Functional relationship between SpoVIF and GerE in gene regulation during sporulation of Bacillus subtilis
Ritsuko Kuwana,
Hiromi Ikejiri,
Satoko Yamamura,
Hiromu Takamatsu and
Kazuhito Watabe
Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan
Correspondence
Kazuhito Watabe
watabe{at}pharm.setsunan.ac.jp
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ABSTRACT
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The sporulation-specific SpoVIF (YjcC) protein of Bacillus subtilis is essential for the development of heat-resistant spores. The GerE protein, the smallest member of the LuxR-FixJ family, contains a helixturnhelix (HTH) motif and is involved in the expression of various sporulation-specific genes. In this study, the gene expression and protein composition of sporulating spoVIF-negative cells were analysed. CgeA, CotG and CotS, which are GerE-dependent coat proteins, were not expressed in the spoVIF-negative cells. Northern blotting showed that SpoVIF regulated the transcription of cgeA, cotG and cotS in a manner similar to that of GerE. In spoVIF-negative cells, gerE mRNA was transcribed normally, but immunoblot analysis using anti-GerE antiserum showed that the quantity of GerE protein was considerably less than that in wild-type controls. Using GFP (green fluorescent protein) fusion proteins, the localization of SpoVIF and GerE was observed by fluorescence microscopy. SpoVIF-GFP was detectable in the mother cell compartment, as was GerE-GFP. These results suggest that SpoVIF directly or indirectly controls the function of the GerE protein, and that SpoVIF is required for gene regulation during the latter stages of sporulation.
Abbreviations: GFP, green fluorescent protein
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INTRODUCTION
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Bacteria such as Bacillus subtilis change from vegetative cells to spores in response to environmental conditions such as nutritional starvation. Spores possess extremely high dormancy, are remarkably resistant to heat, lysozyme and harsh chemicals, and show unique morphologies (Setlow, 1993
). Spore formation is the result of a complex, highly controlled process of macromolecular assembly (Driks, 1999
). The outermost layer of the spore, called the spore coat, consists of an electron-dense thick outer layer and a thinner, lamella-like inner layer (Driks, 2001
). The synthesis and assembly of spore coat proteins is precisely regulated by the sigma factors SigE and SigK, and by regulatory factors such as SpoIIID and GerE (Errington, 1993
; Piggot & Losick, 2001
).
GerE mutant spores of B. subtilis show decreased resistance to heat and lysozyme, their coat development is incomplete, and it is difficult to discriminate between their inner and outer coat layers (Moir, 1981
). The transcription of gerE is controlled by an RNA polymerase that contains SigK (Zheng et al., 1992
), and GerE activates or represses the transcription of genes controlled by SigK in the mother cell compartment (Webb et al., 1995
; Piggot & Losick, 2001
). In addition, GerE is part of a feedback regulation system in which it downregulates the transcription of the sigK gene. In this way, GerE indirectly regulates its own expression (Zheng et al., 1992
; Ichikawa et al., 1999
). GerE is the smallest member of the LuxR-FixJ family, which includes phosphorylation-activated response regulators of the two-component system (Kahn & Ditta, 1991
). The 74 amino acid GerE sequence is similar to the DNA-binding region of other, larger members of this protein family, and it includes a helixturnhelix (HTH) motif (Holland et al., 1987
; Crater & Moran, 2001
).
We previously reported that the transcription of spoVIF (yjcC) is dependent on SigK, and that spoVIF mRNA was detectable from 4 h after the onset of sporulation (T4) (Kuwana et al., 2003
). SpoVIF is involved in spore coat assembly and is required for the development of resistant spores. Transmission electron microscopy of spoVIF-negative spores showed that they closely resembled the phenotype of the gerE mutant spores. Accordingly, we examined the effect of spoVIF deletion on the expression of spore coat proteins during cell development in B. subtilis, and used this mutant to investigate the functional relationships between SpoVIF and GerE.
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METHODS
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Bacterial strains, plasmids, media and general techniques.
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 JM109 was used for the production of plasmids. The oligonucleotides used for PCR amplifications are listed in Table 2
.
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Table 2. Oligonucleotide primers used in this study
Oligonucleotides used for PCR amplifications are listed. The T7 promoter sequence is underlined. PCR products were restricted at the primer-induced enzyme sites and inserted into each enzyme-restricted plasmid for the plasmid constructions listed in Table 1 .
