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Genetics and Molecular Biology |
subunit of mammalian signal recognition particle receptor, is controlled by different promoters in vegetative and sporulating cells of Bacillus subtilis
Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1
Author for correspondence: Kunio Yamane. Tel: +81 298 53 6680. Fax: +81 298 53 6680. e-mail: kyamane{at}sakura.cc.tsukuba.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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subunit of the receptor for mammalian signal-recognition particle (SRP) and is essential for protein secretion and vegetative cell growth. The ftsY gene is expressed during both the exponential phase and sporulation. In vegetative cells, ftsY is transcribed with two upstream genes, rncS and smc, that are under the control of the major transcription factor 
A. During sporulation, Northern hybridization detected ftsY mRNA in wild-type cells, but not in sporulating cells of K and gerE mutants. Therefore, ftsY is solely expressed during sporulation from a K- and GerE-controlled promoter that is located immediately upstream of ftsY inside the smc gene. To examine the role of FtsY during sporulation, the B. subtilis strain ISR39 was constructed, a ftsY conditional mutant in which ftsY expression can be shut off during spore formation but not during the vegetative state. Electron microscopy showed that the outer coat of ISR39 spores was not completely assembled and immunoelectron microscopy localized FtsY to the inner and outer coats of wild-type spores.
Keywords: Bacillus subtilis, FtsY (Srb), gene expression, K and GerE, immunoelectron microscopy
Abbreviations: SRP, signal recognition particle
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Schatz & Dobberstein, 1996 ; Fekkes & Driessen, 1999 ). Bacillus subtilis is a Gram-positive bacterium that secretes high levels of extracellular enzymes into the culture medium. In B. subtilis, small cytoplasmic RNA and Ffh are homologues of SRP 7S RNA and SRP54 protein (a 54 kDa subunit of SRP), respectively (Honda et al., 1993 ; Struck et al., 1989 ). These are essential for protein translocation and the normal growth of B. subtilis (Honda et al., 1993 ; Nakamura et al., 1992 ). We cloned a gene for a homologue of the subunit of the SRP receptor (SR) and designated it srb (Oguro et al., 1995 ). The srb gene was renamed ftsY in the B. subtilis genome project since the amino acid sequence of Srb has 49·7% identity to that of E. coli FtsY (Kunst et al., 1997 ). During the vegetative stage, ftsY is transcribed with the upstream genes, rncS (ribonuclease III) and smc (a homologue of the SMC family protein), under the control of the major transcription factor A. Depleting ftsY in Bacillus subtilis inhibits normal cell growth and leads to a substantial loss of ß-lactamase translocation (Oguro et al., 1995 , 1996 ), indicating that FtsY is essential for protein translocation.
B. subtilis generates a heat-resistant endospore under poor nutrient conditions. During sporulation, the forespore and mother cell each contain a chromosome and engage in a specific and genetic program via four compartment-specific subunits of RNA polymerase. Forespore-specific gene expression is controlled by F and G. Activation of E in the mother cell is followed by the synthesis and activation of K. In addition, two small DNA-binding proteins, SpoIIID and GerE, activate or repress the transcription of many mother cell-specific genes. Mother-cell transcription factors form a hierarchical regulatory cascade in which the synthesis of each factor depends upon the activity of the prior factor, in the order E, SpoIIID, K and GerE (Losick & Stragier, 1992 ; Stragier & Losick, 1996 ). During the assembly of the cortex and coat proteins in the forespore, a number of polypeptides and proteins are synthesized within the mother cell and deposited on the forespore (Stragier & Losick, 1996 ). However, little is known about the role of the protein-secretion machinery in spore formation.
