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

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 β-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 Φ10394.4 (2). Further sequence analysis revealed that Φ10394.4 and Tn1207.3 are essentially identical in nucleotide sequence except that Φ10394.4 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 Φ10394.4 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 Φ10394.4 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 Φ10394.4. We classified this MTase gene as spyIM and its gene product as M.SpyI, following the updated nomenclature of Roberts et al. (45).