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Cloning and structural characterization of the mcrA locus of Escherichia coli
1Genetics Division, National Institute for Medical Research, Mill Hill, London, Great Britain.
Abstract:
Escherichia coli has DNA restriction systems which are able to recognize and attack modified cytosine residues in the DNA of incoming bacteriophages and plasmids. The locus for the McrA/RglA system of modified cytosine restriction was located near the pin gene of the defective element, e14. Hence, loss of the e14 element through abortive induction after UV irradiation caused a permanent loss of McrA restriction activity. e14 DNA encoding McrA restriction was cloned and sequenced to reveal a single open reading frame of 831 bp with a predicted gene product of 31 kDa. Clones expressing the complete open reading frame conferred both McrA and RglA phenotypes; however, a deletion derivative was found which complemented RglA restriction against nonglucosylated T6gt phage but did not complement for McrA restriction of methylated plasmid DNA. Possible explanations for this activity and a comparison with the different organization of the McrB/RglB restriction system are discussed.
Insights
The Escherichia coli McrA restriction system, responsible for degrading modified DNA, was localized to the e14 element. Cloning and sequencing revealed a gene encoding this system, with distinct domains responsible for McrA and RglA activities.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Escherichia coli possesses DNA restriction systems targeting modified cytosine residues in foreign DNA.
- The McrA/RglA system is one such mechanism, recognizing specific DNA modifications.
- The e14 element in E. coli was previously known to be associated with McrA activity.
Purpose of the Study:
- To identify and characterize the genetic locus responsible for the McrA/RglA restriction phenotype.
- To elucidate the molecular basis of McrA and RglA activities.
- To investigate the relationship between the e14 element and McrA restriction.
Main Methods:
- Genetic mapping to locate the McrA/RglA restriction locus near the pin gene on the e14 element.
- Cloning and sequencing of the e14 DNA region encoding McrA restriction.
- Functional analysis of cloned DNA fragments using complementation assays with modified phage and plasmid DNA.
Main Results:
- Loss of the e14 element resulted in permanent loss of McrA restriction activity.
- A single open reading frame (831 bp) encoding a 31 kDa protein was identified on e14.
- Expression of the complete open reading frame conferred both McrA and RglA phenotypes, while a deletion derivative showed differential activity.
Conclusions:
- The e14 element encodes the McrA restriction system.
- The identified gene product likely contains distinct functional domains for McrA and RglA activities.
- Further investigation is needed to fully understand the functional organization and comparison with the McrB/RglB system.