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Biophysics Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700 032, India
Correspondence
Rukhsana Chowdhury
rukhsana{at}iicb.res.in
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Methylation occurs at the C-5 and N-4 positions of cytosine and at the N-6 position of adenine, and is catalysed by enzymes called DNA methyltransferases (MTases). DNA MTases can broadly be divided into those that methylate the exocyclic amino group of adenines and cytosines (N-MTases) and those that methylate the endocyclic C-5 of cytosine (m5C-MTases). In general, both types of MTases are two-domain proteins consisting of one large and one small domain with a DNA-binding cleft located in the domain interface. They contain amino acid motifs that differ between N- and m5C-MTases (Wilson, 1992). All m5C-MTases, irrespective of their phylogenetic origin, contain 10 conserved amino acid motifs separated by regions variable in size and amino acid sequence in different enzymes (Kumar et al., 1994). The N-terminal region of the enzyme includes conserved motifs I–VIII containing the binding site for the universal methyl group donor S-adenosyl methionine (AdoMet) and the catalytic centre of the enzyme. Located beyond motif VIII is a variable region containing the target recognition domains, followed by the C-terminal motifs IX and X.
In prokaryotes, a majority of DNA MTases are components of restriction-modification (R-M) systems, in conjunction with cognate restriction endonucleases. The sequence context of methylation serves to distinguish between self and non-self DNA and protects bacteria from ‘foreign’ invaders like bacteriophages, transposons and other genomic parasites. However, not all MTases are components of R-M systems. Certain adenine-MTases (Dam and CcrM) control gene expression and regulate cellular processes like post-replicative mismatch repair, DNA replication timing and cell cycle regulation (Messer & Noyer-Weidner, 1988; Reisenauer et al., 1999). In some bacteria, cytosine-MTases (Dcm) are associated with very short patch repair (Vsr) endonucleases (Sohail et al., 1990). Spontaneous hydrolytic deamination of m5C to form thymine can lead to the production of T : G mismatches. The mutagenic potential of T : G mismatches is counteracted by the Vsr endonuclease which recognizes and removes T from T : G mismatches in DNA.
Analysis of more than 300 bacterial and archaeal genomes in the REBASE database (Roberts et al., 2005; http://rebase.neb.com/rebase) has revealed that about 90 % of the genomes have at least one R-M system and about 80 % contain multiple R-M systems, most of which have not been biochemically characterized. In silico genome analysis suggests that in many of the R-M systems, the restriction endonuclease component is inactive but the cognate MTase retains full activity. However, since very few such ‘orphan’ methylases have been characterized, their function is not known and it is not clear why they are maintained in the genome. In this context, the single orphan MTase in V. cholerae has been characterized in this study.
V. cholerae is a Gram-negative enteric pathogen that causes the diarrhoeal disease cholera (Kaper et al., 1995). DNA adenine methylation has been reported in V. cholerae, and a role of the V. cholerae DNA adenine methyltransferase (Dam) in virulence (Julio et al., 2001) and chromosome replication (Egan & Waldor, 2003) has been demonstrated. Although V. cholerae possesses an efficient Dam-directed mismatch repair pathway (Bera et al., 1989), it lacks homologues of Escherichia coli Dcm and Vsr endonuclease (Bhakat et al., 1999), known to be associated with the very short patch (VSP) repair system. An examination of the V. cholerae strain N16961 genome sequence database (www.tigr.org) revealed the presence of a single ORF annotated as a putative cytosine MTase. No gene encoding a restriction endonuclease or a Vsr-like endonuclease was detected in the vicinity of the cytosine MTase. In this report we present data demonstrating that V. cholerae strain O395 also contains an ‘orphan’ or solitary cytosine MTase that methylates the first C in the sequence 5'-RCCGGY-3' and contributes to the generation of spontaneous mutants in a non-dividing cell population.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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vchM) and other V. cholerae strains listed in Table 1 were used in the present study. The strains listed in Table 1 were obtained from the National Institute of Cholera and Enteric Diseases, Calcutta, India. All strains were grown in LB medium containing 1 % tryptone (Difco), 0·5 % yeast extract (Difco), 0·5 % NaCl and stored at –70 °C in 20 % (v/v) glycerol. The suicide vector pCVD442, used for construction of the deletion mutant, was maintained in E. coli strain SM10 
pir (Donnenberg & Kaper, 1991).
