Localized mutagenesis for the isolation of temperature-sensitive mutants of Escherichia coli affected in protein synthesis

Abstract

Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44 degrees, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44 degrees, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44 degrees. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.