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Journal of Bacteriology, February 2007, p. 1044-1054, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01411-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

M.SpyI, a DNA Methyltransferase Encoded on a mefA Chimeric Element, Modifies the Genome of Streptococcus pyogenes

Chad W. Euler,^1* Patricia A. Ryan,¹ Judith M. Martin,² and Vincent A. Fischetti¹

Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, New York,¹ Department of Pediatrics, Division of Infectious Diseases, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania²

Received 6 September 2006/ Accepted 18 October 2006

Top Abstract Introduction Materials and Methods Results Discussion Addendum in proof References

 
While screening the clonality of Streptococcus pyogenes isolates from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh, PA, we found a correlation between the presence of the chimeric element 10394.4 (carrying the macrolide efflux gene, mefA) and genomic DNA being resistant to cleavage by SmaI restriction endonuclease. A search of the open reading frames in 10394.4 identified a putative type II restriction-modification (R-M) cassette containing a cytosine methyltransferase gene (spyIM). Heterologous expression of the cloned spyIM gene, as well as allelic-replacement experiments, showed that the action of this methyltransferase (M.SpyI) was responsible for the inhibition of SmaI digestion of genomic DNA in the 10394.4-containing isolates. Analysis of the methylation patterns of streptococcal genomic DNA from spyIM-positive strains, a spyIM deletion mutant, and a spyIM-negative strain determined that M.SpyI specifically recognized and methylated the DNA sequence to generate 5'-C^mCNGG. To our knowledge, this is the first methyltransferase gene from S. pyogenes to be cloned and to have its activity characterized. These results reveal why pulsed field gel electrophoresis analysis of SmaI-digested genomic DNA cannot be used to analyze the clonality of some streptococci containing 10394.4 and may explain the inability of previous epidemiological studies to use SmaI to analyze DNAs from macrolide-resistant streptococci. The presence of the SpyI R-M cassette in 10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in macrolide-resistant streptococci.

Top Abstract Introduction Materials and Methods Results Discussion Addendum in proof References

 
Streptococcus pyogenes is a gram-positive bacterial pathogen of humans that has the ability either to asymptomatically colonize the upper respiratory tract and skin or to cause a wide variety of diseases that differ in severity and tissue tropism. Such diseases range from relatively mild infections, such as pharyngitis and impetigo, to the more severe forms of invasive disease, like streptococcal toxic shock syndrome and necrotizing fasciitis. The treatment of choice for streptococcal pharyngitis infections is penicillin V (8), but in recent years, macrolide antibiotics, such as azithromycin and erythromycin, have been increasingly prescribed as alternative treatments (14, 16, 23, 33). Concomitantly, there has been an increase in macrolide-resistant strains of S. pyogenes (5, 12, 14, 25, 36), posing a global health problem for patients who are hypersensitive to ß-lactam antibiotics (8, 48, 57).

Macrolide resistance in streptococci is usually associated with the presence of one of three genes: (i) ermB and (ii) ermTR, both of which encode methyltransferases (MTases) that confer resistance to macrolide, lincosamide, and streptogramin B antibiotics by modifying a conserved adenine residue on the drug target site of the 23S rRNA (32, 51), and (iii) mefA, which encodes a macrolide efflux pump that imparts resistance to 14- and 15-membered macrolides, such as erythromycin and azithromycin, but not to the 16-membered macrolide, lincosamide, and streptogramin B antibiotics (classified as the "M phenotype" of macrolide resistance) (15). The mefA gene is present in a number of phylogenetically unrelated serotypes of S. pyogenes (5, 12, 25, 36) and in some regions of the world is becoming the predominant erythromycin resistance determinant in streptococcal isolates (25, 27, 44, 52). Earlier studies suggested that erythromycin resistance and the mefA gene may be acquired through horizontal gene transfer; however, at that time, the mechanisms were not clearly identified in S. pyogenes (17, 27, 34).

Recently, the mefA gene was identified on three different DNA chimeric elements, which have both transposon and prophage characteristics. Santagati et al. partially characterized a 52-kb chimeric element carrying the mefA gene, which was integrated into the comEC gene of an erythromycin-resistant strain of S. pyogenes (49). They described 7.2 kb of the 52-kb chimeric element as 100% identical to a defective mefA conjugative transposon, Tn1207.1, from Streptococcus pneumoniae (49, 50). Additionally, they demonstrated that the S. pyogenes 52-kb chimeric element could be transferred by filter conjugation to different strains of S. pyogenes, S. pneumoniae, and Streptococcus gordonii, conferring erythromycin resistance on these species. For these reasons, the 52-kb chimeric element was classified as conjugative transposon Tn1207.3 (49).

