Institute of Microbiology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland
Correspondence
Andrzej Piekarowicz
anpiek{at}biol.uw.edu.pl
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ABSTRACT |
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INTRODUCTION |
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The presence of m5C-MTases is the source of the T-G mismatch through spontaneous deamination of 5-methylcytosine residues (Bhagwat & Lieb, 2002). These T-G mismatches are repaired by a very short patch (VSP) repair system. The presence of an active VSP system was described in Escherichia coli K-12 (Lieb & Bhagwat, 1996
) and in Bacillus stearothermophilus (Laging et al., 2003
). Nucleotide sequence homologous to genes ecoKDcmV and ecoKDcmM of E. coli K-12 has been reported in several enteric pathogens, e.g. Shigella sonnei, Salmonella typhimurium, Salmonella enteritidis, Enterobacter cloacae (Lieb & Bhagwat, 1996
) and Haemophilus parainfluenzae (http://rebase.neb.com/rebase). In two studied cases, the specificity of the mismatch nicking endonuclease is the same as found for the associated DNA m5C-MTase (Hennecke et al., 1991
; Laging et al., 2003
).
The genus Neisseria belongs to a group of bacteria encoding as many as 10 or more different MTases, including at least several m5C-MTases (Roberts & Macelis, 2002). Claus et al. (2000)
, using representational difference analysis (RDA), isolated from Neisseria meningitidis the DNA fragments encoding three m5C-MTases. These DNA fragments were isolated from strain MC58 (MTase M.NmeBI), strain Z2491 (MTase M.NmeAI) and strain 2120 (MTase M.NmeDI). The MTase M.NmeBI was cloned and expressed by these authors. The sequence of the DNA fragments isolated from strains Z2491 and 2120 was used for comparison with other sequences, which allowed identification of these ORFs as m5C-MTases. However, neither their biological activity nor their recognition specificity was determined, although the authors showed that the M.NmeAI does not recognize the sequence 5'-GTCAGC-3' recognized by M.HgiDII despite the strong homology between these two MTases.
In the vicinity of the genes encoding these two m5C-MTases, we have found the presence of ORFs encoding putative Vsr-like enzymes (A. Piekarowicz, unpublished data). Since the specificity of these Vsr-like enzymes should be the same as the associated DNA MTases we have cloned the genes encoding the M.NmeDI and M.NmeAI MTases into expression vectors and purified both enzymes. In this paper we present data indicating that these genes encode biologically active enzymes that recognize the specific DNA sequences 5'-RCCGGB-3' (M.NmeDI) and 5'-CCGG-3' (M.NmeAI).
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METHODS |
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Cloning of N. gonorrhoeae and N. meningitidis DNA fragments carrying the mismatch nicking endonuclease and accompanying R-M system genes.
DNA fragments carrying the mismatch nicking endonuclease and accompanying R-M system genes were amplified by PCR. The fragment of the chromosomal DNA of N. meningitidis strain 2120 that encodes the M.NmeDI MTase was amplified using primer 3 (5'-GCAGGGATCCTCGTTAAAATACAACC-3') and primer 4 (5'-GCAGAAGCTTTTAGCAGCCGTCAG-3'). This amplicon of 1257 bp was cloned into the R.BamHI and R.HindIII sites of pQE-30, resulting in the formation of plasmid pAK3. This plasmid contains the sequence encoding the M.NmeDI MTase fused into the His-Tag sequence encoded by pQE-30 DNA. The 2784 bp fragment of the chromosomal DNA of N. meningitidis strain 2120 encoding ORFs of the NmeDI R-M system together with the endonuclease V.NmeDI was amplified using primer 5 (5'-GACTCTAGACTTCCTGCGCGATTG-3') and primer 6 (5'-CGCGAAGCTTGGTAACCGAGTGTA-3') and cloned into the R.XbaI and R.HindIII sites of pUC19 DNA, creating plasmid pAK4. The fragment of the chromosomal DNA of N. meningitidis strain Z2491 encoding the M.NmeAI MTase was amplified using primer 1 (5'-CGGCCCTCGAGATGAAAAACAGTAAG-3') and primer 2 (5'-GCGCGGATCCCTAACATTCGATATTATC-3'). This amplicon of 1056 bp was cloned into the R.XhoI and R.BamHI sites of pET15b, resulting in the formation of plasmid pAK1. This plasmid contains the sequence encoding the M.NmeAI MTase fused to the His-Tag sequence encoded by pET15b. All the oligonucleotides used to amplify the above fragments are located in the same regions of the chromosomes of N. meningitidis strains used in studies by Claus et al. (2000). The oligonucleotides used to amplify the fragment of the chromosomal DNA of N. gonorrhoeae strain FA1090 encoding M.NgoAORFC713P (nomenclature according to REBASE) together with the ORFs encoding the putative Vsr-like enzyme (V.NgoAORFC713P) and NgoAORFC713P were primer 7 (5'-ATAGCATGGATCCTCAGACGGCATCTTTTATTTCCTC-3') and primer 8 (5'-CACGCCTGCAGATAGAAATGAAAAACAGTAAGTTAAAG-3'). The amplicon of 3148 bp was cloned into the R.BamHI and R.PstI sites of pUC19 DNA, creating plasmid pAK5.
