFP fusion was detected by fluorescence microscopy around the outside of forespores but not in the mother cell compartment (Fig. 4B, D, and F). In contrast, the green fluorescence of the YabG-GFP fusion in spoIVA178 cells did not condense onto the outside of forespores (Fig. 4H). The wild-type cells which do not have a yabG-gfp fusion gave no green fluorescence under the same conditions (Fig. 4J). These results suggested that YabG was a spore protein whose assembly was independent of GerE and CotE.

View larger version (80K): [in this window] [in a new window]   FIG. 4.   Detection of YabG-GFP fusion in sporulating cells. The transformants of strain 168 (A and B) and cotE (C and D), gerE36 (E and F), and spoIVA178 (G and H) strains carrying yabG-gfp fusion and the wild-type strain (168), which does not carry the yabG-gfp fusion (I and J), were grown in DS medium, and the sporulating cells at T8 were analyzed by use of phase-contrast microscopy (A, C, E, G, and I) and fluorescence microscopy (B, D, F, H, and J). Bars = 1 µm.

Properties of mutant spores. We characterized yabG mutant cells; the vegetative growth rate of the mutant in DS medium was the same as that of wild-type cells (data not shown). Mature spores prepared from the medium after 24 h of cultivation at 37°C also showed resistance to heat, chloroform, and lysozyme, as did the wild-type spores (data not shown). The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores (data not shown).

Analysis of the proteins extracted from yabG spores. We analyzed the spore proteins by SDS-PAGE. Proteins were solubilized from the spores which had been collected and purified at T₁₈ of sporulation. The protein profile of the yabG spores on SDS-PAGE was significantly different from that of wild-type spores (Fig. 5). Proteins with molecular masses of 15, 18, 21, 23, 31, 45, and 55 kDa were extracted from yabG spores (lane 2) but were less well extracted or not extracted from wild-type spores under the same conditions (lane 1). To determine the identity of these polypeptides, the electrophoretically resolved peptides were transferred to PVDF membranes and the NH₂-terminal sequences of these polypeptides were determined. The polypeptides with molecular masses of 15, 18, 21, 23, 31, 45, and 55 kDa were identified as CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively (Table 2). We found other minor differences in the bands on the gel but failed to determine their amino acid sequences (data not shown).

View larger version (54K): [in this window] [in a new window]   FIG. 5.   SDS-PAGE analysis of proteins solubilized from spores. Spores were prepared from T18 sporulating cells. The protein samples were solubilized from the spores by being boiled with SDS and 2-mercaptoethanol and analyzed by SDS-PAGE (15% polyacrylamide gel). The protein samples were from wild-type spores (lane 1) and yabG spores (lane 2). The arrowheads indicate the bands significantly increased or decreased in the protein extract of yabG spores.

	DISCUSSION

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Developmental gene expression during sporulation is unique to each of the two compartments (mother cell and forespore) and is regulated by compartment-specific sigma factors (29). yabG has a predicted SigK promoter (Fig. 3) and is transcribed from T₄ of sporulation, when SigK is activated (Fig. 1). The mRNA of yabG was detectable in the samples prepared from wild-type cells and the GerE mutant but not in those prepared from SigE, SigF, SigG, or SigK mutants (Fig. 1). The quantity of yabG transcript extracted from a gerE36 mutant at T₆ of sporulation was almost the same as that extracted from wild-type cells (Fig. 1). From these observations, we conclude that yabG is expressed under the regulation of SigK RNA polymerase in the mother cell compartment.

Analysis using a YabG-GFP fusion and fluorescence microscopy showed that YabG-GFP was detectable in a ring around the forespores (Fig. 4). This and above results indicated that YabG was synthesized in the mother cell compartment and assembled on the surface of the forespores. We introduced the yabG-gfp fusion into cotE and gerE mutants, which impair coat formation, and found that the assembly of YabG was normal in these mutant spores. We performed the same analysis for a strain carrying a spoIVA mutation, which abolishes cortex synthesis and interferes with the assembly of spore coat. SpoIVA-GFP fusion coats the outer surface of the mother cell and surrounds the forespore (22). In the spoIVA178 cells, YabG was dispersed in the mother cell cytoplasm and was not present in a tightly packed focus. These results suggest that YabG assembly around the forespore is dependent on SpoIVA. We prepared anti-YabG antiserum and detected a 33-kDa band, which corresponded to the deduced molecular mass of YabG protein, in the protein extract of sporulating cells but not in that of mature spores of the wild type by immunoblot analysis (H. Takamatsu et al., unpublished data). The 33-kDa band was detectable from T₄ but almost disappeared at T₇ of sporulation. Therefore, we speculate that YabG transiently associates with the forespore and is degraded by proteolysis during maturation of spores.

