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The ylbO gene product of Bacillus subtilis is involved in the coat development and lysozyme resistance of spore

Ritsuko Kuwana , Takashi Okumura , Hiromu Takamatsu , Kazuhito Watabe
DOI: http://dx.doi.org/10.1016/j.femsle.2004.10.038 51-57 First published online: 1 January 2005


The Bacillus subtilis YlbO protein is a Myb-like DNA binding domain-containing protein that is expressed under the control of SigE. Here, we analyzed gene expression and protein composition in ylbO-negative cells. SDS–PAGE analysis revealed that the protein profile of ylbO- negative spores differed from that of wild-type. Specifically, the expression of coat proteins CgeA, CotG, and CotY, which are controlled by SigK and GerE, was reduced in ylbO -negative cells. Northern blot analysis revealed that YlbO regulated the transcription of cgeA, cotG, and cotY. These results suggest that YlbO regulates the expression of some coat proteins during sporulation in B. subtilis directly or indirectly.

  • Bacillus subtilis
  • Sporulation
  • Spore coat
  • Transcription
  • Regulation

1 Introduction

Bacilli and Clostridia produce a dormant cell type (a spore) that is resistant to heat, lysozyme digestion, and other stresses [1]. The Bacillus subtilis spore consists of a spore coat, cortex, and core [1], and endospore formation involves a series of temporally and spatially ordered changes in cell morphology and gene expression [2]. During sporulation in B. subtilis, differential gene expressions in the prespore and/or forespore and the mother cell compartments are governed by four sporulation-specific sigma factors (SigF, SigE, SigG and SigK). SigF and SigG are active specifically in the prespore or forespore, while SigE and SigK control transcription in the mother cell [3]. In addition to the sigma factors, various regulators activate or repress transcription of many different genes during sporulation. RsfA, which is under the control of SigF and SigG, acts as a transcriptional regulator in the prespore [4]. SpoVT, which is controlled by SigG, positively and negatively regulates SigG-dependent gene expression in the forespore [5]. A small DNA binding protein, SpoIIID activates or represses transcription of many SigE and/or SigK dependent genes; the spoIIID gene is transcribed by SigE-containing RNA polymerase in the mother cell [3,68]. GerE is another small DNA binding protein which also activates or represses transcription of many SigK dependent genes. gerE gene is transcribed by SigK-containing RNA polymerases in the mother cell [3,7,8]. Previously, we identified transcriptional factor SpoVIF [9], which is controlled by SigK and regulates expression of genes controlled by SigK and GerE [9,10]. All of the known proteins involved in spore coat development are synthesized by SigE- and/or SigK-containing RNA polymerases in the mother cell [13]. The spore coat functions as a barrier to protect the cortex and core from various environmental conditions; spore coat formation is generally measured in terms of spore sensitivity to lysozyme treatment [1]. Previous DNA microarray analysis studies have shown that many heretofore uncharacterized genes are transcribed by SigE [11,12]. Here, we obtained B. subtilis strains mutated at SigE-responsive genes of unknown function and analyzed the protein profiles of lysozyme-treated spores to search for factors involved in the synthesis and assembly of spore coat proteins.

2 Materials and methods

2.1 Bacterial strains and growth conditions

The B. subtilis and Escherichia coli strains and plasmids used in this study are listed in Table 1. The B. subtilis strains are all derivatives of strain 168; those constructed in this work were prepared by transformation with plasmid DNA and confirmed by PCR. E. coli strain JM109 was used for the production of plasmids. For construction of the cotY mutant (MTB913) a segment of the cotY gene was PCR amplified with primers COTY21 (5′-AAAAAGCTTGGCCGGCATGAGAACTG-3′) and COTY184R (5′-GAAGGATCCGCCTTTTTTATCAAAAAC-3′), cut with HindIII and BamHI at primer-based sites, and inserted into HindIII/BamHI-restricted pMutin3 vectors to obtain plasmids pCOTY5E (Table 1). This plasmid was transformed into B. subtilis 168 by a single crossover recombination and selected by erythromycin resistance (0.5 μg/ml erythromycin) to yield strain MTB913 (Table 1).