Oligonucleotides used to amplify the fragment of the chromosomal DNA of N. gonorrhoeae strain FA1090 encoding M.NgoAORFC703P, together with the ORFs encoding the putative Vsr-like enzyme (V.NgoAORFC703P) and restriction endonuclease (NgoAORFC703P), were primer 9 (5'-CGTCTCTAGACTTGAAGCAGTTTG-3') and primer 10 (5'-GCGTGTCGACGATATTTACTCAAC-3'). The amplicon of 1767 bp was cloned into the R.XbaI and R.SalI restriction sites of pLMPMT4 DNA (Mayer, 1995
) creating plasmid pAK6.
The primers for PCR amplification were obtained from IBB Poland. All the PCR reactions were carried out using Pfu DNA polymerase (MBI Fermentas) and used according to the manufacturer's recommendations. All routine cloning procedures were carried out in accordance with protocols described by Sambrook et al. (1989). The expression vectors (pQE-30 or pET15b) carrying the cloned genes were always isolated from cultures induced by IPTG. The published sequences of pQE-30 (www.qiagen.com), pET15b (www.novagen.com), pUC19 (www.neb.com) and the cloned fragments of the chromosomal DNA of N. meningitidis and N. gonorrhoeae used for construction of the plasmids pAK1, pAK3, pAK4, pAK5 and pAK6 served to determine their genetic and restriction maps using the computer program CLONE (Scientific & Educational Software, Durham, NC 27722-2045, USA).
Purification of M.NmeAI and M.NmeDI m5C-MTases.
To purify the M.NmeAI or M.NmeDI MTases single colonies of fresh transformants of E. coli ER2566(pAK1) or of E. coli XL-1 Blue MRF'(pAK3) were used to inoculate 100 ml LB broth (Sambrook et al., 1989) and incubated at 37 °C. When the OD600 of the culture reached 0·6, IPTG (MBI Fermentas) was added to a final concentration of 1 mM and incubation was continued for an additional 4 h at 30 °C. The culture was then used to isolate the plasmid DNA and for purification of the MTase. The culture was centrifuged and bacteria suspended in 1 ml buffer containing 50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole. After sonication the cellular debris was removed by centrifugation at 40 000 g for 1 h and then the supernatant was applied to a 5 ml Ni-NTA agarose column previously equilibrated with 100 ml of the above buffer. Proteins were eluted according to the manufacturer (Qiagen). The M.NmeAI MTase was eluted at 0·20·25 M imidazole and the M.NmeDI MTase at 0·250·3 M. The homogeneity of the enzymes was determined by 10 % SDS-PAGE. Proteins used as standards were
-galactosidase (116 kDa), bovine serum albumin (66·2 kDa), ovalbumin (45 kDa) and lactate dehydrogenase (35 kDa).
MTase assays.