Concurrently, during genomic sequencing of S. pyogenes strain MGAS10394, isolated from an outbreak of erythromycin-resistant pharyngitis in elementary school children in Pittsburgh, PA (36), Banks et al. identified a similar 58.8-kb chimeric element (3). The element was inserted into the same comEC site as Tn1207.3 and also contained mefA on the 7.2-kb defective transposon Tn1207.1. Nucleotide sequence analysis of the entire chimeric element revealed that it was also composed of conserved lysogenic bacteriophage genes. Additionally, chimeric-element DNA was detected in bacteriophage particles released into the culture supernatant after induction with mitomycin C (3). These results suggested that this chimeric element was composed of the transposon Tn1207.1 inserted into a functional prophage, and thus, it was classified as 10394.4 (2). Further sequence analysis revealed that 10394.4 and Tn1207.3 are essentially identical in nucleotide sequence except that 10394.4 contains an additional 6-kb variable region upstream of the Tn1207.1 transposon (2, 43). Lately, a third chimeric element containing mefA and tetO, a tetracycline resistance gene, was described as having both conjugative-transposon and bacteriophage characteristics (21). These tetO-mefA chimeric elements are different from the other two elements, as they are composed of a varying number of genes similar to those of Tn1207.1 that have combined with a different set of bacteriophage genes, resulting in elements of various sizes (52 to 60 kb), which are integrated into chromosomal locations outside of the comEC gene (9, 21, 22).

All of the studies mentioned above suggest that recent increases in the "M phenotype" of macrolide resistance may have been influenced by the acquisition and dissemination of these mefA chimeric elements within the streptococcal population (21, 22, 43). One of the most common methods used to examine the clonality of such macrolide-resistant and -sensitive streptococcal isolates involves restriction of genomic DNA with the SmaI endonuclease, followed by pulsed field gel electrophoresis (PFGE) analysis (6, 44, 55). Recently, however, a number of epidemiological studies have reported that the DNA from a diverse group of erythromycin-resistant streptococci could not be digested with SmaI (7, 22, 35, 36, 44). This finding prompted the use of alternative restriction enzymes, such as ApaI, EagI, and SfiI, in the analysis, making it difficult subsequently to directly compare the clonalities of strains that differ in macrolide susceptibility and to relate the results of different epidemiological studies (7, 35, 36).

While analyzing the clonality of erythromycin-resistant S. pyogenes strains from the pharyngitis outbreak in Pittsburgh, PA, referenced above (36), we found that the presence of 10394.4 in the genome was associated with streptococcal genomic DNA being refractory to SmaI restriction. Since SmaI cleavage is blocked by CpG methylation at the enzyme recognition site (REBASE [http://rebase.neb.com]) (46), we hypothesized that a 5-methylcytosine MTase encoded on 10394.4 may be methylating the genomic DNA, thereby inhibiting SmaI restriction. In the work presented here, we confirmed this hypothesis through the allelic replacement, cloning, and characterization of the gene encoding the MTase from 10394.4. We classified this MTase gene as spyIM and its gene product as M.SpyI, following the updated nomenclature of Roberts et al. (45).

Top Abstract Introduction Materials and Methods Results Discussion Addendum in proof References

 
Bacterial strains and growth conditions. S. pyogenes isolates obtained from a longitudinal study on the epidemiology of streptococcal infections of elementary school children in Pittsburgh, PA (36, 37), are described in Table 1. These isolates were previously characterized based on their emm types, macrolide susceptibilities, mechanisms of macrolide resistance, and clonalities (36, 37). In subsequent analyses, the isolates were screened for the presence of the mefA gene and the 10394.4 element and its insertion into the comEC gene by PCR and Southern blot hybridization. The primers used for this analysis are described under the chimeric element heading in Table 2. Escherichia coli One Shot Top10 (Invitrogen) was used as the host strain for plasmid construction and recombinant-protein expression.

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TABLE 1. Strains and plasmids

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TABLE 2. PCR and sequencing primers

E. coli was cultured in Luria-Bertani (LB) broth and on LB agar at 37°C. S. pyogenes strains were grown at 37°C in brain heart infusion broth (Difco) and on proteose peptone agar (Difco) supplemented with 4% defibrinated sheep blood (Cleveland Scientific). When required, media were supplemented with antibiotics at the following concentrations: ampicillin at 100 µg/ml, erythromycin at 2 µg/ml, kanamycin at 50 µg/ml for E. coli and 250 µg/ml for S. pyogenes, and spectinomycin at 20 µg/ml for E. coli and 100 µg/ml for S. pyogenes.

