GerE-independent expression of cotH leads to CotC accumulation in the mother cell compartment during Bacillus subtilis sporulation

Loredana Baccigalupi1,{ddagger}, Gaetano Castaldo1,{dagger},{ddagger}, Giuseppina Cangiano1, Rachele Isticato1, Rosangela Marasco2, Maurilio De Felice1 and Ezio Ricca1

1 Dipartimento di Fisiologia Generale ed Ambientale, Università Federico II, via Mezzocannone 16, 80134 Napoli, Italy
2 Dipartimento di Scienze della Vita, Seconda Università di Napoli, Caserta, Italy

Correspondence
Ezio Ricca
ericca{at}unina.it


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Evidence is presented that expression of the cotH gene, whose product is required for the correct assembly of the Bacillus subtilis spore coat, is negatively controlled by the transcriptional regulator GerE. Mutations in the GerE-box, present in the cotH promoter region, increased expression of this gene, which also remained elevated during late stages of sporulation, when in wild-type cells cotH is normally turned off. Such alterations of cotH expression did not significantly affect spore coat structure or function but caused the accumulation of CotC molecules in the mother cell compartment, most likely as a consequence of CotH-mediated protection of CotC.


Abbreviations: DMS, dimethyl sulfate

{dagger}Present address: The School of Pharmacy, University of London, London, UK.

{ddagger}These two authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Bacillus subtilis spore coat is a complex multilayered protein structure which has long been considered to play a most important role in the resistance of spores to toxic chemicals and for their efficient germination (Henriques & Moran, 2000; Driks, 2002). More recent studies have shown that enzymic activity is associated with a specific coat component (Enguita et al., 2002, 2003; Martins et al., 2002) and that the coat surface presents peculiar structures (ridges) that seem to be formed when the spore volume decreases (during sporulation) and to disappear when the spore swells (during germination) (Chada et al., 2003). These findings suggest that the coat is a dynamic structure that may sense the external environment through active enzymes present on its surface and adapt to changes in the spore volume by expanding and contracting in response to dehydration and rehydration occurring during the B. subtilis life cycle (Driks, 2003).

The coat is composed of a heterogeneous group of over 30 polypeptides arranged into three main structural layers: a diffuse undercoat, a laminated lightly staining inner layer and a thick electron-dense outer coat. Several of these polypeptides have been studied and their structural genes (cot genes) identified. Expression of all cot genes is governed by a cascade of four transcription factors, acting specifically in the mother cell compartment of the sporangium in the sequence {sigma}E>SpoIIID>{sigma}K>GerE, with {sigma}E and {sigma}K being RNA polymerase {sigma} factors and SpoIIID and GerE being DNA-binding proteins acting in conjunction with RNA polymerase associated with {sigma}E (E{sigma}E) and {sigma}K (E{sigma}K) (Zheng & Losick, 1990; Henriques & Moran, 2000; Driks, 2002). GerE, in conjunction with {sigma}K-directed RNA polymerase, regulates either positively or negatively the final class of coat genes. Therefore, a gerE null mutant shows an altered expression of several mother cell genes and produces spores with a strongly altered inner coat, no longer resistant to noxious chemicals and enzymes, and defective in late stages of germination (Moir, 1981; Henriques & Moran, 2000; Driks, 2002).

In addition to the transcriptional control, a variety of post-translational modifications have been shown to occur during coat formation. Some coat-associated polypeptides appear to be glycosylated (Henriques & Moran, 2000), while others are derived from proteolytic processing of larger precursors (Aronson et al., 1988; Cutting et al., 1991; Serrano et al., 1999). Cross-linking of structural proteins is also believed to occur and results in the insolubilization of specific components (Kobayashi et al., 1994). At least two coat components assemble in the mature coat in either homo- or hetero-multimeric forms (Isticato et al., 2004; Zilhao et al., 2004).