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For construction of a series of B. subtilis strains, segments of the cgeA, cotE, cotG, cotH, cotS and gerE genes were PCR amplified (primers listed in Table 2
), cut with HindIII and BamHI at primer-based sites, and inserted into HindIII/BamHI-restricted pMutin3 vectors to obtain plasmids pCGEA5E, pCOTE5E, pCOTG5E, pCOTH5E, pCOTS5E and pGERE5E, respectively (Table 1
). These plasmids were transformed into B. subtilis 168 by a single crossover recombination for erythromycin resistance (0·5 µg erythromycin ml-1) to yield strains MTB945, MTB905, MTB907, MTB908, MTB910 and MTB862, respectively (Table 1
).
For construction of a plasmid carrying the E. coli malE gene, oligonucleotide primers MALEM30 and MALE1188R (Table 2
) were used to amplify a 1218 bp malE gene segment from E. coli JM109. The PCR product was cut at the primer-based KpnI and SacI sites and inserted into KpnI- and SacI-restricted pTUE1122 to create plasmid pMALEH6 (Table 1
).
For construction of a series of plasmids, DNA fragments encoding cgeA, cotG, cotH, gerE and yaaH were PCR amplified (see Table 2
for primers), restricted, and inserted into BamHI/XhoI-cut pMALEH6 or XbaI/BglII-cut pTUE1122 (which contains a 6xHis tag) to give the recombinant plasmids pMCGEA1A, pMCOTG1A, pMCOTH1A, pMGERE1A and pYAAH1A, respectively (Table 1
).
For construction of plasmids carrying gfp fusion, gerE and spoVIF sequences were amplified by PCR (primers listed in Table 2
), restricted, and inserted into BamHI/XhoI-cut plasmid pGFP7C [which contains an integrated a green fluorescent protein (GFP) gene] to give the recombinant plasmids pGERE8G and pYJCC9G, respectively (Table 1
). These plasmids were introduced into strain 168 by transformation, and a single crossover was selected for via chloramphenicol resistance, yielding strains GERE8G and YJCC9G, respectively (Table 1
). The authentic gerE and spoVIF genes, respectively were replaced by the gfp fusions in these strains. GERE8G was transferred to the chromosome of B. subtilis YJCCd and the resulting strain was named DCRE8G (Table 1
).
B. subtilis strains were grown in Difco Sporulation (DS) medium (Schaeffer et al., 1965
). The conditions for sporulation of B. subtilis were as previously described (Takamatsu et al., 1999
). Recombinant DNA techniques were carried out according to standard protocols (Sambrook et al., 1989
). Preparation of competent cells, transformation, and preparation of chromosomal B. subtilis DNA was carried out as previously described (Cutting & Vander Horn, 1990
).
Purification of recombinant MalE-CgeA, MalE-CotG, MalE-CotH, MalE-GerE and YaaH proteins from E. coli.
The E. coli transformants carrying pMCGEA1A, pMCOTG1A, pMCOTH1A, pMGERE1A or pYAAH1A were grown in 200 ml L broth supplemented with ampicillin (50 µg ml-1) at 37 °C for 3 h, at which time the culture was supplemented with 1 mM IPTG, 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 (Takamatsu et al., 1998
; Kodama et al., 1999
).
Preparation of antisera against MalE-CgeA, MalE-CotG, MalE-CotH, MalE-GerE and YaaH.
One millilitre of purified MalE-CgeA, MalE-CotG, MalE-CotH, MalE-GerE or YaaH (0·2 mg ml-1) and 16 mg of killed Mycobacterium tuberculosis cells (Difco) were mixed with 2 ml complete Freund's adjuvant (Difco), and 3 ml of each emulsion was injected into healthy rabbits. After 2 weeks, MalE-CgeA, MalE-CotG, MalE-CotH, MalE-GerE and YaaH solutions prepared with incomplete Freund's adjuvant (Difco) were injected; 2 weeks after the second immunization, antisera for MalE-CgeA, MalE-CotG, MalE-CotH, MalE-GerE and YaaH were isolated as previously described (Takamatsu et al., 1998
). Preparation of rabbit antisera against CotE and CotS was performed as previously described (Kakeshita et al., 2001
; Takamatsu et al., 1998
).
SDS-PAGE and immunoblotting.