DNA manipulations. Streptococcal genomic DNA was isolated with either the DNeasy Tissue Kit or the Blood and Cell Culture DNA Kit (QIAGEN), following the manufacturer's protocols, except for the substitution of a modified lysis buffer (50 mM Tris-Cl, pH 6.6, 50 mM EDTA, 0.5% Tween 20, 0.5% Triton X-100) supplemented with 500 U of PlyC, a streptococcal bacteriophage lysin (40), and 250 µg/ml of RNase A (QIAGEN). Plasmid DNA was isolated from E. coli using the QIAprep Spin Miniprep Kit or HiSpeed Plasmid Midi Kit (QIAGEN). DNA fragments were gel purified from 1% agarose gels using the QIAquick Gel Extraction Kit (QIAGEN). T4 DNA ligase, M.HpaII MTase, and all restriction enzymes were purchased from New England Biolabs and used according to the manufacturer's instructions. Oligonucleotides were obtained from Sigma-Genosys. PCR was performed using AmpliTaq Gold DNA polymerase, Gold Buffer, 1.5 mM MgCl₂, and 200 µM deoxynucleotide triphosphates (Applied Biosystems) following standard protocols with the Eppendorf Mastercycler. DNA sequencing was performed by GENEWIZ, Inc. (North Brunswick, NJ). DNA sequence analysis, comparison, and manipulation required Lasergene software modules (DNASTAR Inc.) and the CLUSTALX (56) and BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html) programs. DNA primers were designed with MacVector software (Accelrys Inc.).

PFGE molecular analysis. Agarose disks of genomic DNA from the Pittsburgh isolates (Table 1) were prepared according to a protocol modified from that of Chung et al. (13), in which 500 U of PlyC was substituted for lysozyme and lysostaphin enzymes in the cell lysis solution (13, 40). DNA was digested with SmaI or XmaI (40 U) overnight at 25°C or 37°C, respectively, and subjected to PFGE with the CHEF-DR II system (Bio-Rad) as previously described (13). DNA bands were visualized by staining the gel with ethidium bromide, and the images were captured by an Alpha imager (Alpha Innotech Corp.). DNA banding patterns were analyzed by previously described methods (13, 55).

Construction of the pBadTOPO-spyIM E. coli expression plasmid. The spyIM gene was PCR amplified from genomic DNA of strain 3PM6 using the MetR and MetFI primers described under the M.SpyI E. coli expression heading in Table 2. The 1,227-bp PCR product was gel purified, ligated into the pBAD-TOPO vector, and transformed into E. coli One Shot TOP10 using the pBAD TOPO TA Expression Kit (Invitrogen). Transformants were screened for proper insert orientation by colony PCR and DNA sequencing. Positive clones were screened for MTase expression by the SmaI protection assay described below.

SmaI endonuclease protection assay. Expression and activity of the recombinant M.SpyI was tested using a combination of protocols described previously (11, 29) following the instructions for the pBADTOPO TA Expression Kit. Briefly, recombinant M.SpyI expression was induced by the addition of 0.2% or 2% L-arabinose to cultures at an optical density at 600 nm of 0.5, and growth then continued for 2 or 4 h at 37°C, respectively. L-Arabinose concentrations and induction times were varied to determine the most efficient expression conditions. The cultures were centrifuged at 3,000 x g for 10 min. The cell pellets were washed once in 50 mM Tris-HCl, pH 7.5, resuspended in MTase reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM 2-mercaptoethanol, 10 mM EDTA), and frozen at –20°C overnight. Cells were lysed by two freeze-thaw cycles, followed by sonication for 5 min with a W-380 Sonicator (Heatsystems-Ultrasonics Inc.). The crude lysate was centrifuged at 16,000 x g for 20 min at 4°C to collect the supernatant. The lysate supernatant (10 µl), supplemented with 80 µM of S-adenosylmethionine (SAM) (New England Biolabs), was then incubated for 2 h at 37°C with 1 µg of phage DNA (New England Biolabs) as the substrate for the methylation reaction. The reaction mixture was heat inactivated for 20 min at 65°C, and then the DNA was subjected to SmaI digestion for 2 h at 25°C by the addition of NEBuffer 4 (40 µl) supplemented with 10 mM MgCl₂ and 20 U SmaI. Digestion or protection of DNA was analyzed by 1% agarose gel electrophoresis.

