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In vivo methylation of bacteriophage phi X174 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 lambda 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 phiX174. phiX. 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, phiX.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 phiX.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 phiX174 map. Mature singlestranded virion DNA was isolated from phiX174 propagated in C mec(+) or C mec(-) in the presence of l-[methyl-(3)H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that phiX.mec(+) DNA had a 10-fold-higher ratio of [(3)H]5-methylcytosine to [(3)H]cytosine compared to phiX.mec(-). Since phiX.mec(+) contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of phiX174 is mediated by the host mec(+) enzyme only. These results are not consistent with the conclusions of previous reports that phiX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.
Insights
A mutant Escherichia coli strain lacking DNA-cytosine methylase activity was studied. This methylase is crucial for protecting bacteriophage phiX174 DNA from restriction enzymes, indicating host control over phage DNA modification.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Escherichia coli possesses DNA-cytosine methylase (mec) activity essential for protecting phage DNA from host restriction systems.
- A mutant strain of E. coli C (mec(-)) was isolated, exhibiting loss of DNA-cytosine methylase activity.
- This phenotype is analogous to previously studied E. coli K-12 mec(-) mutants, suggesting overlapping enzyme specificities.
Purpose of the Study:
- To investigate the role of the host DNA-cytosine methylase in modifying bacteriophage phiX174 DNA.
- To determine if phage phiX174 methylation is mediated by a host or phage-induced enzyme.
- To assess the impact of host methylation on phage DNA resistance to restriction enzymes.
Main Methods:
- Isolation and characterization of an Escherichia coli C mec(-) mutant.
- Infection of mec(+) and mec(-) E. coli strains with bacteriophage phiX174.
- Analysis of covalently closed, supertwisted double-stranded DNA (RFI) using restriction endonucleases R.EcoRII and R.BstI.
- Isolation and chromatographic analysis of radiolabeled mature single-stranded virion DNA.
Main Results:
- phiX174 DNA isolated from mec(-) cells was sensitive to R.EcoRII cleavage, while DNA from mec(+) cells was resistant.
- R.BstI cleaved both mec(+) and mec(-) RFI, confirming the presence of R.EcoRII recognition sites.
- Radiolabeling experiments showed a 10-fold higher ratio of 5-methylcytosine to cytosine in phiX174 DNA from mec(+) cells.
- Each phiX174 DNA molecule from mec(+) cells contained approximately one 5-methylcytosine residue.
Conclusions:
- Host DNA-cytosine methylase activity in E. coli is responsible for the methylation of bacteriophage phiX174 DNA.
- Methylation of phiX174 DNA by the host mec(+) enzyme protects it against cleavage by the R.EcoRII restriction enzyme.
- These findings contradict previous reports suggesting phage-induced methylation and its essential role in phage development.