The initial stages of coat assembly occur early after the onset of sporulation and involve functional interactions among at least two morphogenetic proteins, both made under {sigma}E control, SpoIVA and CotE. Initially, SpoIVA localizes at the outer forespore membrane and then directs the assembly of CotE in a ring-like structure that surrounds the forespore at a distance of about 75 nm (Driks et al., 1994). The gap generated by the localization of SpoIVA and CotE is thought to become the site of assembly of the inner coat components. In contrast, the outer coat proteins are assembled on the outside of the CotE structure (Henriques & Moran, 2000; Driks, 2002). Additional proteins with morphogenetic functions are needed for coat formation: SpoVID, produced under {sigma}E control, has the dual role of directing SafA to the forming spore and maintaining the CotE ring around the forespore (Henriques & Moran, 2000; Ozin et al., 2001; Driks, 2002); CotH, produced under {sigma}K control, plays a role in the assembly of various outer coat components and in the development of the lysozyme resistance typical of the mature spore (Naclerio et al., 1996; Zilhao et al., 1999). Additionally, CotH, in conjunction with CotE, is required for efficient spore germination (Naclerio et al., 1996). Recent studies have shown that the previously observed role of CotH in the assembly of the outer coat components CotC, CotG and CotB (Naclerio et al., 1996) is to stabilize CotC (Isticato et al., 2004) and CotG, which in turn is needed for dimerization and assembly of CotB (Zilhao et al., 2004).

Here we analyse the expression of the cotH gene and the effects of its deregulation on spore structure and on the assembly of the CotH-controlled coat component CotC.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and transformation.
B. subtilis strains utilized are listed in Table 1. Plasmid amplification for DNA sequencing, subcloning experiments and transformation of Escherichia coli competent cells were performed with E. coli strain DH5{alpha} (Sambrook et al., 1989). Bacterial strains were transformed by previously described procedures: CaCl2-mediated transformation of E. coli competent cells (Sambrook et al., 1989) and two-step transformation of B. subtilis (Cutting & Vander Horn, 1990).


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Table 1. B. subtilis strains

 
Sporulation and germination.
Sporulation was induced by the exhaustion method in Difco Sporulation (DS) Medium as previously described (Cutting & Vander Horn, 1990; Nicholson & Setlow, 1990). Sporulation and germination efficiencies were measured as previously reported (Cutting & Vander Horn, 1990; Naclerio et al., 1996).

Construction of cotH : : lacZ translational fusion and {beta}-galactosidase assays.
A 405 bp DNA fragment was PCR-amplified from the B. subtilis chromosome, priming the reaction with oligonucleotides G and H (Table 2). The purified fragment was cloned into a pGEM-Teasy vector (Promega), yielding plasmid pGT-GH. The genomic fragment contained in pGT-GH was then excised by BamHI digestion, gel purified and cloned into pNM482 vector (Minton, 1984), in-frame with the lacZ gene of E. coli, to obtain a cotH : : lacZ translational fusion. The resulting plasmid was linearized by digestion with SmaI and was ligated to a chloramphenicol-resistance (cat) cassette excised from plasmid pIM1101 (Ricca et al., 1992), yielding plasmid pGC24. Plasmid pGC24 was used to transform competent cells of B. subtilis strain PY79, generating the B. subtilis strain GC237 by single reciprocal recombination (Campbell-like) at the cotH locus. Chromosomal DNA of this strain was then used to transform the congenic gerE null mutant KS450, generating strain GC238. This and all other primary recombination products were confirmed by PCR.


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Table 2. Synthetic oligonucleotides

 
The specific {beta}-galactosidase activity was determined using ONPG as substrate as previously reported (Ricca et al., 1992). Samples (1 ml each) of cotH : : lacZ-bearing cells were collected, during sporulation, at the times indicated and assayed as previously described (Ricca et al., 1992).

Construction of mutations in the GerE-box.
Mutations in the GerE-box were introduced using oligonucleotides Mut1 and Mut 2 (Table 2), both containing a mutated version of the GerE-box (GG to TT and T to G changes, respectively; see Fig. 3a). Mut1 and Mut2 were separately used, in conjunction with oligonucleotide H (Table 2), to prime a PCR reaction with B. subtilis chromosomal DNA as a template. The PCR products of the expected size (180 bp) were cloned into pGEM-Teasy vector (Promega) and sequenced to confirm the presence of the expected nucleotide changes. The 180 bp DNA fragments were sequentially digested with EcoRV and BglII and used to replace the equivalent region in pGT-GH. The modified BamHI restriction fragments were then cloned in pGC24, generating plasmids pLB34 (GerE-box/mut2) and pLB35 (GerE-box/mut1) with the translational fusion cotH : : lacZ mutated at the GerE-box sequence. These plasmids were then used to transform competent cells of B. subtilis strain PY79 by single reciprocal recombination (Campbell-like) at the cotH locus, yielding the B. subtilis recombinant strains NC37 (cotH : : lacZ pLB34) and NC38 (cotH : : lacZ pLB35).