View this table:

Bacterial strains, plasmids, and oligonucleotides used in this study

For construction of pMCOTY1A, a DNA fragment encoding cotY was PCR amplified with COTY3 (5′-GAA GGA TCC GAG CTG CGG AAA AAC CCA-3′) and COTY485R (5′-AGT CTC GAG CCA TTG TGA TGA TGC TTT TTA-3′), restricted, and inserted into BamHI/XhoI-cut pMALEH6 (which contains a 6× His tag) to give the recombinant plasmid pMCOTY1A (Table 1).

B. subtilis strains were grown in Difco Sporulation (DS) medium [13] and sporulated under the previously described conditions [14]. Recombinant DNA techniques were carried out according to standard protocols [15]. Preparation of competent cells, transformation, and preparation of chromosomal B. subtilis DNA was carried out as previously described [16].

2.2 LacZ assay for evaluation of gene expression

The Japanese and European Consortia for Functional Analysis of the B. subtilis Genome constructed and maintained the pMutin strains used in this study. Each strain contains a lacZ transcriptional fusion protein that can be used to monitor gene expression [17]. Chromosomal DNA from the strains was extracted and introduced into the spoIIAC (sigF) mutant by competent cell transformation. The resultant cells were grown on DS agar medium including X-gal for 48 h at 37 °C, and the colony colour was monitored to detect gene expression, as described previously [18].

2.3 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 previously described [14]. The cultures were either heated at 80 °C for 30 min, or treated with lysozyme (250 μg/ml final concentration) at 37 °C for 10 min. After the cultures were serially diluted in steps of 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 colony counting.

2.4 Preparation of spores

The B. subtilis strains were grown in DS medium at 37 °C as previously described [19]; mature spores were harvested 18 h after the cessation of exponential growth and washed once with 10 mM sodium phosphate buffer (pH 7.2). For removal of 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. Cells were then washed repeatedly with buffer (10 mM sodium phosphate, pH 7.2, 0.5 M NaCl) at room temperature [19].

2.5 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 [18]. The proteins were separated by 14% SDS–PAGE and visualized by Coomassie brilliant blue R-250 staining [19].

2.6 Purification of recombinant CotY proteins from E. coli.

The recombinant plasmid pMCOTY1A was transformed into E. coli cells. The resulting transformants were grown in 200 ml of L broth supplemented with ampicillin (50 μg/ml) at 37 °C for 3 h, at which time the culture was supplemented with 1 mM isopropyl-β-d-thiogalactopyranoside, and the cells were incubated for a further 3 h at 37 °C. The His-tagged recombinant proteins were purified by affinity chromatography on Ni-NTA agarose beads (Qiagen) and were further purified by electro-elution from an SDS gel after SDS–PAGE, as previously described [20,21].

2.7 Preparation of antisera against CotY

One milliliter of purified CotY protein (0.2 mg/ml) and 16 mg of killed Mycobacterium tuberculosis cells (Difco) were mixed with 2 ml of complete Freund's adjuvant (Difco), and 3 ml of the emulsion was injected into healthy rabbits. After two weeks, CotY solutions prepared with incomplete Freund's adjuvant (Difco) were injected; two weeks after the second immunization, antisera for CotY were isolated as previously described [21]. The preparation of rabbit antisera against CgeA, CotG, and YaaH was as described previously [9].

2.8 SDS–PAGE and immunoblotting

Whole proteins were prepared from sporulating cells as described previously [21], and protein samples (10 μg) were analyzed by 14% SDS–PAGE as previously reported [18]. Bio-Rad RC DC proteins assay kit was used to measure quantity of protein sample (Bio-Rad). Immunoblotting was performed with rabbit immunoglobulin G (IgG) against CgeA, CotG, CotY, and YaaH as described [9,21].

2.9 RNA preparation and Northern analysis

Total RNA was prepared from B. subtilis cells as described [22], and Northern analysis, hybridization and detection was performed with the DIG Northern Starter Kit (Roche) according to the previous report [10]. RNA probes for the Northern hybridization were synthesized with T7 RNA polymerase using PCR products as templates. The oligonucleotides for the cgeA and cotG templates were as previously described [9]. The 0.4 kb probe for cotY, corresponding to nt 40–458 downstream of the translation initiation codon of cotY, was prepared by PCR with primers COTY40 (5′-GAG GGA TCC GTA TGC GAT GCA GTG GAA A-3′) and COTY458RT7 (5′-TAA TAC GAC TCA CTA TAG GGC GAG GTG AGG CGA TGT TCT GTC AAC-3′). The underlined regions in the primers represent the T7 promoter sequence. RNA probes specific for cgeA, cotG, and cotY were labeled with the Roche digoxigenin labeling system as previously described [9,10].

