Complementation analysis of temperature-sensitive host specificity mutations in Escherichia coli

A selection procedure was devised for the isolation of temperature-sensitive mutants of Escherichia coli Kl2 unable to restrict foreign DNA. Many of the non-restricting mutants isolated also displayed temperature sensitivity in the modification of DNA. The mutations were shown to map in the hs cluster of genes which determine the host specificity of DNA in E. coli. The kinetics of inactivation of restriction showed that a short exposure to high temperature was sufficient to impair restriction of phage λ DNA and that after shift to low temperature restriction did not return to the wild-type level until a period of growth had occurred. One of the mutants was used as a starting strain from which further mutants were then selected for their inability to host-modify DNA. Many of the mutants thus isolated, in addition to being impaired in modification, were found to be non-restricting at both high and low temperatures. A complementation analysis of the mutants was carried out using an F′ donor strain derived from E. coli B and carrying the host specificity genes hssB+ hsr- hsm+. In all but one of the F′ merodiploids constructed between this F′ and the temperature-sensitive host specificity mutants of E. coli K. the temperature sensitivity of the mutant phenotype was complemented and the merodiploids displayed K- and B -specific restriction and K- and B-specific modification. From these results it is concluded that all of the temperature-sensitive mutants carry mutations in the hsm gene, and are hssK + her+hsmts. In addition it is argued that an hsm-directed polypeptide is required for restriction in addition to polypeptides directed by hssK and hsr. These results are discussed in terms of models based on the interaction of subunits to form oligomeric enzymes.