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Fig. 3. (a) PCR-induced mutations in the GerE-box of the cotH promoter region. (b) cotH-directed {beta}-galactosidase synthesis in an otherwise-wild-type strain ({square}), in a gerE null background ({blacksquare}) and in a wild-type background carrying the mut1 ({bullet}) or mut2 ({circ}) mutation. Samples were collected at various times after the onset of sporulation. The data are means of three independent experiments.

 
Mutant GerE-box at the cotH locus.
Oligonucleotides Mut1 (bearing a mutated GerE-box sequence and the BglII genomic site) and H13 (annealing in the cotH coding region and containing the EcoRI genomic site) were used to prime a PCR reaction with B. subtilis chromosomal DNA as a template. The PCR product was cloned in pGEM-Teasy vector (Promega), yielding plasmid pNC11. The insert was sequenced to confirm the presence of the expected mutation in the GerE-box. pNC11 was digested with BglII and EcoRI; the fragment of 850 bp was gel purified and used to substitute the same fragment in pER112, a pER110 derivative (Naclerio et al., 1996) containing the entire cotG gene and a truncated form of cotH extending to the EcoRI site. The resulting pNC13, with a mutated GerE-box upstream of the truncated form of cotH, was used to transform ER203 (cotG{Delta}erm), generating strain NC5 by a Campbell-type single cross-over integration. To insert a wild-type copy of cotG at the amyE locus, a PCR fragment containing cotG coding and regulatory sequences (Sacco et al., 1995) was amplified with oligonucleotides H and Del3 and cloned in pGEM-Teasy vector (Promega). The plasmid was digested with SphI and SalI and the 1·3 kb fragment containing the cotG sequence was transferred into pDG364 (Cutting & Vander Horn, 1990) linearized with the same enzymes. The resulting plasmid pNC15 was linearized and used to transform NC5 competent cells, selecting chloramphenicol resistance and yielding recombinant strain NC6, by double cross-over recombination in the non-essential amyE gene on the B. subtilis chromosome.

Western blotting.
Spores, prepared as previously described (Isticato et al., 2004), were collected after a 30 h incubation at 37 °C, washed four times and purified by lysozyme treatment as previously described (Cutting & Vander Horn, 1990; Nicholson & Setlow, 1990). The number of purified spores obtained was measured by direct counting with a Bürker chamber under an optical microscope (Olympus BH-2 with 40x lenses). Aliquots of 1010 spores suspended in 0·3 ml distilled water were used to extract coat proteins by 0·1 M NaOH treatment at 4 °C as previously reported (Isticato et al., 2004). The concentration of the extracted coat proteins was determined by the Bio-Rad DC (Detergent Compatible) Protein Assay to avoid potential interference by the NaOH present (0·2–0·6 mM final concentration) in the extraction buffer and 15 µg of total proteins was fractionated on 18 % denaturing polyacrylamide gels. Proteins were then electrotransferred to nitrocellulose filters (Bio-Rad) and used for Western blot analysis by standard procedures. The analysis of sporulating cells was performed as previously described (Isticato et al., 2001, 2004). Samples were harvested at various times during sporulation and disrupted by sonication in 25 mM Tris (pH 7·5), 0·1 M NaCl, 1 mM EDTA, 15 % (v/v) glycerol and 0·1 mg phenylmethylsulfonyl fluoride ml–1. Sonicated material was then fractionated by centrifugation at 12 000 g for 20 min. The pellet, containing the forespores resistant to the sonication treatment, was solubilized in 0·1 M NaOH at 4 °C and the total protein concentration determined as described above. 50 µg (mother cell extract) or 15 µg (forespore extract) of total proteins were fractionated on 18 % denaturing polyacrylamide gels. Western blot filters were visualized by the SuperSignal West Pico Chemiluminescence (Pierce) method as specified by the manufacturer.

