In Vivo Methylation of Bacteriophage φX174 DNA

Abstract

A mutant (designated mec − ) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage λ against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec − mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec + and C mec − cells infected with bacteriophage φX174. φX· mec − RFI is sensitive to in vitro cleavage by R· Eco RII and is cut twice to produce two fragments of almost equal size. In contrast, φX· mec + RFI is relatively resistant to in vitro cleavage by R· Eco RII. R· Bst I, which cleaves mec + /RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R· Eco RII. These results demonstrate that φX· mec + RFI is methylated in vivo by the host mec + enzyme and that this methylation protects the DNA against cleavage by R· Eco RII. This is consistent with the known location of two mec + / RII sequences (viz., [Formula: see text]) on the φX174 map. Mature singlestranded virion DNA was isolated from φX174 propagated in C mec + or C mec − in the presence of l -[ methyl - 3 H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that φX· mec + DNA had a 10-fold-higher ratio of [ 3 H]5-methylcytosine to [ 3 H]cytosine compared to φX· mec − . Since φX· mec + contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of φX174 is mediated by the host mec + enzyme only. These results are not consistent with the conclusions of previous reports that φX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.