Allelic replacement of the restriction-modification (R-M) cassette in 10394.4. The strategy for allelic replacement of the R-M cassette is outlined in Fig. 1. First, DNA regions flanking spyIM and the adjacent restriction enzyme genes (944 bp upstream and 1,016 bp downstream) were PCR amplified from the genomic DNA of strain 3PM6 using the two primer sets described under the R-M allelic replacement heading in Table 2. The PCR products were treated with a combination of either EagI and XhoI or AvrII and AgeI and were purified with a Qiaquick PCR Purification Kit (QIAGEN). The upstream and downstream fragments were individually cloned into shuttle vector pFW13 (42). The two resulting vectors, pFW13EX and pFW13AA, were then digested with NheI and NcoI and religated to produce the R-M allelic replacement vector, pFW13EA. The pFW13EA vector was treated with the M.HpaII MTase, following the manufacturer's suggestions (New England Biolabs), to methylate 5'-CCGG residues and to prevent vector digestion by the cognate restriction enzyme of the R-M cassette within the streptococci. The methylated vector was electroporated into 3PM6 following the streptococcal transformation protocol of Kimoto and Taketo (28), and transformants were selected on proteose peptone blood agar supplemented with kanamycin. Allelic replacement of the R-M cassette with the Kan^r gene (aacA/aphD) was confirmed by PCR, Southern blot analysis, and DNA sequencing. The resulting mutant strain (3PM6RM) lacked the genes that encoded M.SpyI and the adjacent restriction enzyme subunit.

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FIG. 1. Construction of the allelic-replacement vector pFW13EA. The diagram outlines the steps to construct vector pFW13EA. At the top are shown the locations of the adjacent regions upstream and downstream of the R-M cassette that were PCR amplified and digested with the appropriate restriction enzymes by methods detailed in the text. Each amplicon was separately ligated into plasmid pFW13 to create pFW13EX and pFW13AA. Both plasmids were subsequently digested with NheI and NcoI and ligated to produce pFW13EA, the allelic-replacement vector, which contains the kanamycin resistance gene (aacA/aphD) flanked by the regions adjacent to the R-M cassette. The gray arrows and blocks indicate the regions PCR amplified for insertion into the pFW13 vector. The R-M bracket indicates the region of the restriction-modification cassette that was replaced with the kanamycin resistance gene (aacA/aphD). The plasmid diagrams indicate relevant restriction sites, the kanamycin resistance gene (aacA/aphD), and the E. coli origin of replication (ori).

Construction of pLZ12spec-spyIM S. pyogenes expression plasmid. A 1,409-bp region encompassing both the spyIM gene and the upstream promoter region was PCR amplified from the genomic DNA of strain 3PM6 using the primers described under the M.SpyI S. pyogenes expression heading of Table 2. The amplicon was then digested with BamHI and SphI and cloned into pLZ12spec, an E. coli-streptococcal shuttle vector (10), to produce pLZ12spec-spyIM (Table 1). This construct was electroporated into the mutant strain 3PM6RM to produce the complemented strain 3PM6RM-C (Table 1). trans-Complementation of the MTase knockout by pLZ12spec-spyIM was tested by PFGE molecular analysis as described above.

Bisulfite analysis of 5-methylcytosine residues. Streptococcal genomic DNA (500 ng) was modified by bisulfite treatment with the EZ DNA Methylation-Gold kit (Zymo Research) according to the manufacturer's instructions. The bisulfite-modified DNA was used as the template for PCR using primer pairs specific for the sense (bi-F and bi-R) or antisense (bi-botF and bi-botR) strands of the modified DNA, as described in Table 2 under the bisulfite reaction heading. The amplicons were then purified and sequenced in both the 5' and 3' directions, using the primers listed under the bisulfite reaction heading in Table 2. The DNA sequences were aligned with the published sequence of S. pyogenes strain MGAS10394 using the MegAlign program (DNAstar Inc.) to help identify 5-methyl cytosine residues.

Top Abstract Introduction Materials and Methods Results Discussion Addendum in proof References

 
Genomic DNA harboring 10394.4 is resistant to SmaI restriction. To further differentiate the clonality of the S. pyogenes strains isolated from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh school children (36), PFGE was performed on SmaI-digested genomic DNA from isolates that were positive or negative for the chimeric element 10394.4 (Table 1 and Fig. 2A). Analysis of the PFGE patterns revealed that SmaI was able to digest DNA from strains that did not contain 10394.4. Conversely, genomic DNA from strains containing 10394.4 was resistant to SmaI digestion (Table 1 and Fig. 2A). Furthermore, SmaI did not digest the genomic DNA of three erythromycin-susceptible isolates (Table 1 and Fig. 2A). PCR and sequence analysis showed that these strains also contained the 10394.4 chimeric element but had mutations in the mefA gene (Table 1 and data not shown). These observations suggested that resistance to SmaI cleavage of genomic DNA was not directly related to erythromycin resistance, but rather, to the presence of the 10394.4 chimeric element.

