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 Phi10394.4 (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 Phi10394.4 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 Phi10394.4-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(m)CNGG. 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 Phi10394.4 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 Phi10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in
macrolide-resistant streptococci.