The present study shows that, in addition to the expression of ftsY in vegetative cells by the A promoter, ftsY is expressed solely at t8 during sporulation (times are given as hours after the onset of sporulation; i.e. t8 is 8 h after the onset of sporulation), from a promoter that is controlled by K and GerE, and located immediately upstream of ftsY and inside the smc gene. Electron microscopy showed that the outer coat of the ftsY mutant spores is composed of thin layers and immunoelectron microscopy localized FtsY to the coat.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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were maintained and cultured in Luria–Bertani (LB) medium. Bacterial cells were cultivated in Schaeffer medium (Schaeffer et al., 1965 ) with vigorous shaking to induce sporulation. B. subtilis ISR39 (trpC2 srb::pMT3ftsY) was constructed from B. subtilis 168 by homologous recombination between the chromosome and the plasmid pMT3ftsY, carrying another truncated ftsY gene, as described below.
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), which contains a plasmid origin of replication that functions only in Escherichia coli, the spac-1 promoter, the lacI gene expressed by the penP promoter, the ermC gene and three 
-independent transcriptional terminators in front of spac-1. A 434 bp DNA fragment containing the flanking and N-terminal portions of ftsY (134 aa) was synthesized by the PCR using the synthetic oligonucleotides PS-1 (5'-CTATACAGCCAAGCTTGAATTCGTTCAGTAACGAGG-3', generating a HindIII restriction site) and PS-2 (5'-CGCATTATGGGGATCCGTTTTCCCGACGCCGTTTAC-3', generating a BamHI restriction site) at positions 4915–4933 and 5330–5349, respectively, in the DNA sequence reported by Oguro et al. (1996) . The amplified fragment was digested with HindIII/BamHI, then ligated into pMutinT3 that had been digested with the same enzymes. The construct in which the ribosome-binding sequence and the truncated ftsY gene were positioned downstream of three -independent transcriptional terminators and the spac-1 promoter was designated pMT3FtsY.
RNA preparation and Northern hybridization.
Total RNAs of B. subtilis cells cultured in Schaeffer medium were extracted at various vegetative and sporulating stages as described by Igo & Losick (1986) . Northern hybridization proceeded according to a modification of the method described by Sambrook et al. (1989) . Total RNA (10 µg) was resolved by electrophoresis through a 1·5% agarose gel containing 2·2 M formaldehyde, then transferred to Gene Screen Plus nylon membranes (NEN Research Products). Prehybridization and hybridization proceeded at 65 °C in hybridization buffer (0·9 M NaCl, 0·09 M sodium citrate, 2xDenhardt’s reagent, 0·1% SDS, 100 µg salmon sperm DNA ml-1). To isolate DNA probes for ftsY, a 1·0 kb DNA region of ftsY was amplified by PCR using the synthetic oligonucleotides PS-3 (5'-AAAGAGGTTAAAAGATGAGCTT-3') and PS-4 (5'-GCCTATCAAGTAAGAAGATA-3') at positions 4935–4956 and 5995–5976, respectively, of the DNA sequence reported by Oguro et al. (1996) . A 1·1 kb fragment of cotYZ was amplified using PC-1 (5'-ATGATGTGTACGATTGATTA-3') and PC-2 (5'-ATATATAGACGTTCACCCAC-3') at positions 2720–2701 and 1571–1590 of the sequence described by Zhang et al. (1993) , and 0·6 kb of cotZ was amplified using PC-1 and PC-3 (5'-AAACACTTGTAAAGAGGAAT-3') at position 2151–2170 of the latter sequence (Zhang et al., 1993 ). The PCR template was chromosomal DNA of B. subtilis 168. After purification by agarose gel electrophoresis, the amplified DNA fragments were labelled with 32P using a random primer DNA labelling kit (Takara Shuzo) and used as hybridization probes.
Mapping the 5' terminus of ftsY mRNA during sporulation.
