The Location of a Temperature-sensitive trans-Dominant Mutation and Its Effect on Restriction and Modification in Escherichia coli K12

Institute of Microbiology, Czechoslovak Academy of Sciences, Víde ská 1083, 142 20 Prague 4, Czechoslovakia Institute of Biochemistry and Physiology of Micro-organisms, USSR Academy of Sciences, Puschino on the Oka, Moscow Region, USSR ABSTRACT Summary: An Escherichia coli K12 chromosomal Eco RI- Bam HI 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 T4 gt57βgt 14 and vir. Pvu II 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 Bgl II fragment of phage X carrying the p R promoter was inserted into the Bam HI 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 X phage Bgl II fragment into the Bgl II site within this gene resulted in the disappearance of the trans -dominant effect. When the cloned Bam HI- Eco RI fragment was shortened by Hpa I and Eco RI restriction enzymes, the trans -dominant effect was fully expressed. The results indicate that the X ts +d mutation is located in the hsd S 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 HsdS ts +d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.