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). 5-Methyl deoxycytidine and deoxycytidine (Sigma) were used as standard markers.
Immunoblotting.
Chromosomal or plasmid DNA (5 µg) was denatured by boiling for 5 min and cooled rapidly on ice. The DNA was spotted onto nitrocellulose paper (Millipore) pre-soaked in 10x SSC, dried at room temperature and baked for 2 h at 80 °C. The blot was blocked with 5 % BSA and incubated overnight with mouse polyclonal antibody to m5C (1 : 10 000; Calbiochem), washed in Tris-buffered saline (TBS) and detected using HRP-conjugated rabbit anti-mouse antibody (1 : 5000; Jackson Laboratory) and enhanced chemiluminescence.
Expression and purification of His6-M.Vch.
The complete ORF of the V. cholerae vchM gene was PCR-amplified and inserted in-frame at the BamHI/KpnI sites of the expression vector pQE30 (Qiagen) to create an N-terminal His6-tagged protein. The resulting plasmid, designated pQEvchM, was introduced into E. coli M15 and the His-tagged fusion protein was isolated following the manufacturer's instructions.
In vitro methylation assay.
Plasmid pBR322 DNA isolated from E. coli strain GM31 (dcm–; Sohail et al., 1990) was linearized with EcoRI and used as substrate for an in vitro methylation reaction (Marks et al., 2003) with V. cholerae M.Vch. Briefly, 250 ng linearized pBR322 DNA was treated with M.Vch in the presence of 200 µM AdoMet for 2 h. Plasmid DNA was purified from the reaction mixture, cleaved with BsrF1 (5 U, 3 h) and electrophoresed on an 8 % polyacrylamide gel. Ethidium bromide stained gels were visualized using a Gel Doc 1000 system (Bio-Rad).
Construction of V. cholerae vchM mutant.
The V. cholerae vchM gene was cloned as a 1523 bp fragment in vector pBluescript KS to give plasmid pSB319. An internal 935 bp region was deleted from the cloned gene by digestion with NdeI and NsiI followed by religation. The resulting plasmid was digested with SalI and SacI, and a 651 bp fragment was obtained that contained –105 bp to +177 bp of the vchM ORF fused to a 306 bp region downstream of the ORF. This fragment was cloned into the positive selection suicide vector pCVD442 (Apr, sacB) and transformed into E. coli SM10 pir. The plasmid was conjugally transferred into V. cholerae O395 (streptomycin-resistant, Smr) and ampicillin-resistant (Apr) Smr colonies arising due to a single recombination event were selected and spread on plates containing 10 % sucrose. Smr Aps and sucrose-resistant colonies were selected and Southern hybridization and nucleotide sequencing was used to confirm that deletion had occurred in the vchM gene.
RNA isolation and RT-PCR.
For isolation of RNA, V. cholerae strains O395 or CM319 were grown to exponential phase (3 h) or overnight in LB at 37 °C. Total RNA was extracted and purified using guanidinium isothiocyanate (Ausubel et al., 1989). The RNA was treated with RNase-free DNase 1 (1 U µg–1, amplification grade; Invitrogen) in the presence of an RNase inhibitor (RNasin; Gibco-BRL) and RT-PCR was performed using a single tube RT-PCR kit (Gibco-BRL). DNase-treated RNA (200 ng) was used in all reactions. Samples were removed after 20, 25, 30 and 35 cycles (94 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s; followed by 7 min extension at 72 °C), electrophoresed on 2 % agarose gels with ethidium bromide and analysed using a Gel Doc 1000 system (Bio-Rad). The data obtained in the linear range of PCR amplification were considered for analysis. Genomic DNA served as a positive control and RNA that had been treated with DNase, but not reverse transcribed, was used as negative control. Each set of experiments was performed at least thrice.
Determination of spontaneous mutation frequency.
