Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling

Abstract

We report here that the high-temperature sensitivity of a dnaA46 mutant was suppressed by addition of high concentrations of NaCl into the culture medium. This suppression was also observed with other high-temperature-sensitive dnaA mutants, except dnaA167 and dnaA508 mutants, which have mutations in the N-terminal region of DnaA protein. Since high concentrations of NaCl in the medium increased negative DNA supercoiling in a dnaA46 mutant, we hypothesized that the increase in DNA supercoiling is involved in the suppression of the temperature-sensitivity of the dnaA46 mutant by high concentrations 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 high 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 conditions of high temperatures and high concentrations of NaCl.

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism

“…In line with this proposal, as a stimulating effect of high negative superhelicity on the DnaA46 activity was shown in vitro [59]. Such mechanism of suppression was also proposed in the case of decreased temperature sensitivity of the dnaA46 mutant grown in a medium containing 0.51M salt, which also results in an increased negative superhelicity of the chromosomal DNA [59]. Addition of nalidixic acid, an inhibitor of the DNA gyrase which introduces negative supercoils at the expense of ATP, was shown to reduce growth of the dnaA46 strain at the restrictive temperature [59].…”

“…The lack of topA or seqA increases the overall negative supercoiling of chromosomal DNA which could assist a defective DnaA protein in DNA unwinding and allow for initiation of DNA replication. In line with this proposal, as a stimulating effect of high negative superhelicity on the DnaA46 activity was shown in vitro [59]. Such mechanism of suppression was also proposed in the case of decreased temperature sensitivity of the dnaA46 mutant grown in a medium containing 0.51M salt, which also results in an increased negative superhelicity of the chromosomal DNA [59].…”

“…Such mechanism of suppression was also proposed in the case of decreased temperature sensitivity of the dnaA46 mutant grown in a medium containing 0.51M salt, which also results in an increased negative superhelicity of the chromosomal DNA [59]. Addition of nalidixic acid, an inhibitor of the DNA gyrase which introduces negative supercoils at the expense of ATP, was shown to reduce growth of the dnaA46 strain at the restrictive temperature [59]. Due to involvement of the pta and ackA genes in cellular energetics, we hypothesized that the suppression of DnaA46 thermolability by deletion of these genes could result from an increase in the negative DNA supercoiling.…”

Heterologous expression of a histone-like protein fromStreptococcus intermediusinEscherichia colialters the nucleoid structure and inhibits the growth ofE. coli

“…The extent of DNA supercoiling of these plasmids was examined using agarosegel electrophoresis in the presence of the DNA intercalating agent chloroquine at a low concentration (2 mg mL À1 ). In the presence of this low level of chloroquine, negative DNA supercoiling become less negative, and migrated slower, and positive DNA supercoiling become more positive and migrated faster (Kondo et al, 2000). As shown in Fig.…”

“…The samples were analyzed using electrophoresis in 1% agarose gel in the absence or presence of chloroquine (1.2 or 2 mg mL À1 ). Electrophoresis was carried out in TBE buffer [0.089 M Tris/borate (pH 8.0) and 2 mM EDTA] at 60 V for 16 h, as described previously (Kondo et al, 2000).…”