In vivo methylation and primer extension analysis.
Sporulating cells of strains PY79 (wild-type) and KS450 (gerE), 6 h after the onset of sporulation, were used for methylation experiments. Methylation was performed as previously reported (Marasco et al., 1994), by adding freshly diluted dimethyl sulfate (DMS) (Aldrich) to a final concentration of 0·1 % and incubating for 3 min at 37 °C with shaking. The reaction was stopped by adding an equal volume of ice-cold saline phosphate (SP) buffer (150 mM NaCl, 40 mM K2HPO4, 22 mM KH2PO4; pH 7·2). Cells were harvested by centrifugation at 10 000 g for 10 min and washed twice with SP buffer. Chromosomal DNA was purified as previously described (Cutting & Vander Horn, 1990) and contaminating RNA removed by treatment with RNase A and T1 followed by polyethylene glycol 6000 precipitation (Marasco et al., 1994). Breakage points of the modified DNAs were induced by heat-treatment at 95 °C, as previously reported, and revealed by primer extension as described by Marasco et al. (1994). Chromosomal DNA was primer extended in a linear PCR using Taq polymerase with separate oligonucleotides E1 and H8 (Table 2). End-labelling, sequencing reactions and PCR programme were as described previously (Marasco et al., 1994). Gels were fixed, dried and autoradiographed on Kodak X-Omat AR films with an intensifying screen at –70 °C for 24–48 h.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
cotH expression is negatively regulated by GerE
Primer extension experiments have previously shown that cotH is expressed during sporulation under the control of {sigma}K-directed RNA polymerase (Naclerio et al., 1996). To analyse cotH expression in more detail, a 405 bp DNA fragment containing the cotH promoter and 15 N-terminal codons of the cotH open reading frame was PCR amplified using oligonucleotide primers G and H (Table 2) and fused in-frame to the E. coli lacZ gene, yielding plasmid pGC24. The resulting cotH : : lacZ translational fusion was introduced into the chromosome by single reciprocal (Campbell-like) recombination between homologous DNA sequences in pGC24 and on the B. subtilis chromosome. Chromosomal DNA containing the integrated fusion-bearing plasmid was then used to transform a congenic null mutant in the structural gene for the transcriptional regulator GerE (Table 1). The time-course experiment in Fig. 1(a) shows that cotH-directed {beta}-galactosidase production began in wild-type cells 4 h after the onset of sporulation (t4), reached a maximum 1 h later (t5) and then slowly decreased. In cells that did not contain a wild-type copy of the gerE gene, cotH-directed {beta}-galactosidase production increased until t7. These results suggest that the transcriptional factor GerE negatively regulates cotH expression.



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Fig. 1. (a) cotH-directed {beta}-galactosidase synthesis in an otherwise-wild-type strain, GC237 ({square}), and in a gerE null background, GC238 ({blacksquare}). Samples were collected at various times after the onset of sporulation. (b) Schematic representation of the cotG-cotH region of the B. subtilis chromosome. Arrows indicate transcription orientation and initiation sites. Boxed triplets are the translational start sites of the two genes. The highlighted sequences are that of the GerE-box in this promoter region and its consensus.

 
GerE binds to the cotH promoter region
A putative GerE-box, matching 9 out of 10 positions of the consensus GerE-box (Zhang et al., 1994), has been previously identified in the cotH-cotG promoter region (Sacco et al., 1995; Naclerio et al., 1996) (Fig. 1b). To check whether this putative box is a binding site for the transcriptional regulator GerE, we performed an in vivo footprinting analysis of that part of the cotH promoter region. Sporulating cells of a wild-type (PY79, Table 1) and a congenic gerE null mutant (KS450, Table 1) of B. subtilis were treated with DMS 6 h after the onset of sporulation, as described in Methods and the methylated chromosomal DNA purified. Two end-labelled oligonucleotides, E1 and H8 (Table 2), were each used to prime a linear (non-exponential) PCR amplification of the two strands of the methylated chromosomes and the PCR products were separated by electrophoresis on denaturing sequencing gels. As shown in Fig. 2(a), bands corresponding to two adjacent G residues were less intense in the wild-type than in the gerE null mutant strain when the amplification reaction was primed with oligonucleotide E1. On the other DNA strand, a G residue was also protected when the amplification reaction was primed with oligonucleotide H8 (Fig. 2b). These results suggested that in the wild-type strain, those positions were protected by GerE and less efficiently methylated than the same residues in the gerE null mutant. Analysis of the DNA sequence reactions performed with the same oligonucleotides as used for the in vivo footprinting experiments allowed us to localize the GerE-protected residues within the putative GerE-box present in the cotH promoter region.



