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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

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

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.

Published ahead of print on 3 November 2006.

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