During the purification, MTase activity was detected by measurement of transfer of -CH3 groups from [methyl-3H]AdoMet to DNA. One microlitre of the prepared enzyme was used in a total volume of 20 µl to methylate 1 µg
DNA in the presence of 50 mM Tris/HCl (pH 7·5), 10 mM EDTA, 10 mM 2-mercaptoethanol and 7·4x104 Bq [methyl-3H]AdoMet. After 2 h incubation at 37 °C, reaction mixtures were spotted onto DE81 paper (Whatman), washed as described by Sambrook et al. (1989)
, then dried and their radioactivity measured in a Wallac 1400 liquid scintillation counter. To methylate oligonucleotides (ODNs), the reaction mixtures contained a total volume of 20 µl: 1 µg ODN substrate, 50 mM Tris/HCl (pH 7·5), 10 mM EDTA, 10 mM 2-mercaptoethanol, 7·4x104 Bq [methyl-3H]AdoMet and 1 µg of the purified enzyme. The extent of methylation was assayed as described by Renbaum & Razin (1995)
.
Determination of the methylation of the separate DNA strands.
Samples (5 µg) of the synthetic double-stranded ODN were methylated as described for MTase assays and separated on a denaturing polyacrylamide gel (Landry et al., 1992). The bands were excised, and the gel slices were crushed in microfuge tubes and incubated overnight at room temperature in 300 µl 0·5 M ammonium acetate, 1 mM EDTA. The slices were microfuged and the supernatant collected. The ammonium acetate supernatants were mixed with water washes and the 3H radioactivity was measured using 10 ml scintillation cocktail (Rotiszint).
Enzymes and chemicals.
Restriction enzymes were purchased from MBI Fermentas and New England Biolabs. T4 DNA ligase, Pfu DNA polymerase and DNA and protein size markers were purchased from MBI Fermentas. Kits for the DNA clean-up and plasmid DNA isolation were purchased from A&A Biotechnology, Gdansk, Poland. Ni-NTA agarose was purchased from Qiagen and [methyl-3H]AdoMet from PerkinElmer Life Sciences. All the chemicals used were reagent grade or better and they were obtained from Sigma and ICN, unless otherwise noted.
Computer analysis.
DNA and protein sequences were compared with the GenBank and SWISS-PROT databases on the BLAST server hosted by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast). The N. gonorrhoeae strain FA1090 genomic sequence was obtained from the University of Oklahoma's Advanced Center for Genome Technology (http://www.genome.ou.edu/gono.html) and the N. meningitidis strain Z2491 (serogroup A) genomic sequence from the Sanger Institute (http://www.sanger.ac.uk/Projects/N_meningitidis). We used the genomic sequence of the N. meningitidis strain FAM 18 (http://www.sanger.ac.uk/Projects/N_meningitidis), which like strain 2120 belongs to serogroup C (complex ST-11), and the DNA sequence (accession no. AJ238948) published by Claus et al. (2000) to characterize the organization of the R-M NmeDI region.
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RESULTS |
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The DNA sequence analysis of the chromosome of N. meningitidis strain Z2491 (serogroup A) showed the presence of the ORF NMA0427 encoding MTase (M.NmeAI) (position 398 752 bp to 399 807 bp) associated with the ORF NMA0429 encoding the potential Vsr-like protein (V.NmeAI) and the ORF NMA0428 (Fig. 1). This region (position 398 752 bp to 401 893 bp) is flanked by ORF NMA0426 (position 398 238 bp to 398 486 bp) on one side and by ORF NMA0430 (position 401 900 bp to 405 041 bp), encoding the putative DNA helicase, on the other. ORF NMA0427 encodes a 351 aa protein that shares similarity with various m5C-MTases. The M.NmeAI MTase shares homology with various m5C-MTases recognizing the sequence 5'-GTCGAC-3', such as M.HgiDII (38 % identity, 53 % similarity; accession no. CAA38941), M.LmoAP (38 % identity, 53 % similarity; accession no. CAC22275) and M.TerORFS122P (34 % identity, 48 % similarity; accession no. ZP00072165). It also shares a high homology with m5C-MTases recognizing different sequences, such as M.Kpn2I (recognition sequence 5'-TCCGGA-3'; 32 % identity, 48 % similarity; accession no. CAC41108), M.NspI (recognition sequence 5'-RCATGY-3'; 30 % identity, 45 % similarity; accession no. AAC97190) and M.HphI (recognition sequence 5'-TCACC-3'; 30 % identity, 45 % similarity; accession no. CAA59690).