The total soluble proteins were prepared from sporulating cells as described previously (Takamatsu et al., 1998
). Protein samples were analysed by 14 or 15 % SDS-PAGE as described previously (Kuwana et al., 2002
). Immunoblotting was performed with rabbit immunoglobulin G (IgG) against MalE-CgeA, CotE, MalE-CotG, MalE-CotH, CotS, MalE-GerE and YaaH as described previously (Takamatsu et al., 1998
). We tested the activity of anti-E. coli MalE antiserum for the spore proteins extracted under our experimental conditions, and concluded that it did not react with any spore proteins of B. subtilis (data not shown).
RNA preparation and Northern analysis.
Total RNA was prepared from B. subtilis cells as described by Igo & Losick (1986)
. RNA probes for the Northern hybridization were synthesized with T7 RNA polymerase using PCR products as templates. Templates were prepared using primers in which the T7 promoter sequence (underlined) was added to the specific sequences for amplification (see Table 2
for primers). RNA probes specific for cgeA, cotG, cotS and gerE were PCR amplified and labelled with the Roche digoxigenin labelling system. For Northern analysis, hybridization and detection was performed with the DIG Northern Starter Kit (Roche) as previously described (Kuwana et al., 2003
).
Phase-contrast and fluorescent microscopy.
Strains harbouring integrated gerE-gfp or spoVIF-gfp fusion constructs were sporulated in DS medium, transferred to microscope slides and observed under a phase-contrast microscope. Fluorescence obtained from the GFP fusion was observed under a Leica fluorescence microscope (DMRE HC) with a GFP mirror cube unit (Leica). The images were captured with a cooled charge-coupled camera (model RTE/CCD-1300Y; Roper Scientific) and analysed with MetaMorph ver 4.6 r7 image-processing software (Universal Imaging Corporation).
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RESULTS AND DISCUSSION
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Lack of coat protein expression in spoVIF-negative cells
We previously reported transmission electron microscopic observations that the coat layers are altered in spoVIF-negative spores. The phenotype of these spores resembled that of loss-of-function mutants of gerE, a gene which is involved in spore morphogenesis and resistance to heat and lysozyme (Moir, 1981
; Kuwana et al., 2003
). Thus, we investigated whether SpoVIF might act up- or downstream of GerE. We confirmed that a plasmid having the wild-type spoVIF gene could complement the phenotype of a spoVIF-negative strain, suggesting that this strain had no second-site mutation (data not shown). We first focused on examining coat protein expression in spoVIF- and gerE-negative spores. We harvested wild-type, sigK-, gerE- and spoVIF-negative cells at 18 h after the onset of sporulation (T18), extracted total protein contents, and analysed the samples by immunoblotting using antisera against CgeA, CotE, CotG, CotH, CotS and YaaH (Fig. 1
). CotE, which is controlled by SigE, is a morphogenic protein involved in the proper assembly of some spore coat proteins (Zheng & Losick, 1990
; Driks et al., 1994
; Driks, 1999
). YaaH, which is transcribed by SigE, is involved in germination (Kodama et al., 1999
), while CotH, which is controlled by SigK, is an inner coat protein (Naclerio et al., 1996
). CgeA, CotG and CotS are synthesized under the regulation of SigK and GerE (Roels & Losick, 1995
; Sacco et al., 1995
; Takamatsu et al., 1998
). We used these proteins as markers to estimate the effect of spoVIF deletion on the expression of sporulation-related proteins. We confirmed the specificity of antisera using strains that were null for expression of cgeA, cotE, cotG, cotH, cotS or yaaH (Table 1
, and data not shown). The molecular masses of the CgeA, CotE, CotH, CotS and YaaH proteins estimated from the immunoblotting were in fairly good agreement with the deduced molecular masses and previous results (Bauer et al., 1999
; Takamatsu et al., 1998
). The detected CotG was larger than the deduced molecular mass, which was consistent with previous findings (Sacco et al., 1995
). CotE, CotH and YaaH were detected in gerE- and spoVIF-negative cells as well as in wild-type controls (Fig. 1B, D, F
). CotE and YaaH were found in sigK-negative cells, which is consistent with their genetic control by SigE, not SigK (Zheng & Losick, 1990
; Kodama et al., 1999
) (Fig. 1B, F
, lane 2). In contrast, the SigK-regulated protein, CotH, was not detected in sigK-negative cells (Naclerio et al., 1996
) (Fig. 1D
, lane 2). CgeA, CotG and CotS were not detected in sigK- and gerE-negative cells (Fig. 1A, C, E
, lanes 2 and 3). CgeA was not detectable in spoVIF-negative cells (Fig. 1A
, lane 4), and CotG and CotS were hardly detectable in spoVIF-negative cells (Fig. 1C, E
, lane 4). These results are consistent with reports that these proteins are controlled by SigK- and GerE (Roels & Losick, 1995
; Sacco et al., 1995
; Takamatsu et al., 1998
), and further suggest that SpoVIF is involved in the expression of proteins that are regulated by SigK and GerE.