2.10 Transmission electron microscopy

Mature 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 [10].

3 Results and discussion

3.1 Properties of mutant spores

We have searched for factors involved in the expression and the assembly of the B. subtilis spore coat. Previously, two other groups reported DNA microarray analyses indicating that about 300 genes are controlled by SigE [11,12]. Of these, 171 of these were genes of unknown function. To assess these genes of unknown function, we obtained a series of mutant strains from the Japanese and European Consortia for Functional analysis of the B. subtilis genome. Of the 171 genes, 24 were not available from the Consortia stocks. Thus, 147 strains were analyzed in terms of their gene expression during sporulation, using a LacZ assay. We found that 116 of the 147 genes showed LacZ activity on sporulation medium, whereas the remaining 31 genes had no LacZ activity under our tested sporulation conditions (data not shown). Of these 116 genes, 72 were specifically transcribed during sporulation, as shown by LacZ assay (data not shown). For further analysis of the 72 sporulation-specific genes, we prepared purified spores from each mutant and analyzed their spore protein profiles by SDS–PAGE. Many of the mutants showed wild-type or nearly wild-type protein profiles (data not shown). However, only three disruptions out of 72, deficiencies in the ylbO, ykvV and yqfD genes caused considerable changes in the composition of spore proteins (Fig. 1 and data not shown). It has already been reported that disruption of ykvV and yqfD led to defects in spore formation [11,12]. However, disruption of ylbO had previously been reported as having no obvious effect on sporulation phenotype, it produced heat-resistant spores [4]. YlbO does not participate in forming heat-resistant spore but it is possibly involved in spore coat formation. We then examined the lysozyme sensitivity and the protein profile of the ylbO mutant in detail.


SDS–PAGE analysis of proteins solubilized from spores. Cells were cultured for 24 h in DS medium, and protein samples were solubilized from the spores by boiling with SDS and 2-mercaptoethanol. The solubilized proteins were separated by SDS–PAGE (14% gel) and stained with Coomassie brilliant blue. Lane 1, wild-type spores; lane 2, ylbO-negative spores.

3.2 Resistance of ylbO-negative spores

The vegetative growth of the ylbO-negative cells in DS medium was the same as that of wild-type cells (data not shown), and consistent with a previous report [4], deficiency of the ylbO gene had no apparent effect on production of heat-resistant spores (Table 2). However, the ylbO mutation led to a considerably reduced spore resistance to lysozyme (Table 2), suggesting that some coat proteins could be improperly expressed in ylbO-negative spores and that YlbO is involved in spore coat morphogenesis.

View this table:

Resistance of mutant spores

3.3 The expression of coat proteins in ylbO-negative cells

We harvested wild-type and ylbO-negative cells 8 h after onset of sporulation, extracted total proteins and immunoblotted the samples with antisera against the spore coat proteins CgeA, CotG, CotY, and YaaH (Fig. 2). CgeA, CotG, and CotY are synthesized under the regulation of SigK and GerE [2325], while YaaH is synthesized under the control of SigE [20]. We used these proteins as markers to estimate the effect of ylbO deletion on the expression of spore coat proteins. To ensure accuracy, we confirmed the specificity of the antisera using strains that were null for expression of cgeA, cotG, cotY and yaaH (Fig. 2, lane 3) [9]. The molecular masses of the CgeA, CotG, CotY and YaaH proteins estimated from the immunoblotting were in fairly good agreement with the deduced molecular masses and previous results [9]. The expression levels of CgeA and CotG were reduced in the ylbO-negative cells compared to the wild type cells, and CotY expression was reduced to nearly undetectable levels in the ylbO-negative cells (Fig. 2). In contrast, the levels of YaaH were similar in the ylbO-negative and wild-type cells (Fig. 2). These results suggest that YlbO is involved in the expression of some, but not all, spore coat proteins.


Immunoblotting of coat proteins in ylbO-negative cells. Eight hours after cessation of sporulation, protein samples (10 μg) were solubilized from wild-type cells (lane 1), and ylbO-negative cells (lane 2). As a negative control, the protein sample from each null mutant cell was loaded in lane 3: cgeA-negative cells (a), cotG-negative cells (b), cotY-negative cells (c), or yaaH-negative cells (d). The samples were resolved by 14% SDS–PAGE and immunoblotting was performed with anti-CgeA (a), anti-CotG (b), anti-CotY (c), and anti-YaaH (d) antisera. The arrowheads show the positions of each protein.

