Mutations in the DNA gyrB gene that are temperature sensitive for lambda site-specific recombination, Mu growth, and plasmid maintenance

Abstract

We report the isolation of two mutations in the gyrB gene of Escherichia coli K12 obtained from an initial selection for resistance to coumermycin A1 and a subsequent screening for bacteria that fail to support site-specific recombination of phage lambda, i.e., Him-. These two mutations have a temperature-sensitive Him- phenotype, supporting site-specific recombination efficiently at low temperature, but inefficiently at high temperatures. Like other Him mutants, the gyrB-him mutants fail to plate phage Mu; again this defect is observed only at high temperatures. Additional thermally sensitive characteristics have also been observed; growth of lambda as well as maintenance of the plasmids pBR322 and F' gal are reduced at high temperature. Restriction of foreign DNA imposed by a P1 prophage is also reduced in these mutants. The temperature-sensitive phenotypic characteristics imposed by both the gyrB-him-230(Ts) and gyrB-him-231(Ts) mutations correlate with in vitro studies that show decreased gyrase activity, especially at higher temperatures, and in vivo studies showing reduced supercoiling of lambda DNA in the mutants at high temperature.