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Fig. 1. Immunoblotting of coat proteins in gerE- and spoVIF-negative spores. The protein samples were solubilized at T18 from the following strains: wild-type (lane 1), sigK- (lane 2), gerE- (lane 3) and spoVIF- (lane 4). The samples were analysed by 14 % SDS-PAGE and immunoblotting was performed with anti-CgeA (A), anti-CotE (B), anti-CotG (C), anti-CotH (D), anti-CotS (E) and anti-YaaH (F) antisera. The arrowheads show the positions of each protein.
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SpoVIF regulates the transcription of various genes controlled by SigK and GerE
To confirm the effect of spoVIF deletion on the transcription of genes controlled by SigK and GerE, we examined the transcription of cgeA, cotG and cotS (Fig. 2
). Total RNA was isolated from B. subtilis 168 (wild-type), and from spoVIF- and gerE-negative cells, and analysed by Northern hybridization. Probes specific for the cgeA and cotS transcripts detected appropriately sized bands (Roels & Losick, 1995
; Takamatsu et al., 1998
), as did the cotG probe (0·6 kb; Sacco et al., 1995
). Two specific mRNAs were detected with the cgeA probe in the wild-type cells, but not in spoVIF or gerE null cells (Fig. 2A
). Transcription of cotG and cotS was considerably repressed in spoVIF-negative cells as compared to wild-type controls (Fig. 2B, C
, lanes 10 and 11), and was absent in gerE-negative cells (Fig. 2B, C
, lanes 12 and 13). These results show that the transcription of cgeA, cotG and cotS is dependent on SpoVIF. We speculate that SpoVIF may be directly or indirectly involved in the transcription of genes controlled by SigK and GerE.

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Fig. 2. Northern blot analysis of cgeA, cotG and cotS mRNA. Total RNA was prepared from sporulating cells, and each mRNA was detected by Northern hybridization using probes specific for cgeA (A), cotG (B) and cotS (C). The arrowheads indicate the position of each mRNA hybridizing with the digoxigenin-labelled RNA probe. Lanes 19, total RNA (10 µg) isolated from strain 168. The number of hours after the end of the exponential growth phase is shown at the top. Transcription in spoVIF- (lanes 10, 11) and gerE- (lanes 12, 13) cells at T6 and T8 was also analysed by Northern hybridization.
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Transcription of gerE in spoVIF-negative cells
The above results suggested that spoVIF was involved in the transcription of gerE. To confirm this possibility, the expression of gerE mRNA in spoVIF-negative cells was examined by Northern analysis. Transcripts of gerE were detected in spoVIF-negative cells at wild-type levels (Fig. 3
), suggesting that transcription of gerE was not controlled by SpoVIF.

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Fig. 3. Northern blotting of gerE mRNA expression in spoVIF-negative cells. Total RNA was prepared from sporulating cells, and each mRNA was detected by Northern hybridization using a probe specific for gerE. The arrowhead indicates the position of gerE mRNA hybridizing with the digoxigenin-labelled RNA probe. Total RNA (10 µg) was isolated from wild-type cells (lanes 19) and spoVIF-negative cells (lanes 1018). The number of hours after the end of the exponential growth phase is shown at the top.
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Detection of GerE protein in spoVIF-negative cells
To examine the expression of the GerE protein in spoVIF-negative cells, we performed immunoblotting with anti-GerE antiserum (Fig. 4
). GerE was detected in protein samples solubilized from wild-type cells at T4 and later periods of sporulation, with GerE expression levels increasing as sporulation progressed. In contrast, although spoVIF-negative cells showed GerE expression beginning at T4, the levels were reduced. In these experiments, the GerE-positive band coincided with the deduced molecular mass (Fig. 4
) and was not detected in gerE-negative cells (data not shown). A second, larger band (about 14 kDa) was detected in all protein samples, including gerE-negative cells, indicating that it was not GerE-specific. These results suggest that SpoVIF is required for expression or maintenance of the GerE protein.