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FIG. 2. PFGE analysis of S. pyogenes M6 isolates from school children in Pittsburgh, PA. (A) PFGE patterns of SmaI-digested genomic DNAs from isolates that were negative (strains 1-, 7-, and 8PM6) or positive (strains 2-, 3-, 4-, 5-, 6-, 9-, and 10PM6) for the 58-kb mefA-carrying chimeric element 10394.4 (Table 1). , Lambda Ladder PFG Marker (New England Biolabs). (B) Comparison of PFGE patterns of SmaI- and XmaI-digested genomic DNA from the 10394.4-positive isolate 3PM6.

Genomic DNA harboring 10394.4 is partially digested by XmaI. SmaI restriction of DNA can be blocked by CpG methylation at the restriction enzyme cleavage site, 5'-CCCGGG (REBASE [http://rebase.neb.com]) (46). To determine if this type of methylation was protecting the genomic DNAs of 10394.4-containing isolates, DNA preparations were treated with XmaI, an isoschizomer of SmaI that cleaves DNA at the sequence 5'-CCCGGG. Unlike SmaI, XmaI is not fully blocked but partially impaired by CpG methylation overlapping the SmaI recognition site (REBASE [http://rebase.neb.com]) (46). PFGE analysis showed that while SmaI was inhibited, XmaI partially cleaved the genomic DNA from the 10394.4-containing strain 3PM6 (Fig. 2B). Taken together, the differences in digestion patterns between XmaI and SmaI may be explained by the locations of their distinct cleavage sites within the same methylated recognition sequence. These results suggest that the partial digestion by XmaI and the inability of SmaI to cleave 3PM6 DNA may indeed be caused by methylation.

5-Methylcytosine MTase identified on 10394.4. Analysis of the published sequence of 10394.4 revealed that the bacteriophage-like region of this chimeric element contains a putative type II R-M cassette encoding a type II restriction endonuclease (in two subunits) and an adjacent MTase gene (spyIM) (Fig. 3) (2, 3). A BLASTP comparison of the predicted amino acid sequence of this MTase (M.SpyI) to those of proteins in the REBASE database [http://tools.neb.com] (1, 46), revealed high sequence similarities to other 5-methylcytosine MTases that recognize the DNA sequence 5'-CCNGG (Table 3). The comparison also identified M.Spy1207ORFAP, which is located in the DNA sequence of Tn1207.3, as an open reading frame (ORF) identical to that of M.SpyI (49). Of note, no ORF corresponding to M.SpyI has yet been identified in the tetO-mefA chimeric elements (18). A CLUSTALW alignment of the amino acid sequences from the BLASTP search showed that M.SpyI contains the 10 conserved motifs that are present in the 5-methylcytosine MTases, in the proper order, and significant homology within the variable region making up the target recognition domain of these enzymes (Fig. 4). These conserved features suggested that spyIM may encode a 5-methylcytosine MTase that could recognize the DNA sequence 5'-CCNGG. This recognition sequence is included within the DNA sequence recognized by SmaI (5'-CCCGGG). Therefore, we were interested in determining if M.SpyI methylates CpG sites on the streptococcal chromosome, such as those contained in the SmaI DNA recognition sequence.

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FIG. 3. Schematic of a 28-kb segment of the chimeric element 10394.4. At the top are shown the positions of the mefA gene and the restriction-modification cassette, which contains the spyIM gene, within the predicted ORFs of 10394.4. The numbers below the sequence indicate relative sizes in kb. The black arrows denote regions that have transposon- and bacteriophage-like characteristics, as detailed in the text. At the bottom is a close-up of the predicted ORFs and their corresponding Spy numbers from the genome sequence of S. pyogenes MGAS10394, GenBank accession number CP000003.

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TABLE 3. Comparison of M.SpyI with other cytosine MTases by BLASTP search of the REBASE protein database

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FIG. 4. Alignment of the amino acid sequence of M.SpyI with the five closest 5-methylcytosine MTases from a REBASE BLASTP search. Alignment of the sequences M.SpyI, M1.ScrFI, M.StyD41, M.Ecl18kI, M.SenPI, and M.SsoII was carried out using the ClustalX program, and the amino acids were shaded with the Box Shade program. The black boxes indicate identical residues, while gray boxes highlight conserved residues. Gaps are indicated by dashes. The locations of the 10 conserved amino acid motifs present in 5-methylcytosine MTases are indicated by black lines above the amino acid sequences and are numbered I to X. The variable region making up the target recognition domain (TRD) of 5-methylcytosine MTases is indicated by a gray line above the amino acid sequences. M.Spy1207ORFAP was not included in the alignment because the ORF is identical to that of M.SpyI in the Tn1207.3 chimeric element.