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Fig. 2. In vivo methylation of the GerE-box in the cotH promoter region, bottom (a) and top (b) strands. Cells of strains PY79 (wild-type, lanes 1) and KS450 (gerE, lanes 2) were treated with DMS to methylate the G residues 6 h after the onset of sporulation. Lanes G, A, T and C: nucleotide sequencing reactions for the strand being extended (Gs in the methylated strand correspond to Cs in the sequencing lane). Oligonucleotides utilized for primer extension were E1 (a) and H8 (b). Arrows indicate protected G residues. In vivo footprinting experiments on both DNA strands were performed in duplicate with identical results.

 
Mutations in the GerE-box alter cotH expression
cotH and cotG genes are divergently oriented and their promoter regions partially overlap (Fig. 1b). While cotG is positively regulated by GerE (Sacco et al., 1995), cotH is negatively controlled by the same transcriptional factor as shown above (Fig. 1a). The GerE-box found in the cotH-cotG promoter region could, therefore, be involved in the regulation of cotG and/or cotH. To verify whether the GerE binding site identified by the in vivo footprinting of Fig. 2 is involved in the control of cotH expression, we constructed two mutations in the GerE-box localized upstream of the cotH : : lacZ gene fusion carried by plasmid pGC24. The two mutations, indicated in Fig. 3(a), were introduced into plasmid pGC24 by PCR (see Methods) by using oligonucleotides Mut1 and Mut2 (Table 2) with mis-matched sequences. The recombinant plasmids pLB38 and pLB37, carrying respectively the GG to TT (mut1) and the T to G (mut2) replacements (Fig. 3a), were introduced into the chromosome by single reciprocal (Campbell-like) recombination between B. subtilis DNA sequences in the plasmids and the corresponding homologous region on the chromosome. To check whether the recombination had occurred upstream or downstream of the mutation, transformed cells were plated on a sporulation-inducing medium supplemented with X-Gal. For both pLB37 and pLB38, two colony phenotypes were observed with respect to cotH-directed {beta}-galactosidase production: one developing a pale blue colour, identical to that of wild-type cells, and one developing a stronger blue colour. Two clones showing the latter phenotype were used for plasmid rescue experiments upon digestion of their chromosomal DNA with BglII. Subsequent DNA sequencing allowed us to locate the mutations upstream of the lacZ coding region in all four cases analysed (data not shown). One clone for each transformation was selected, named NC38 (mut1) and NC37 (mut2), and used for further analysis. The time-course experiment in Fig. 3(b) shows that the cotH directed {beta}-galactosidase production of strains NC38 (mut1) was similar to that observed for strain KS450 (gerE36), while that of strain NC37 (mut2) was only slightly affected compared to the gerE+ strain. These results indicate that the GerE-box found in the cotH-cotG promoter region controls the expression of the cotH gene. Whether the same GerE-box also controls cotG expression was not investigated here since we decided to focus this study exclusively on cotH expression.

Alteration of cotH expression does not affect spore coat structure or function
One of the mutations we obtained in the GerE-box of cotH (mut1) relieved the GerE control on cotH expression in the presence of a wild-type gerE allele (Fig. 3). Therefore, we decided to use this mutation to study the effects of the deregulated expression of cotH on spore coat structure and/or function without the interference due to the deregulation of all other GerE-controlled genes, as would occur in a gerE null mutant. With this aim, we transferred the mut1 mutation from upstream of the cotH : : lacZ fusion to upstream of the cotH coding region: the mutation was transferred to plasmid pER112, which carries a truncated copy of cotH (cotH*) and an entire cotG, yielding plasmid pNC13 (Fig. 4; see Methods). The recombinant plasmid was then introduced into the chromosome of the B. subtilis strain ER203 (Table 1; Sacco et al., 1995) by single reciprocal (Campbell-like) recombination, yielding strain NC5. Since strain ER203 has a deletion of the cotG gene extending up to a BglII site (Sacco et al., 1995) and since in plasmid pNC13 the mut1 mutation is 14 bp from that BglII site (on the cotH side), the single cross-over event was more likely to occur in the cotH coding region, thus positioning the mut1 mutation upstream of an otherwise wild-type copy of cotH (Fig. 4). A plasmid rescue experiment upon digestion of the NC5 chromosomal DNA with EcoRI, and subsequent DNA sequencing analysis, allowed us to confirm the presence of the mut1 mutation upstream of the cotH coding region in strain NC5 (not shown).