Overnight cultures of V. cholerae strains O395 and CM319 were diluted 1 : 106 in fresh LB medium (approx. 500 c.f.u. ml–1) and grown at 37 °C. Samples were removed in the exponential phase of growth, the number of c.f.u. ml–1 was assayed and 6 ml culture was concentrated by centrifugation and plated on LB agar plates containing 10 µg rifampicin (Rif) ml–1 or 3 µg novobiocin (Nov) ml–1. Similarly, at 24 h, the number of c.f.u. ml–1 was determined and 1 ml culture was plated on antibiotic plates. Spontaneous mutation frequency was determined as the ratio of the number of colonies obtained on antibiotic plates to the total number of cells plated.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). However, plasmid pBR322 isolated from V. cholerae could be transformed into E. coli strains carrying functional McrAB at a very low frequency (less than 10–9), but could be transformed into an E. coli mcrA–B– strain, XL1Blue MRF', at frequencies greater than 10–7. Since the E. coli McrAB system restricts DNA containing m5C (Raleigh & Wilson, 1986), this result indicated that pBR322 isolated from V. cholerae contains m5C. Estimation of m5C in V. cholerae genomic DNA by HPLC analysis of nucleotides released by extensive digestion of genomic DNA with nucleases indicated the presence of m5C (Fig. 1a). The presence of m5C in V. cholerae chromosomal DNA and plasmids isolated from V. cholerae was also demonstrated by immunoblotting using anti-m5C antibodies (Fig. 1b). The results obtained indicated that, although V. cholerae O395 genomic DNA and plasmid pBR322 DNA isolated from strain O395 contained m5C, the proportion is much lower than in calf thymus DNA and only slightly lower than in E. coli K-12 DNA used as controls in these experiments.
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Most bacterial cytosine MTases reported are associated with cognate restriction endonucleases and some Dcm-like cytosine MTases are associated with VSP repair nicking endonucleases like Vsr. An unidentified ORF is present adjacent to the putative cytosine MTase gene of strain N16961 (TIGR locus VCA0199; www.tigr.org) and also the vchM cytosine MTase gene of strain O395 (see below), but it bears no homology with vsr. Indeed, dcm-vsr homologues have been reported to be absent in V. cholerae (Bhakat et al., 1999). Also, the unidentified ORF is unlikely to encode a restriction enzyme since an in-frame deletion mutation in the vchM cytosine methylase gene could be constructed (described below). Thus, the V. cholerae M.Vch is an ‘orphan’ or solitary cytosine MTase. However, a phylogenetic tree for homologues of V. cholerae M.Vch (Fig. 2) indicates that many homologues of M.Vch are components of R-M systems.
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Cloning of V. cholerae vchM gene and construction of mutant
The vchM gene from V. cholerae strain O395 was PCR-amplified as a 1523 bp fragment and cloned into plasmid pBluescript KS to give plasmid pSB319. The plasmid could be transformed into E. coli strain XL1Blue MRF' (mcrAB–) but no transformants were obtained using E. coli strains XL1Blue or DH5
. Nucleotide sequencing of the cloned fragment revealed that it contained a 1152 bp complete ORF identical to the cytosine-MTase gene of N16961 (locus VCA0198) and the 5' segment of an ORF identical to a hypothetical gene of strain N16961 (locus VCA0199) located immediately downstream. Thus, both the gene sequence and genomic organization of the vchM gene of strain O395 are identical to the cytosine-MTase encoding gene of strain N16961 (VCA0198) in the V. cholerae genome sequence database (www.tigr.org).
An in-frame deletion in the vchM gene was constructed in the positive selection vector pCVD442 as described in Methods. The resulting plasmid was used for allelic exchange in strain O395 to generate strain CM319 (vchM). The mutation was confirmed by Southern hybridization, nucleotide sequencing and by PCR using primers flanking the vchM gene (data not shown). RT-PCR analysis also indicated that vchM-specific transcripts were not present in the mutant strain CM319 (Fig. 3a, lanes 2 and 4). Furthermore, m5C could not be detected in genomic DNA from the mutant strain CM319 by HPLC (Fig. 1a
), and immunoblotting with m5C-specific antisera did not give a signal with chromosomal and plasmid DNA isolated from strain CM319, although a specific signal was obtained with DNA from the wild-type parent strain (Fig. 1b). These results indicated that M.Vch is the only m5C-MTase in V. cholerae strain O395.
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, lanes 1 and 3). Expression of the housekeeping gene dapA decreased in the stationary phase (Fig. 3b, lanes 1 and 2).