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Fig. 4. Immunoblot analysis of GerE expression. Wild-type (lanes 14) and spoVIF-negative cells (lanes 58) were harvested at 2 h intervals during sporulation (T2 to T8). Whole-protein samples were solubilized from the sporulating cells and analysed by 15 % SDS-PAGE. Immunoblotting was performed with an anti-GerE antiserum. The arrowhead indicates the position of GerE. The number of hours after the end of the exponential growth phase is shown at the top.
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The localization of GerE- and SpoVIF-GFP fusions
To examine the localization of GerE and SpoVIF proteins during sporulation, we prepared sporulating cells with in-frame fusions of gfp to gerE or spoVIF and observed these cells by phase-contrast and fluorescence microscopy. In spoVIF+ cells, GerE-GFP localized to the mother cell at T6 (Fig. 5
A), which was consistent with previous data (Webb et al., 1995
). Next, we introduced GerE-GFP into the spoVIF-negative cells to examine the effect of spoVIF deletion on the synthesis and localization of GerE. In the spoVIF-negative cells, the level of GerE-GFP appeared as high as wild-type, and it localized to the mother cell in the same manner as in wild-type cells (Fig. 5B
). These results indicated that SpoVIF was not required for either expression or localization of GerE-GFP. We assume that GerE-GFP is more stable than the native GerE in spoVIF-negative cells. Based on the results shown in Figs 4 and 5
, we speculate that in spoVIF-negative cells, the native GerE protein may be degraded by proteolysis or incorporated into insoluble materials. Detection of a SpoVIF-GFP fusion revealed that this protein localized to the mother cell compartment at T6 (Fig. 5C
). In contrast, other proteins that are essential for coat development, such as SpoIVA, SpoVID, YrbA (SafA) and CotE, are localized on the outer surface of the forespore membrane (Price & Losick, 1999
; Ozin et al., 2001
; Webb et al., 1995
). These distinctive localization patterns suggest that SpoVIF is related to the function of GerE in the mother cell compartment.

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Fig. 5. Detection of GFP fusion proteins in sporulating cells. The cells expressing GerE- and SpoVIF-GFP fusions were allowed to sporulate in DS medium. Samples were taken 6 h after the onset of sporulation. (A) Sporangia of strain GERE8G, bearing an in-frame gerE-gfp fusion. (B) Sporangia in the spoVIF-negative strain YJCCd, which also contains an in-frame gerE-gfp fusion. (C) Sporangia of strain YJCC9G, bearing an in-frame spoVIF-gfp fusion. Phase-contrast (left panel) and fluorescence (right panel) microscope images are shown. Arrowheads indicate mother cell (MC) and forespore (FS).
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Possible functions of the SpoVIF protein in sporulating cells
Both SpoVIF and GerE are highly conserved proteins in Bacillus. Sequence alignment showed high degrees of conservation between GerE and the C-terminal DNA-binding domains of the B. subtilis proteins YfiK, YhcZ, YvqC and YxjL, which are proteins of unknown function showing similarities to the two-component response regulators (Ducros et al., 2001
). GerE is the smallest member of the LuxR-FixJ family and lacks an N-terminal signalling domain (Crater et al., 2002
; Ducros et al., 2001
). These facts suggest a possibility that SpoVIF works upon GerE protein as the N-terminal region of a two-component response regulator and also supports the GerE function. Indeed, there are several possible explanations for the functional relationship between SpoVIF and GerE: (i) SpoVIF regulates GerE indirectly by inhibiting the enzymes responsible for degradation or inactivation of GerE; (ii) SpoVIF activates GerE directly; and (iii) SpoVIF is a novel gene regulator that controls the expression of genes involved in the regulation of GerE function. At least a dozen GerE-dependent genes have been identified in B. subtilis (Piggot & Losick, 2001
), but we examined only three of them, cgeA, cotG and cotS, in this study. More detailed studies are needed to clarify the above possibilities. Overall, this work adds to our understanding of the B. subtilis SpoVIF-GerE gene control system, which has probably developed as a method for the coordination of spore protein synthesis for the purpose of constructing complex coat layers.
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ACKNOWLEDGEMENTS
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We thank Kazuo Kobayashi and Naotake Ogasawara of the Nara Institute of Science and Technology, Japan, for their technical support and advice. 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.
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Received 7 August 2003;
revised 22 September 2003;
accepted 7 October 2003.