3.4 Transcriptions of some coat proteins are controlled by YlbO

We examined the transcription of cgeA, cotG and cotY in YlbO-negative and wild-type control cells using Northern hybridization. Probes specific for the cgeA, cotG and cotY transcripts detected appropriately-sized bands [9,2325]. We confirmed the specificity of each probe using strains that were null for expression of cgeA, cotG, or cotY (Fig. 3, lanes 11–12). Two mRNAs were detected with cgeA-specific probes in the wild-type cells, but their levels were considerably reduced in ylbO-negative cells (Fig. 3(a)). Both transcripts originate from a promoter just upstream of cgeA, and cgeB (located just downstream of cgeA) is co-transcribed with cgeA[23]. The 1.0 kb cgeA transcript is assumed to be arise via transcription termination within cgeB, or from processing and/or degradation of the more abundant and presumably full-length, 1.4 kb transcript [23]. YlbO likely participate in the synthesis and/or the stabilization of two cgeAB mRNAs. Our analysis also revealed that levels of the cotG mRNA were reduced in the ylbO-negative cells (Fig. 3(b)), as was transcription of cotY (Fig. 3(c)). These results show that the transcription of cgeA, cotG and cotY is associated with YlbO.


Northern blot analysis of cgeA, cotG, and cotY mRNA. Total RNA (10 μg) was prepared from sporulating strain 168 (lanes 1–5) and ylbO- negative (lanes 6 to 10) cells. As a negative control, total RNA samples (10 μg) from each null mutant cell were loaded in lanes 11–12: cgeA-negative cells (a), cotG-negative cells (b), or cotY-negative cells (c). The number of hours after the end of the exponential growth phase is shown at the top. Total RNA was and analyzed by Northern hybridization using probes specific for cgeA (a), cotG (b) and cotY (c). The arrowheads indicate the position of each mRNA hybridizing with the digoxigenin-labeled RNA probe.

3.5 Morphology of ylbO mutant spores

We analyzed the ultrastructure of ylbO spores by transmission electron microscopy (Fig. 4). 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. 4(a)) [1]. Some changes in coat morphology were observed in ylbO mutant spores. The development of outer and inner coat layers was not completed (Fig. 4(b)). The core of ylbO mutant spore cleary appeared, which suggests that the condensation and dehydration of the core is impaired.


Electron microscopy of ylbO spores. Wild-type 168 (a) and ylbO mutant spores (b) were collected at T18 of sporulation and analyzed by electron microscopy. Bars, 0.5 μm.

3.6 The transcriptional system in the mother cell

The YlbO protein has a Myb-like DNA binding motif in its N-terminus [4]. RsfA, which has a leucine zipper motif, is a B. subtilis paralogue protein of YlbO that is controlled by SigF and SigG to regulate transcription in the prespore [4]. YlbO is controlled by SigE-containing RNA polymerases in the mother cell [4,11,12], suggesting that YlbO may function as a transcriptional regulator in the mother cell in the same manner that RsfA acts in the prespore.

In addition to YlbO, there are three other known transcriptional regulators in the mother cell: SpoIIID, GerE and SpoVIF. The genes shown in this study to be regulated by YlbO, cgeA and cotG, are controlled by GerE and SpoVIF [9]. The cotY gene is regulated by GerE, though it is unknown whether SpoVIF regulates transcription of cotY[25]. Our data indicate that YlbO controls the expression of some SigK- and GerE-dependent spore coat proteins. It is also possible that YlbO may regulate the SigE-dependent genes, because YlbO is dependent on SigE-containing RNA polymerases. We have found that at least two sigma factors (SigE and SigK) and four regulators (SpoIIID, GerE, SpoVIF, and YlbO) affect the expression of genes in the mother cell. SpoIIID and GerE have feedback regulation systems involving sigK and genes coding some spore proteins [7]. Thus, these four regulators likely monitor and adjust the mRNA and protein expression levels of coat proteins in response to environmental conditions. The expression and regulation of spore proteins during sporulation is complex, and further work will be necessary to fully understand this complicated process.


This work was supported by Grant-in-Aids for Young Scientists (B) and Scientific Research on Priority Areas (C) ‘Genome Biology’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


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