Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine
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Abstract:
Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.
Insights
Acholeplasma laidlawii strains K2 and JA1 exhibit distinct DNA restriction mechanisms. Strain K2 targets the GATC sequence, while strain JA1 uniquely restricts DNA containing 5-methylcytosine, revealing novel restriction-modification systems.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Mycoplasma bacteriophages L51 and L2 DNA are subject to host cell modification and restriction upon transfection into Acholeplasma laidlawii strains JA1 and K2.
- The L51 genome contains a single MboI restriction site (GATC) that is methylated in K2-grown phage DNA and unmethylated in JA1-grown phage DNA.
Purpose of the Study:
- To elucidate the specific DNA restriction and modification mechanisms employed by Acholeplasma laidlawii strains K2 and JA1.
- To characterize the nature of the restriction systems, particularly focusing on sequence and methylation specificity.
Main Methods:
- Transfection of wild-type and mutant Mycoplasma bacteriophage DNA into Acholeplasma laidlawii strains.
- Analysis of DNA methylation status using restriction endonuclease digestion (MboI).
- Isolation and characterization of restriction-deficient variants.
Main Results:
- Strain K2 restricts DNA containing the GATC sequence, protecting its own DNA via cytosine methylation at this site.
- Strain JA1 restricts DNA containing 5-methylcytosine, irrespective of the surrounding nucleotide sequence, representing a novel restriction activity.
- JA1 modification involves the absence of cytosine methylating activity, and a restriction-deficient variant confirmed specificity for 5-methylcytosine.
Conclusions:
- Acholeplasma laidlawii strain K2 possesses a restriction-modification system targeting the GATC sequence.
- Acholeplasma laidlawii strain JA1 exhibits a newly discovered restriction-modification system specific for 5-methylcytosine, independent of sequence context.
- This study reports the first instance of DNA restriction activity directed against a single methylated base.