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The Location of a Temperature-sensitive trans-Dominant Mutation and Its Effect on Restriction and Modification in Escherichia coli K12
Abstract
An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4αgt57βgt14 and λvir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage λ carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdX ts +d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the λ phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the X ts +d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of X ts +d mutation was studied. The results of complementation experiments, using F′-merodiploids or plasmid pBR322 with an inserted X ts +d mutation, support the idea that the HsdSts +d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.
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© Society for General Microbiology 1989