5-Methylcytosine is one of the major epigenetic marks of DNA in living organisms. Some bacterial species possess DNA methyltransferases that modify cytosines on both strands to produce fully-methylated sites or on either strand to produce hemi-methylated sites. In this study, we characterized a DNA methyltransferase that produces two sequences with different methylation patterns: one methylated on both strands and another on one strand. M.BatI is the orphan DNA methyltransferase of Bacillus anthracis coded in one of the prophages on the chromosome. Analysis of M.BatI modified DNA by bisulfite sequencing revealed that the enzyme methylates the first cytosine in sequences of 5'-GCAGC-3', 5'-GCTGC-3', and 5'- GCGGC-3', but not of 5'-GCCGC-3'. This resulted in the production of fully-methylated 5'- GCWGC-3' and hemi-methylated 5'-GCSGC-3'. M.BatI also showed toxicity when expressed in E. coli, which was caused by a mechanism other than DNA modification activity. Homologs of M.BatI were found in other Bacillus species on different prophage like regions, suggesting the spread of the gene by several different phages. The discovery of the DNA methyltransferase with unique modification target specificity suggested unrevealed diversity of target sequences of bacterial cytosine DNA methyltransferase.
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