Primer extension proceeded using the synthetic oligonucleotide Pr (5'-ACCCTCTCAAACTCATCTAT-3') at position 4358–4339 of the nucleotide sequence reported by Oguro et al. (1996) . The RNAs to be tested (40 µg) and 5x104 c.p.m. 32P-labelled oligonucleotide primer were hybridized at 40 °C overnight. Rous-associated virus-2 reverse transcriptase was added and the mixture was incubated at 42 °C for 1 h. The reaction products were resolved on DNA sequencing gels. The 5' ends of ftsY-specific mRNAs were determined by comparison with sequencing ladders generated from an M13 clone that included a 1·6 kb DNA fragment of the upstream gene (smc) of ftsY using the Pr oligonucleotide primer. A 1·6 kb DNA fragment was synthesized by PCR using synthetic oligonucleotides PS-5 (5'-CCTCTGTATCAGGCACC-3') and PS-6 (5'-CAGGAGGATCCAGTTTTGCAG-3', generating a BamHI restriction site) at positions 3279–3295 and 4635–4615, respectively, in the DNA sequence reported by Oguro et al. (1996) . The amplified fragment was digested by DraI/BamHI and ligated into M13 digested with HincII/BamHI.
Preparation of cell lysates from sporulating cells.
Sporangia of B. subtilis growing in Schaeffer medium were harvested, washed once in TBS (25 mM Tris/HCl, pH 7·5, 135 mM NaCl, 2·7 mM KCl) and frozen at -70 °C until use. Frozen cells suspended in 100 µl GTE (25 mM Tris/HCl, pH 7·5, 50 mM glucose, 10 mM EDTA) were lysed with lysozyme at a final concentration of 2 mg ml-1 at room temperature for 5 min, then boiled in 0·4 M Tris/HCl, pH 6·8, 2% SDS, 0·5% ß-mercaptoethanol and 10% (v/v) glycerol for 5 min, and separated by centrifugation. The supernatants were used as cell-lysate preparations.
SDS-PAGE and immunoblotting.
Protein samples were resolved by SDS-PAGE (10% acrylamide), and electrotransferred to a PVDF membrane (Immobilon; Millipore). The membrane was incubated overnight at room temperature in phosphate buffered saline/Tween 20 (8 mM sodium phosphate, pH 7·5, 150 mM NaCl, 0·1% Tween 20), containing 5% low-fat milk. The membrane was then incubated for 1 h at room temperature with an anti-FtsY antiserum (at a dilution of 1:5000), in phosphate-buffered saline/Tween 20, followed by an incubation with a secondary antibody conjugated to horseradish peroxidase (Amersham Biotech) at a 1:5000 dilution for 1 h. Immunoblots were washed and visualized using enhanced chemiluminescence reagents, as described by the manufacturer (Amersham Biotech).
Electron microscopy.
Wild-type cells (168) and ISR39 grown in Schaeffer medium and harvested at t24 were fixed and embedded as described by Nishiguchi et al. (1994) , then stained with 1% uranyl acetate for 30 min and Reynold’s lead (Hayat, 1972 ) for 30 min. Stained cells were examined using a JEOL 2000EXII electron microscope.
Immunoelectron microscopy.