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Fig. 4. Strategy to obtain strain NC5 by Campbell-like integration of the circular plasmid NC13 (this study) into the chromosome of strain ER203. The most important restriction sites are indicated as well as the position of the mut1 mutation.

 
The GerE-box mutation carried by strain NC5 (mut1) is likely to affect not only cotH but, in a negative way, also cotG expression, which strictly requires GerE (Sacco et al., 1995). To analyse the effects on spore coat structure and/or function exclusively due to the increased expression of cotH, we constructed a congenic B. subtilis mutant strain, NC6, carrying a wild-type copy of cotG and necessary upstream regions at the amyE locus (see Methods). Wild-type PY79 and its congenic mutants NC5 and NC6 showed similar sporulation and germination efficiencies (not shown). Purified spores of these two strains showed similar levels of resistance to chloroform and lysozyme (not shown) and, based on a transmission electron microscopy analysis, similar spore coat ultrastructure (not shown).

Alteration of cotH expression leads to CotC accumulation in the mother cell
Analysis of SDS-DTT- or NaOH-extracted spore coat proteins by SDS-PAGE did not reveal any major difference among the Cot protein profiles of PY79, NC5 and NC6 spores (data not shown). We used a Western blot approach to analyse whether the alteration in cotH expression affected CotC, an outer coat component assembling in the coat in multiple forms, ranging in size between 12 and 30 kDa, all strictly dependent on CotH (Naclerio et al., 1996; Isticato et al., 2004). Sporulating cells of strains PY79, NC5 and NC6 were harvested at various times during sporulation, lysed by sonication as described in Methods, and the forming spores surviving the sonication treatment were separated by centrifugation. The forming spores were then extracted by alkali treatment and the proteins released compared with those present in the mother cell cytoplasm. For each time point both protein fractions were analysed by Western blot with CotC-specific antibodies (Isticato et al., 2004). No differences were found between NC5 and wild-type in the coat protein profile in all forespore fractions analysed (Fig. 5a, b). Analysing the mother cell fractions, we observed that only NC5 spores revealed the presence of two CotC polypeptides, of 12 and 21 kDa (Fig. 5a, b). These CotC forms were both present 10 h after the onset of sporulaton while only the 12 kDa form was also found 2 h earlier (Fig. 5b). It was previously reported that CotG has no effect on CotC assembly (Isticato et al., 2004); we observed that with respect to CotC assembly, NC5 and NC6 spores were indistinguishable (data not shown).



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Fig. 5. Western blot of proteins extracted 6 h (t6), 8 h (t8) and 10 h (t10) after the onset of sporulation from the mother cell and the forespore of sporulating cells of strain PY79 (wild-type; a, c) and NC5 (mut1; b, d). 50 µg (mother cell extract) or 15 µg (forespore extract) of total proteins were fractionated on 18 % (a, b) or on 12·5 % (c, d) polyacrylamide gels. After electrotransfer onto nitrocellulose membranes, filters were reacted with CotC-specific rabbit antibodies (a and b) or with CotA-specific rabbit antibodies (c and d), then with peroxidase-conjugated secondary antibodies, and visualized by the Pierce method. Proteins extracted from mature spores of a wild-type strain are shown. The estimated sizes of CotC- and CotA-dependent polypeptides, as previously determined by Isticato et al. (2004), are also reported.

 
Mother cell and forespore samples of wild-type and NC5 strains were also tested with anti-CotA antibody. As shown in Fig. 5(c, d), unregulated expression of cotH does not affect CotA, either in the mother cell or in the forespore compartment, suggesting that the effect of CotH on CotC is most probably specific.