The B. subtilis yabG gene is deduced to encode a 33-kDa protein which has no obvious signal sequence or hydrophobic regions. Because the YabG protein showed no similarity with other proteins in the protein database SWISS-PROT, we could not deduce its molecular function from the sequence. An SDS-PAGE analysis showed that the protein sample solubilized from yabG mutant spores in the presence of SDS and 2-mercaptoethanol contained increased levels of CotT (15 kDa), YeeK (18 kDa), YxeE (21 kDa), CotF (23 kDa), YrbA (31 and 45 kDa), and SpoIVA (55 kDa), which were not visible or barely visible in the preparation from wild-type spores (Fig. 5). The 15- and 23-kDa polypeptides were probably the precursor proteins of CotT and CotF, respectively (4, 7). The processing enzyme(s) involved in maturation of these spore coat proteins has not yet been identified. We speculated that the 18-kDa polypeptide might be generated by proteolysis of the primary YeeK protein because the B. subtilis yeeK gene would potentially encode a 43-kDa protein. The B. subtilis yxeE gene would encode a 15-kDa protein, and the YxeE protein extracted from yabG mutant spores was estimated to be 18 kDa. Both yeeK and yxeE were functionally unknown, but our preliminary results suggest that these genes were expressed during sporulation (T. Kodama et al., unpublished data). The 45-kDa protein extracted from yabG spores corresponds to the entire YrbA protein whose molecular mass was estimated as 43 kDa. The 31-kDa polypeptide appears to be generated by proteolysis of YrbA protein because its N-terminal sequence corresponds to that from Met-164 to Met-175 of YrbA (14, 33, 34). The spoIVA gene product (55 kDa) is essential for assembly of coat proteins onto the coat layers (8, 23, 28). Since the protein profile of the yabG spores did not change after an additional 24-h incubation (data not shown), the altered coat protein composition of yabG spores as shown in Fig. 5 indicates that YabG protein is related to protein processing in sporulating cells directly or indirectly.

We analyzed the transcripts of spoIVA and yrbA in yabG cells and found that they were not affected by the mutation (data not shown). Moreover, immunoblot analyses showed that the synthesis of SpoIVA and YrbA in sporulating cells was not affected by the yabG mutation. These proteins become undetectable as forespores mature in the wild type (H. Takamatsu et al., unpublished data). Therefore, we assume that the YabG protein is not involved in gene regulation or protein synthesis. CotE is a major factor which is required for assembly of some coat proteins into the coat layer of spores (3, 37). Its mutant spores lack outer coat proteins and show defects in lysozyme resistance and germination (37). In contrast, only a few proteins decreased in the preparation from yabG mutant spores (Fig. 5). Electron microscopy showed that the ultrastructure of yabG spores was visibly the same as that of wild-type spores (data not shown). These results suggest that YabG protein is involved in coat protein composition directly or indirectly but that, unlike CotE, it is not essential for acquisition of spore resistance and germination under the experimental conditions. Here we did not deny the possibility that yabG spores lack some functions which are not able to be examined in laboratory conditions. A preliminary experiment revealed that YabG protein potentially had a protease activity, since YrbA protein was cleaved in the presence of YabG in vitro (H. Takamatsu et al., unpublished data). SpoIVA, YrbA, and CotT probably localize to forespores prior to YabG functions in the sporulating cells because the yabG gene is transcribed 2 h after the start of synthesis of these proteins (4, 23, 34). We speculate that YabG limits the quantity of some coat proteins by proteolysis. Further experiments are required to understand why and how some coat proteins are processed and/or removed by YabG on the surface of forespores.