Identification of the M.Vch methylation site
The recognition sequence of M.Vch was identified by analysis of cytosine methylation sensitive restriction endonuclease digestion patterns of plasmid and chromosomal DNA from the V. cholerae strains O395 and CM319 (vchM). EcoRII, which recognizes unmethylated 5'-CCWGG-3' sites, the target for Dcm methylation, completely cleaves chromosomal DNA from both strains O395 and CM319 (Fig. 4e), indicating that 5'-CCWGG-3' sites are not methylated in V. cholerae and suggesting that the M.Vch MTase recognizes a different target. The restriction endonucleases MspI and HpaII both recognize the sequence 5'-CCGG-3'. While HpaII will not cleave if either C residue in the recognition sequence is methylated, MspI cleavage is blocked by methylation of the 5' C (Roberts et al., 2005). All HpaII/MspI recognition sites in plasmid pBR322 were cleaved by both HpaII and MspI in pBR322 isolated from E. coli DH5, yielding fragments of expected sizes (Figs 4a and b, lanes 1). However, when pBR322 isolated from strain O395 was digested with the enzymes HpaII or MspI, nine fragments of 622, 404, 201, 160, 123, 90, 76, 34 and 26 bp could not be detected; instead additional fragments of sizes 631, 438, 326, 227 and 147 bp were obtained (Figs 4a and b, lanes 2). Analysis of the molecular masses of the additional fragments obtained indicated that they represent fused fragments. The presence of the fused fragments suggested that certain 5'-CCGG-3' sites in pBR322 from strain O395 were resistant to cleavage by HpaII and MspI. Further analysis of these refractory sites reveal that they fall within the sequence 5'-RCCGGY-3'. Failure of both HpaII and MspI to cleave at 5'-RCCGGY-3' sites suggested that the first 5' C within the sequence 5'-RCCGGY-3' is methylated by M.Vch, since methylation of only this cytosine blocks both MspI and HpaII cleavage (Kwiatek et al., 2004; Roberts et al., 2005). When plasmid pBR322 isolated from strain CM319 (vchM) was digested with HpaII or MspI, all potential sites were completely cleaved, yielding a pattern similar to that obtained in HpaII and MspI digests of pBR322 DNA from E. coli (Figs 4a and b, lanes 1 and 3), indicating absence of m5C in the recognition sites.
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vchM) was completely cleaved by BsrF1 (Fig. 4d), indicating that BsrF1 sites were methylated by M.Vch in the wild-type V. cholerae DNA. To further examine the substrate specificity of M.Vch, plasmid pBR322, which contains seven BsrF1 recognition sites, was isolated from V. cholerae O395 and the vchM mutant strain CM319, and digested with BsrF1. Plasmid pBR322 isolated from strain O395 was not cleaved by BsrF1, indicating the presence of methylation in the recognition sequence (Fig. 4c, lane 2). However, pBR322 isolated from strain CM319 (vchM) was completely digested by BsrF1 (Fig. 4c, lane 3), yielding a pattern similar to that obtained in the BsrF1 digest of pBR322 isolated from E. coli DH5 (Fig. 4c, lane 1). Taken together, the BsrF1, HpaII and MspI digestion patterns suggest that V. cholerae M.Vch recognizes the sequence 5'-RCCGGY-3' and methylates the first 5' C residue within the sequence.
In vitro methylation by M.Vch
To examine if M.Vch directly methylates the 5'-RCCGGY-3' sites in plasmid pBR322, an in vitro experiment was carried out by incubating pBR322 with M.Vch followed by digestion with the restriction endonuclease BsrF1. The results obtained indicate that, in the presence of AdoMet, M.Vch can methylate the 5'-RCCGGY-3' sites in pBR322, rendering them refractory to cleavage by BsrF1 (Fig. 5 lane 1). pBR322 incubated without M.Vch was cleaved by BsrF1 (Fig. 5, lane 2).
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). The gene was present in all six strains belonging to V. cholerae serogroup O139 examined and in six of the seven non-O1 non-O139 strains examined (Table 1). Southern blot analysis was used to confirm the absence of vchM in strains E82 and V94 (data not shown). In some non-O1 strains, two or more bands were obtained (Table 1), suggesting that they may contain other cytosine MTases in addition to vchM. To examine if the vchM gene is expressed in all strains containing the vchM gene, RT-PCR analysis was performed with randomly selected strains of both O1 and non-O1 serogroups. vchM expression was detected in all strains carrying the vchM gene, but not in strains in which the gene is absent (Table 1, Fig. 6).