Wild-type cells at the vegetative stage and during sporulation (t18) were harvested by centrifugation and suspended in 1·0 ml phosphate-buffered Karnovsky’s fixative (Karnovsky, 1965 ) at room temperature for 1·5 h. Fixed cells were washed once in 0·5 M NH4Cl, suspended in hot solubilized 1% Bacto agar in water, gelatinized, then sequentially dehydrated at 4 °C for 15 min each in 50, 70, 80, 90 and 95% ethanol, followed by twice in 100% ethanol at -20 °C for 30 min. Thereafter, the cells were washed twice with 100% acetone at -20 °C for 30 min, then placed in Lowicryl HM20/acetone (1:3, 1:1, 3:1) at -50 °C for 1 h each, followed by 100% Lowicryl HM20 at -50 °C overnight. After adding fresh resin, blocks were polymerized by UV irradiation at -50 °C in a gelatinous capsule overnight. The blocks were thin-sectioned (gold-silver sections) using a diamond knife and placed on nickel grids that were subsequently placed on droplets of 1% glycine, 1% gelatin for 30 min, then onto a 1:200 dilution of rabbit anti-FtsY antibody overnight in a hydrated chamber. The grids were then washed five times by floating on droplets of 10 mM Tris/HCl (pH 8·0), 0·1 mM EDTA for 10 min and incubated with a 1:100 dilution of goat anti-rabbit antibodies conjugated to 15 nm gold particles (Bio-Rad) for 1 h. After a second wash, cells were stained with 1% uranyl acetate followed by Reynold’s lead (Hayat, 1972 ) for 30 min each, then examined using a JEOL 2000EXII electron microscope.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The ftsY gene forms an operon with rncS and smc. We investigated ftsY expression during sporulation by Northern hybridization and determined the size of the RNA product as well as the time it appeared in B. subtilis 168 (Fig. 1a). Cultured 168 cells were harvested at various developmental stages and total RNA was extracted for Northern hybridization. The total RNA isolated from cells during vegetative growth, at t-2, contained a band of approximately 5·5 kb that corresponded to a transcript which included three genes (Fig. 1a, lane 1). These results indicated that the three genes are simultaneously transcribed during exponential phase with two upstream genes (rncS and smc) by a putative A-containing RNA polymerase.
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, lane 2) and it was undetectable in cultures 2–6 h after the end of the exponential phase of growth (t2–t6) (Fig. 1a, lanes 3 to 5). This rapid disappearance of the 5·5 kb band may be caused by specific degradation after t0, in addition to reduced RNA production, since no obvious breakdown of 16S and 23S rRNAs was evident in the same samples (data not shown). At t8 on the other hand, a 1·7 kb band containing ftsY mRNA appeared (Fig. 1a, lane 6). We analysed transcripts of spore-coat proteins expressed during sporulation by Northern hybridization to compare the timing of ftsY expression with that of cotY and cotZ as markers, since Zhang et al. (1994) reported that cotY and cotZ are co-transcribed by K-containing RNA polymerase from the PYZ promoter with a smaller cotY mRNA resulting from premature termination or RNA processing. We detected two bands (1·4 and 0·6 kb) in total RNAs at t7, t8 and t9 using the 1150 bp DNA probe for cotYZ, but only one 1·4 kb band using the 569 bp DNA probe for cotZ (Fig. 1b). The density of the 1·4 and 0·6 kb bands indicated that cotYZ expression was maximal at t8 under our culture conditions. This period of cotYZ expression coincided with that of ftsY.
We then analysed the amounts of FtsY in lysates of B. subtilis 168 by immunoblotting. Bands for FtsY were intense at t-2 and t0. However, the density decreased after t0 (Fig. 1c, lanes 3–5). At t8, which is the period of ftsY expression (Fig. 1a), the FtsY band was again detected, but at a density that was 2·5-fold higher than that at t6 (Fig. 1c, lane 6). This result is consistent with the findings of the Northern hybridization (Fig. 1a). After t8, the amount of FtsY again decreased and the band was very faint at t10 (Fig. 1c, lane 7). On the other hand, at t2 (Fig. 1a), the amounts of FtsY protein were substantial, whereas ftsY mRNA is virtually absent (Fig. 1a, lane 3 and Fig. 1c, lane 3). These data suggest that the half-life of FtsY protein is relatively long.
