In Vivo Methylation of Bacteriophage φX174 DNA

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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·EcoRII 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·EcoRII. R·BstI, 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· EcoRII. 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·EcoRII. 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-3H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that φX·mec+ DNA had a 10-fold-higher ratio of [3H]5-methylcytosine to [3H]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. Images