Recombinant M.SpyI protects DNA from SmaI digestion. To investigate whether M.SpyI was capable of modifying DNA to inhibit SmaI, recombinant M.SpyI was expressed in E. coli and tested for MTase activity. Cell lysates from cultures in which M.SpyI was either induced or not induced were separately incubated with DNA as a substrate and then subsequently subjected to SmaI digestion. This analysis revealed that SmaI did not cleave DNA that was incubated with recombinant M.SpyI induced under various conditions (Fig. 5). Conversely, DNA incubated with noninduced cell lysates (i.e., with M.SpyI not present) was cleaved by the action of SmaI (Fig. 5). Taken together, these results indicated that the M.SpyI was able to modify the DNA to render it resistant to SmaI digestion. Furthermore, cell lysates containing M.SpyI that were not supplemented with SAM in the methylation reaction did not protect DNA from SmaI digestion (Fig. 5). Since SAM is an essential cofactor in type II methyltransferase reactions (45, 58), this result suggests that DNA methylation is the modification responsible for SmaI inhibition.

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FIG. 5. Recombinant M.SpyI protects DNA from digestion with the SmaI endonuclease. DNA was first incubated with the crude lysate of E. coli cells that expressed recombinant M.SpyI and then was subjected to SmaI digestion. The digestion patterns were analyzed by electrophoresis on a 1% agarose gel. Lane 1, undigested DNA; lane 2, non-lysate-treated DNA alone with SmaI; lanes 3 and 4, DNA incubated with crude lysate (supplemented with 80 µM of SAM) in which M.SpyI was induced with 0.2% arabinose for 2 h or 2% arabinose for 4 h, respectively; lane 5, DNA incubated with M.SpyI-induced crude lysate without SAM; lane 6, DNA plus crude lysate from a noninduced culture with 0% arabinose for 4 h; M, 1-kb Plus DNA Ladder (Invitrogen).

Allelic replacement of the R-M cassette allows cleavage of genomic DNA by SmaI and XmaI. Deletion of the spyIM gene in 10394.4 would confirm the role of the MTase in methylation of the genomic DNA and provide a useful tool to further dissect the functions of the R-M system genes from the rest of those encoded on the chimeric element. Since an active MTase enzyme is usually necessary to protect host DNA from being digested by the cognate restriction enzyme of the R-M cassette (30), we sought to inactivate both enzyme activities by allelic replacement of part of the R-M cassette with a kanamycin resistance gene from vector pFW13EA (Fig. 1). This resulted in the mutant strain 3PM6RM, which lacked the spyIM gene and the adjacent ORF encoding one of the restriction enzyme subunits (Table 1 and Fig. 1). PFGE analysis of digested genomic DNA from this strain showed that it was completely cleaved by both SmaI and XmaI (Fig. 6A and B). These results indicated that methylation of the genomic DNA by M.SpyI is responsible for the inhibition of SmaI digestion and the partial inhibition of XmaI digestion.

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FIG. 6. PFGE patterns of digested genomic DNAs from 3PM6 (a spyIM-positive strain), 3PM6RM (a spyIM deletion mutant), and 3PM6RM-C (a trans-complemented spyIM mutant). (A) Restriction patterns of SmaI-digested genomic DNA. Lane 1, undigested DNA (3PM6); lane 2, 3PM6; lane 3, 3PM6RM; lane 4, 3PM6RM-C. (B) Restriction patterns of XmaI-digested genomic DNA. Lane 1, 3PM6; lane 2, 3PM6RM; lane 3, 3PM6RM-C. The arrows in both panels identify fragments that are different from the predicted digestion pattern of S. pyogenes strain MGAS10394 due to the allelic replacement of part of the RM cassette with the kanamycin resistance gene (aacA/aphD).

To confirm that the digestion of 3PM6RM DNA was due to a lack of M.SpyI activity in the mutant, the spyIM gene was trans-complemented into 3PM6RM on the pLZ12spec-spyIM vector and the genomic DNA was tested for inhibition of the restriction enzymes. Both the inhibition and the partial inhibition of genomic-DNA digestion by SmaI and XmaI, respectively, were restored in the M.SpyI-complemented mutant (Fig. 6A and B). These results confirmed that M.SpyI was responsible for protecting the DNA from the actions of these restriction enzymes in vitro.