Mapping the 5' terminus of ftsY mRNA expressed during sporulation
To define the 5' terminus of the 1·7 kb transcript of ftsY found at t8 (Fig. 1a), we performed primer-extension analysis using the synthetic oligonucleotide Pr (see Methods). The primer-extension product is indicated by an arrow and two smaller minor products are visible in Fig. 2. These minor products could have resulted from premature termination by the reverse transcriptase. The largest extension product indicated by an arrowhead (Fig. 2) corresponded to the 5' terminus of the 1·7 kb transcript of ftsY mRNA during sporulation. This transcript was more abundant in RNA from cells harvested at t8 than at t9. The 5' terminus of the ftsY mRNA was located 705 bp upstream of the translation-initiation site for the ftsY ORF, inside the smc gene (Fig. 3). These results indicated that ftsY is transcribed solely via the putative promoter (PK) during sporulation, since the ftsY gene is 987 bp long and a -independent transcriptional terminator is located downstream of the stop codon of ftsY. The nucleotide sequence around the PK promoter (4199–4226 region) was similar to the consensus sequence of the -35 (AC) and -10 (CATA---Ta) promoter region recognized by B. subtilis RNA polymerase containing K (Fig. 4a) (Roels & Losick, 1995 ; Zhang et al., 1994 ; Zheng et al., 1992 ). Fig. 4(a) shows that the nucleotide sequences of GerE-independent promoters closely match the consensus sequence, whereas the sequences of GerE-dependent promoters generally have little resemblance (Roels & Losick, 1995 ). The nucleotide sequence of the -10 region of the PK promoter has low identity with the consensus sequence of the K promoter, which is consistent with the fact that ftsY transcription during sporulation is regulated in a GerE-dependent manner. We identified putative GerE-binding sequences (4110–4121 bp and 4160–4171 bp) upstream from the PK promoter (Figs 3 and 4b). These data suggest that ftsY is transcribed by PK promoters during sporulation as shown in the upper part of Fig. 3.
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factor relates to ftsY transcription at t8, RNAs from the sigma factor-deficient strains MO1781 (SigE-), MO719 (SigF-), MO718 (SigG-), MO1027 (SigK-) and the GerE-deficient strain, 1G 12, were extracted at t0 and t8, and hybridized using the 1061 bp DNA fragment encoding ftsY as the probe (Fig. 5a). The 5·5 kb band found in B. subtilis 168 (Fig. 5a, lane 1), including the two upstream genes, was detected in all total RNA samples isolated at t0 (Fig. 5a, lanes 3, 5, 7, 9 and 11). The lower-molecular-mass bands may be degradation products of the 5·5 kb transcript. In contrast, the 1·7 kb bands found in the RNA preparation of wild-type cells at t8 (Fig. 5a, lane 2), were not detected in preparations of the E, F, G, K and gerE mutant cells sampled at t8 (Fig. 5a, lanes 4, 6, 8, 10 and 12). Under these conditions, we detected reduced levels of cotY and cotZ transcripts in RNA preparations derived from gerE mutant cells at t8 (Fig. 5b), compared with the wild-type. Zhang et al. (1994) reported that the expression of cotY and cotZ is under the control of K-containing RNA polymerase and GerE. No obvious bands corresponded to cotY and cotZ in the K mutant. However, lower levels of cotY and cotZ transcripts were detected in the gerE mutant compared with the wild-type. These results are all in good agreement. We detected transcripts of cotY and cotZ in RNA preparations derived from gerE mutant cells at t8, indicating that the disappearance of 1·7 kb band corresponding to ftsY is not due to substantial degradation of RNA by RNases during preparation.
