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
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ABSTRACT |
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Present address: The School of Pharmacy, University of London, London, UK.
These two authors contributed equally to this work.
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INTRODUCTION |
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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 E>SpoIIID>
K>GerE, with
E and
K being RNA polymerase
factors and SpoIIID and GerE being DNA-binding proteins acting in conjunction with RNA polymerase associated with
E (E
E) and
K (E
K) (Zheng & Losick, 1990
; Henriques & Moran, 2000
; Driks, 2002
). GerE, in conjunction with
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 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
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
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.
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METHODS |
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Construction of cotH : : lacZ translational fusion and -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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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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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 (0·20·6 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 ml1. 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 2448 h.
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RESULTS |
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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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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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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).
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DISCUSSION |
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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
-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.
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ACKNOWLEDGEMENTS |
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Received 24 May 2004;
revised 12 July 2004;
accepted 15 July 2004.
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