Computer analysis suggests that ORF NMA0428 shares homology with MutL proteins: e.g. 50 % identity and 60 % similarity to mismatch repair protein MutL of Heliobacillus mobilis (accession no. AAN87382), 42 % identity and 64 % similarity to MutL of Xanthomonas axonopodis (accession no. AAM37257), 41 % identity and 62 % similarity to MutL of Caulobacter crescentus (accession no. AAK22680), and 40 % identity and 66 % similarity to MutL of Ralstonia solanacearum (accession no. CAD16270).
In the chromosome of N. gonorrhoeae strain FA1090 two homologous ORFs encoding potential m5C-MTases associated with mismatch nicking endonuclease were detected. However, both ORFs M.NgoAORFC703P (position 301 718 bp to 302 119 bp) and M.NgoAORFC713P (position 1 112 994 bp to 1 113 970 bp) encode truncated forms of proteins due to the presence of mutations. The truncated forms of putative MTases encoded by M.NgoAORFC713P and M.NgoAORFC703P share a very high level of homology to M.NmeAI and M.NmeDI respectively (around 98 % identity).
Purification and characterization of the M.NmeDI and M.NmeAI MTases
The M.NmeDI and M.NmeAI MTases were purified to near-homogeneity from E. coli XL-1 Blue MRF'(pAK3) or E. coli ER2566(pAK1) using single-step purification on Ni-NTA agarose columns. As shown in Fig. 2 the molecular masses of these proteins (39·6±1 kDa for M.NmeAI and 49±1 kDa for M.NmeDI) are close to those predicted on the basis of the nucleotide sequence.
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R.HpaII and R.MspI generate the same patterns of fragments after digestion of pAK3 DNA (Fig. 3). The complete digestion of the pAK3 DNA should generate 16 fragments, of 1047, 910, 539, 527, 404, 242, 236, 190, 147, 128, 110, 67, 42, 34, 34 and 26 bp. Instead, fused fragments of 1290, 1146, 561 and 434 bp (black arrows in Fig. 3
) and the absence of fragments of 1047, 910, 527 or 404 bp (grey arrows in Fig. 3
) are observed. The analysis of the fragment profile of pAK3 DNA obtained after digestion with R.ScrFI, R.HpaII and R.MspI indicates the common sequence of 5'-RCCGGB-3' that is methylated by M.NmeDI (Table 1
).
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Specificity and properties of the M.NmeAI MTase
The high homology of the M.NmeAI to m5C-MTases recognizing the sequence 5'-GTCGAC-3' suggested that this enzyme recognizes the same sequence. Since the plasmid pAK1 encoding the M.NmeAI does not contain the presumptive specific recognition sequence 5'-GTCGAC-3', the ability to recognize it was tested by the methylation of the specific ODNs. The results presented in Table 3 indicate that an ODN containing this sequence is not methylated by the purified MTase. However, as in the case of M.NmeDI, we have noticed that pAK1 is not cleaved by several restriction enzymes whose activity is inhibited by the presence of C-5 methylated cytosine residues in the DNA (Roberts & Macelis, 2002
). Among the restriction enzymes tested [R.HpaII (recognition sequence 5'-CCGG-3'), R.MspI (recognition sequence 5'-CCGG-3'), R.NciI (recognition sequence 5'-CCSGG-3')], R.HpaII was totally unable to cleave pAK1 DNA, R.NciI showed partial cleavage, while R.MspI cleaved completely (Fig. 4
). These results indicate that M.NmeAI recognizes the sequence 5'-CCGG-3', which was confirmed by the methylation of the ODNs containing this sequence (Table 2
).
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Cloning of the N. gonorrhoeae putative homologues of M.NmeAI and M.NmeDI MTases
The organization and the types of the ORFs in N. gonorrhoeae strain FA1090 present between the pheS and pheT genes in the chromosome sequence are the same as in N. meningitidis strain 2120. The analysis of the amino acid sequence of the ORF encoding a potential MTase homologous to M.NmeAI indicated the presence of an additional stop codon and a frameshift mutation (position 1 113 500 bp, insertion of G: GACCTT[G]GGTCAG) that divided the amino acid sequence into three separate putative proteins. After cloning the chromosome fragments containing these pseudogenes, we were unable to detect any specific methylase activity using different assay methods. Similarly, we have shown the presence of the frameshift mutation in the amino acid sequence of the ORF encoding a potential MTase homologous to M.NmeDI, and cells harbouring pMPMT4, carrying the cloned chromosome fragment encoding this ORF, did not show any specific methylase activity.