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) was determined by Southern hybridization and PCR (data not shown). The strain ISR39 has three -independent transcriptional terminators upstream of the spac-1 promoter to avoid transcription of the ftsY gene from both the A (PA) and PK promoters. Expression of intact ftsY gene in this strain should be regulated by only the IPTG-inducible promoter spac-1, of which the nucleotide sequences of -35 and -10 regions are typical of a A promoter. Therefore, in the presence of a low concentration of IPTG, the ftsY gene can be expressed during the vegetative stage, but not during sporulation. We measured the amount of FtsY and the growth of ISR39 cells cultured in the presence of 0·1 mM IPTG. Immunoblotting detected normal levels of FtsY during logarithmic cell growth when cells were cultured in the presence of 0·1 mM IPTG. (Fig. 6c, lanes 1 and 2). The growth of ISR39 was impaired in the absence of IPTG (data not shown), in agreement with published results showing that FtsY is essential for growth (Oguro et al., 1996 ). We then investigated the expression of ftsY during exponential growth and sporulation by Northern hybridization, to define the size of the RNA product and determine when it appeared in B. subtilis ISR39 in the presence of 0·1 mM IPTG. We detected a 1·0 kb band at t-2 and t0 (Fig. 6b, lanes 1 and 2). This product is the expected size resulting from transcription from the spac-1 promoter. However, the ftsY transcript was not detected at t4–t8 (Fig. 6b, lanes 4–6). We monitored the level of FtsY protein by immunoblotting during sporulation under the same conditions (Fig. 6c, upper panel). In ISR39 cells, FtsY protein was present at t-2–t2 (Fig. 6c, lanes 1–3) and the level of FtsY decreased at t4–t6 (Fig. 6c, lanes 4 and 5). This timing is similar to that seen in the parent strain 168 (Fig. 1c). However, at t8, FtsY was barely detectable in ISR39. These results indicated that in the presence of IPTG, ISR39 cells do not express the ftsY gene which is under control of the K promoter (Fig. 6c, lane 7).
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) demonstrated four steps in spore-coat assembly that proceed in a defined temporal order and these are mainly regulated by the successive appearance of the regulatory proteins E, spoIIID, K and GerE. Spore-coat polypeptides are synthesized only in the mother-cell compartment, starting after 3–4 h of sporulation (t3–t4) and are individually deposited on the surface of the prespore. The finding that transcription of ftsY depends on both K and GerE suggested that FtsY protein is required for inner and outer coat layer assembly that includes post-assembly modification of the coat protein. We examined the ultrastructure of the ftsY mutant spores by electron microscopy. In wild-type 168 spores, the coat appeared to consist of a thick, dense outer multilayer and a lamella inner coat (Fig. 7a and c). Compared with spores produced by the parental strain, 80% of 150 FtsY-depleted mutant spores had a thin and somewhat disorganized outer coat structure (Fig. 7b and d). These morphological changes have also been found in cotXYZ triple mutant and cotM mutant spores (Henriques et al., 1997 ; Zhang et al., 1993 ). At t48, the ftsY mutant spores assumed the same form as they did at t24 (data not shown). This is not due to delayed sporulation in the ftsY mutant cells. Considerably less material appeared to be assembled in the surface layers of the outer coat of ISR39 spores. The appearance of this lamella-type structure of lower electron density was very similar to that typical of the inner coat layers. In addition, we analysed the effect of ftsY upon sporulation by measuring the sporulation frequency of ISR39 cells that were cultured in the presence of 0·1 mM IPTG and harvested at t24. Samples were plated following with or without heating at 80 °C for 15 min. The sporulation frequency of the mutant appeared to be almost identical to that of the wild-type (data not shown).
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). Gold granules were not found in vegetative cells of ISR39 in the absence of IPTG (data not shown). We counted gold granules in the cytoplasm and membrane of 25 cells. FtsY was localized in the cytoplasm and membrane at an approximate ratio of 2:3. This result was similar to that found by cell fractionation (data not shown). In E. coli, FtsY, a homologue of the subunit of the mammalian SRP receptor, is functional at both the cytoplasm and membrane at an approximate ratio of 1:1 (Luirink et al., 1994 ). FtsY in spores was predominantly located on the inner and outer coats (Fig. 8b and c) where they would have been brought to the forespores from mother cells. In ISR39 spores cultured in the presence of 0·1 mM IPTG, gold granules were not localized on the coat regions (Fig. 8d). In contrast, gold granules located in the core region would be expressed before the polar septum is formed and would have remained in the core region during sporulation.
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ACKNOWLEDGEMENTS |
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This study was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture of Japan. Electron microscopy was supported by the TARA (Tsukuba Advanced Research Alliance) project of Tsukuba University.
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Received 21 February 2000;
revised 12 June 2000;
accepted 3 July 2000.
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