M.SpyI recognizes the genomic-DNA sequence CCNGG. Bisulfite modification of DNA converts nonmethylated cytosine residues to uracil, while 5-methylcytosine residues are protected and remain unaltered in the reaction. Subsequent PCR amplification of the modified DNA changes the uracil residues into thymine so that only the 5-methylcytosine residues are identified as cytosine when the amplicon is sequenced (20). To determine the nucleotide sequence that was methylated by M.SpyI, we used the bisulfite reaction to modify the genomic DNAs from the 10394.4-containing strains 3PM6 and 10PM6, the 3PM6RM deletion mutant, and 8PM6, a 10394.4-negative strain (Table 1). A 700-bp DNA sequence inside the 16S rRNA genes of these strains was chosen to be PCR amplified and sequenced because it contained all combinations of the 5'-CCNGG DNA sequence, including the SmaI DNA recognition site and other potentially methylated CpG sequences.

The modified sequences were aligned to 600 bp of the published genome sequence of MGAS10394 to identify the methylation patterns of each strain. The alignment in Fig. 7 shows that in the DNA sequences of strains that do not contain the spyIM gene (the 3PM6RM mutant and 8PM6), all cytosines were converted to thymine. Conversely, DNAs from spyIM-positive strains (3PM6 and 10PM6) contained unchanged cytosines on the inner C residue of the 5'-CCNGG DNA sequences (Fig. 7), suggesting that M.SpyI methylates this specific cytosine residue (5'-C^mCNGG). Additionally, both inner C residues were methylated in the SmaI recognition sequence, 5'-C^mC^mCGGG (Fig. 7). This methylation pattern fits with the above results, because the SmaI recognition sequence actually consists of two overlapping 5'-CCNGG DNA sequences (5'-CCCGG and 5'-CCGGG). Analysis of the DNA sequence from the opposite strand of the same 700 bp showed an equivalent cytosine methylation pattern (data not shown), indicating that these sites have the same cytosine residue methylated on both strands of the DNA, a finding consistent with most type II restriction-modification MTases (46, 58). The specificity and activity of M.SpyI were also confirmed by bisulfite analysis of two other regions outside the 16S rRNA genes and with DNA methylated with recombinant M.SpyI (data not shown).

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FIG. 7. Sequence comparison of PCR products from bisulfite-modified streptococcal genomic DNA to identify cytosines methylated by M.SpyI. The sequences were aligned to the published genomic sequence of the S. pyogenes M6 strain MGAS10394 on the top line of each row. Unmodified nucleotides that were identical to the residues in the MGAS10394 reference sequence (GenBank accession number CP000003) are represented with a dot. Methylated cytosines have been defined as cytosine nucleotides that were not converted to thymine residues by bisulfite treatment and are highlighted by asterisks above the DNA sequences. The boxes indicate the putative DNA recognition sequences for M.SpyI, with the dashed box highlighting the overlapping sequence recognized by SmaI. Line 1, MGAS10394 reference sequence; lines 2 and 3, modified DNAs from 10394.4-containing isolates 3PM6 and 10PM6, respectively; line 4, modified DNA from the spyIM deletion mutant 3PM6RM; line 5, modified DNA from the 10394.4-negative isolate 8PM6. The numbering of the sequence in line 1 corresponds to the published numbered DNA sequence of MGAS10394. The numbering of the other lines corresponds to the positions of the nucleotides from the start of the bi-F primer in the sense strand amplicon of the bisulfite reaction (Table 2).

Taken together, these results confirm that the DNA sequence modified by M.SpyI is 5'-CCNGG, as predicted by the BLASTP comparison of M.SpyI to other 5-methylcytosine MTases described above. These results further indicate that M.SpyI methylation of the DNA is responsible for the inhibition of the SmaI cleavage of the genomic DNA from the 10394.4-containing isolates.

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PFGE analysis of SmaI-digested genomic DNA has been used in many epidemiological studies to successfully characterize the clonalities of multiple serotypes of S. pyogenes (39, 41, 54, 55). However, numerous studies have reported that the DNA from some macrolide-resistant isolates is refractory to SmaI digestion (7, 22, 35, 36, 44), an observation recently associated with the "M phenotype" of erythromycin resistance, which is imparted by Tn1207.3 or the 10394.4 chimeric element (22, 44). The results of our study agree with these reports. Here, we also found that SmaI could not digest genomic DNA from streptococcal isolates containing 10394.4; this led to the identification of a type II R-M cassette on 10394.4, which encodes two subunits of a restriction endonuclease and carries an adjacent MTase gene (spyIM).

