Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNAsupercoiling

We report here that the high-temperature sensitivity of a dnaA46 mutant was suppressed by addition of high concentrations of NaCl into the culture med ium. This suppression was also observed with other high-temperature-sensiti ve dnaA mutants, except dnaA167 and dnaA508 mutants, which have mutations i n the N-terminal region of DnaA protein. Since high concentrations of NaCl in the medium increased negative DNA supercoiling in a dnaA46 mutant, we hy pothesized that the increase in DNA supercoiling is involved in the suppres sion of the temperature-sensitivity of the dnaA46 mutant by high concentrat ions of NaCl. This hypothesis was supported by in vitro and in vivo results as follows. A low DNA replication activity of purified DnaA46 protein at h igh temperatures was increased in line with an increase in DNA supercoiling of template DNA. The dnaA46 mutant showed higher sensitivity to nalidixic acid, a DNA-relaxing drug, than did the wild-type cells under the condition s of high temperatures and high concentrations of NaCl.