Sequence analysis of the variable region (TRD) of M.NmeDI
M.NmeDI shows the presence of characteristics of the 10 conserved motifs (I through X) of m5C-MTases (Posfai et al., 1989; Cheng et al., 1993
; Kumar et al., 1994
; Lauster, 1989
) with the same linear order and the TRD region located between the conserved motifs VIII and IX (data not presented). The TRD region of M.NmeDI MTase contains about 110 aa. In this region residues present in the C-terminal part (TRD-C) (about 75 aa) share almost 90 % identity, while the N-terminal part has only about 28 % identity with its closest neighbours which recognize the sequence 5'-RCCGGY-3' (Fig. 5
), suggesting the possibility that they represent two different TRD sequences. To confirm that the TRD-C is responsible for the recognition of the sequence 5'-RCCGGY-3', while the N-terminal part is responsible for the recognition of the degenerate sequence, we generated mutations in the C-terminal part of TRD (amino acids 323 to 335). However, the two deletion constructs studied had lost the capacity to methylate both the 5'-RCCGGY-3' and the degenerate sequence, arguing strongly against the presence of the two different TRD regions (data not shown).
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DISCUSSION |
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In the most efficiently methylated sequence 5'-RCCGGY-3' and its degenerate form 5'-RCCGGB-3' the M.NmeDI methylates the same cytosine residues. The TRD region of M.NmeDI MTase shows the presence of two distinct regions, of which the carboxyl-terminal part shares very high identity with the MTases recognizing the sequence 5'-RCCGGY-3', while the amino-terminal part shares less then 30 %. We do not know whether the amino-terminal part of TRD regions of such MTases like M.NpuI or M.Cfr10I reflects their ability to recognize different degenerate forms of the same basic 5'-RCCGGY-3' sequence as recognized by M.NmeDI. If this were true then the lack of homology in the N-terminal parts of their TRD regions would be connected to differences in the degeneracy of the recognition sequence. However, it was shown that M.AvaIX, which shares high homology with its C-terminal part of the TRD with M.NmeDI, methylates only within the sequence 5'-RCCGGY-3' (Matveyev et al., 2001). Although DNA MTases are viewed as highly sequence specific (Dryden, 1999
), recent observations suggest that methylation of non-canonical sites may be a common feature of these enzymes (Bandaru et al., 1996
; Beck et al., 2001
; Cohen et al., 2002
; Friedrich et al., 2000
). What could be the biological role of the ability to recognize the non-canonical form of the recognition sequence? If the R.NmeDI REase associated with M.NmeDI makes mistakes and is able to cleave the DNA in the degenerate sequences, then such activity will be suicidal for the cell. The ability of the MTase to recognize the same degenerate form of the sequence will then protect the DNA against the cleavage and suicidal death. Further studies of the activity of the R.NmeDI are needed to determine if this assumption is correct.
M.NmeAI is the only MTase characterized in N. meningitidis and N. gonorrhoeae that recognizes the sequence 5'-CCGG-3' (Roberts & Macelis, 2002). The regions of N. meningitidis Z2491 and N. gonorrhoeae FA1090 encoding the M.NmeAI and its homologue had no genes nearby that could conceivably encode a restriction endonuclease. We can argue that M.NmeAI is a solitary MTase whose function in these bacteria is not known. It could be that this MTase was a part of an R-M system that was transferred horizontally (the region is flanked by the uptake sequences) and later the gene encoding the restriction enzyme was lost. However, the fact that this MTase does not methylate the chromosomal DNA of N. meningiditis uniformly (data not presented) may indicate some important role in the life of these bacteria.
The 100 % identity of the M.NmeAI present in N. meningitidis serogroup C strain 2120 to M.NmeAI in N. meningitidis serogroup A and the same localization in the chromosome strongly suggest that these two MTases recognize the same 5'-CCGG-3' sequence.
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ACKNOWLEDGEMENTS |
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Received 29 December 2003;
revised 24 February 2004;
accepted 5 March 2004.
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