We cloned, expressed, and characterized the spyIM gene, encoding the 5-methylcytosine MTase, which we named M.SpyI. Through the heterologous expression of M.SpyI, we provided evidence that it is capable of protecting DNA from SmaI digestion. The allelic replacement of the spyIM gene in S. pyogenes, combined with bisulfite methylation analysis, confirmed that the inner cytosine was methylated in the DNA sequence C^mCNGG and that M.SpyI alone was responsible for the methylation leading to the partial digestion by XmaI and the inhibition of SmaI digestion of streptococcal DNA in vitro. Although spyIM is carried on a mobile element, to our knowledge, this is the first MTase gene to be cloned and characterized from S. pyogenes.

The possible in situ roles for the MTase and the adjacent restriction enzyme subunits of the type II R-M system are currently being investigated. However, previous studies have shown that type II MTases act in concert with their cognate type II restriction enzymes, which usually recognize and cleave the overlapping DNA sequence that is acted upon by the MTase to protect the bacteria from foreign DNA (reviewed in reference 58). Methylated genomic DNA is protected from the action of the cognate restriction enzyme, while nonmethylated DNA from other lytic or lysogenic bacteriophages is restricted so that it cannot kill or sabotage the survival of the bacteria (58). This suggests another example of how 10394.4 may increase host strain survival in the population and provides another reason why erythromycin-resistant isolates carrying mefA are increasing in the streptococcal population (25, 27, 44, 52). In fact, analysis of DNA sequences from 23 of the published lysogenic bacteriophages of S. pyogenes show that they contain an average of 17 CCNGG sites in their genomes (data not shown) (2, 4, 19, 53). Interestingly, all seven of the other bacteriophages present in the published sequence of the MGAS10394 strain, in which 10394.4 was classified, contain 5'-CCNGG sites (2). The presence of these sites could have allowed the restriction enzyme to cleave its incoming bacteriophage DNA if the R-M cassette was already integrated into the genome, suggesting that 10394.4 might have been the most recent element acquired by this streptococcal strain.

Another potential role for the R-M cassette might be to promote maintenance of the mefA chimeric element in the bacterial chromosome through the postsegregational killing of strains that have had a recombinational event, which cured the strain of 10394.4 (24, 30, 47). Strains that have lost 10394.4 and its associated MTase may initially contain residual restriction enzyme that could digest any nonmethylated regions of genomic DNA, resulting in the suicidal death of the bacteria (26, 31, 38). This process would select for isolates that have preserved 10394.4 and the mefA gene in the population and thus possibly maintain macrolide resistance even when the selective antibiotic is not present in the bacterial environment.

Our results also provide an explanation for a finding from past epidemiological studies that the genomic DNA of some streptococcal isolates with the "M phenotype" of macrolide resistance cannot be digested with SmaI (7, 22, 35, 36, 44). We have shown that the M.SpyI MTase is directly responsible for this phenotype. In the future, we recommend the use of alternative restriction enzymes that recognize DNA sequences identical to those recognized by SmaI but that are not blocked by this type of methylation, such as the Crf9I endonuclease (Fermentas Inc.) recently used to compare the clonalities of macrolide-resistant isolates of S. pyogenes from Portugal (52).

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At the time this article was submitted, Figueiredo et al. (T. A. Figueiredo et al., Antimicrob Agents Chemother 50:3689-3694, 2006; published ahead of print 6 September 2006) reported that the resistance of S. pyogenes genomic DNA to SmaI restriction is a characteristic that could be used as a marker for the presence of the Tn1207.3 and 10394.4 chimeric elements. These results support our finding that these elements contain a methyltransferase and underscore the importance of studying their role in the dissemination of macrolide resistance within the streptococcal population.

 
We thank all of the members of the Laboratory of Microbial Pathogenesis and Immunology, especially Anu Daniel, Mattias Collin, Ray Schuch, Daniel Nelson, and Barbara Juncosa, for their scientific expertise and valuable advice on this and ongoing projects.

This work was supported by USPHS grants AI11822 and AI057472 to V.A.F.

 
* Corresponding author. Mailing address: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, Box 172, 1230 York Avenue, New York, NY 10021. Phone: (212) 327-8168. Fax: (212) 327-7584. E-mail: eulerc{at}mail.rockefeller.edu.

Published ahead of print on 3 November 2006.

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Journal of Bacteriology, February 2007, p. 1044-1054, Vol. 189, No. 3
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