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T mutations. In the exponential phase of growth, T : G mismatches are repaired, together with a variety of other mismatches, by the general sequence non-specific methyl-directed mismatch repair (MMR) pathway, while in the stationary phase T : G mismatches are specifically repaired by the VSP repair system (Bhagwat & Lieb, 2002). The spontaneous mutation frequencies of V. cholerae strains O395 and CM319 to rifampicin resistance and novobiocin resistance were measured in the exponential and stationary phases of growth (Table 2). Nucleotide sequencing has revealed that the sequences of the genes rpoB and gyrB, mutations in which confer resistance to rifampicin and novobiocin respectively, in strain O395 are identical to those of strain N16961, the genome of which has been completely sequenced (www.tigr.org), and contain two and four M.Vch methylation sites, respectively. In the exponential phase, the V. cholerae wild-type and vchM mutant strains had comparable spontaneous mutation frequencies (Table 2), indicating that T : G mismatches arising from deamination of m5C in the parent strain were efficiently repaired. This result was consistent with reports that an efficient MMR repair system is present in V. cholerae (Bera et al., 1989). In the stationary phase of growth, the spontaneous mutation frequency of the parent strain was significantly higher than that of the corresponding vchM mutant strain CM319 (Table 2). One explanation for this difference may be that, since the Vsr-like endonuclease, which corrects T : G mismatches in non-dividing cells, might be absent in V. cholerae (Bhakat et al.), the T : G mismatches arising from deamination of m5C in the parent strain are not efficiently repaired, giving rise to CT mutations within the M.Vch methylation sites (5'-RCCGGY-3') in the rpoB and gyrB genes. Since the T : G mismatches do not arise in the vchM mutant strain, the spontaneous mutation frequency of the mutant is relatively lower. To test this hypothesis, fragments of the rpoB and gyrB genes (carrying the 5'-RCCGGY-3' sites), from RifR and NovR mutants of strain O395 and CM319, were PCR-amplified and sequenced. One of the three RifR mutants of strain O395 examined carried a CT mutation at the first C residue in the first 5'-RCCGGY-3' site within the rpoB gene, while two of five NovR mutants of strain O395 examined contained a CT change at the first C residue of the first and fourth 5'-RCCGGY-3' sites within the gyrB gene. An equal number of RifR and NovR mutants of strain CM319 were examined, but none carried a CT change within the 5'-RCCGGY-3' sites of rpoB or gyrB. Thus, the presence of M.Vch and the absence of a VSP repair-like system impose upon V. cholerae a mutator phenotype. It may be mentioned in this context that the V. cholerae genome (4·03 Mb) contains only 2172 M.Vch methylation sites (5'-RCCGGY-3') while 5906 such sites are present in the E. coli genome (4·64 Mb).
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). The results obtained indicate that in the strains N16961 and C6709 the spontaneous mutation frequency in stationary-phase cells was higher than in cells grown to the exponential phase (Table 2). However, the spontaneous mutation frequency of strains E82 and V94 was similar in the exponential and stationary phases of growth.
What could be the biological role of the M.Vch MTase? It is known that mutations occur in non-dividing microbial populations and this phenomenon provides a source of genetic variants from which individuals with a variety of advantages may be selected in evolution. It is attractive to hypothesize that in V. cholerae the presence of M.Vch and absence of Vsr may contribute to the generation of such mutants in non-growing conditions, under which V. cholerae may be presumed to spend most of the time in its natural aquatic habitats. Furthermore, there is evidence that R-M systems may behave selfishly in that their loss from a cell may lead to cell killing through restriction of the genome. It has been suggested that, in bacteria containing orphan MTases, prior methylation of a specific sequence in the genome may afford protection against parasitism by R-M systems recognizing the same sequences (Takahashi et al., 2002). Whether the M.Vch MTase, which methylates the sequence 5'-RCCGGY-3', has a similar role in conferring protection against parasitism by R-M complexes needs to investigated.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 24 October 2005;
revised 26 December 2005;
accepted 10 January 2006.
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