In addition to the two CotC forms, also a cotU-dependent polypeptide of 23 kDa (Isticato et al., 2004) was observed in the mother cell fractions of NC5 (Fig. 5). As previously reported cotU encodes a CotC homologue, recognized by anti-CotC antibody (Isticato et al., 2004).

A quantitative determination of the amount of CotC present on NC5 or NC6 spores was obtained by dot blot experiments using serial dilutions of purified CotC (Isticato et al., 2004) and of coat proteins extracted from spores of the wild-type and mutant strains. Proteins were reacted with anti-CotC antibody and then with alkaline-phosphatase-conjugated secondary antibodies, and the colour developed by the BCIP/NBT system (Bio-Rad). A densitometric analysis indicated that equal amounts of CotC were extracted from spores of strains PY79 and NC5 (data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CotH is a 42 kDa proteinaceous component of the B. subtilis spore coat that controls the assembly of several coat polypeptides (Naclerio et al., 1996; Zilhao et al., 1999). Assembly of the structural coat component CotC on the forming coat has been recently shown to involve a dual role of CotH, i.e. the stabilization and protection of CotC monomers and homodimers (Isticato et al., 2004). CotH is also required for the stability of CotG, which is needed for CotB assembly in a regulatory cascade (Zilhao et al., 2004).

In the present work we report evidence that the expression of the cotH gene of B. subtilis is negatively regulated by the transcriptional regulator GerE. A GerE binding site, previously identified by sequence homology in the cotH promoter region (Sacco et al., 1995; Naclerio et al., 1996), was protected by in vivo methylation in the presence of a wild-type allele of gerE (Fig. 2), thus suggesting that GerE binds to the cotH promoter region and controls its expression. Moreover, PCR-derived mutations in the GerE-box of cotH located upstream of the lacZ reporter gene, which reduced its homology to the GerE-box consensus sequence (Zhang et al., 1994), caused an increased and prolonged production of cotH-directed {beta}-galactosidase, similar to that observed in a gerE null mutant (Fig. 3). Our results do not allow us to exclude the possibility that a GerE-dependent factor and not GerE itself binds to the cotH promoter region. However, we consider this possibility unlikely since the protected GerE-box identified in this study (Fig. 2) is homologous to GerE-boxes identified upstream of other cot genes and shown to be directly regulated by GerE (Zhang et al., 1994; Ichikawa & Kroos, 2000).

To analyse the effects of the GerE-independent expression of cotH in the presence of a gerE wild-type allele, on the structure and/or function of the spore coat, the mut1 mutation was moved upstream the cotH coding region. EM analysis, as well as the analysis of resistance and germination properties of wild-type and mutant spores, showed that a GerE-independent expression of cotH does not significantly affect spore structure or function. The only observed effect due to the cotH deregulation was the accumulation of CotC polypeptides in the mother cell, possibly due to protection of CotC molecules not assembled in the coat. This effect is most likely specific since at least one other coat component, CotA, is not affected by the unregulated expression of cotH. This hypothesis is based on the following observations: (i) CotH does not affect the expression of the CotC structural gene (Naclerio et al., 1996; Ichikawa & Kroos, 2000); (ii) CotH stabilizes and allows assembly of CotC multiple forms on the forming spore coat (Isticato et al., 2004); and (iii) CotC accumulation in the mother cell does not prevent the assembly of normal amounts of CotC molecules on the forming spore since equal amounts of CotC were found in wild-type and NC5 spore coats. Therefore, while unassembled CotC molecules would be rapidly eliminated in wild-type conditions, they are protected and accumulate at their site of synthesis because of the non-physiological conditions generated by the presence of CotH during late sporulation.

Consequences of this hypothesis would be that: (i) CotC is normally produced in the mother cell compartment of the sporulating cell in excess of the amount that can be assembled; and (ii) the amount of CotC molecules that can be assembled on the forming spore is somehow regulated.


   ACKNOWLEDGEMENTS
 
We thank L. Kroos and M. Ciaramella for critical reading of the manuscript, L. Di Iorio for technical assistance and A. Henriques for the generous gift of anti-CotA antibody. This work was supported by the European Union grant No. QLK5-CT-2001-01729 to E. R., by MIUR (Cofin 2002; FIRB 2002) grants to E. R. and by MIUR (Cofin 2002) grant to M. D. F.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 24 May 2004; revised 12 July 2004; accepted 15 July 2004.



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