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 temperaturesensitivity of the dnaA46 mutant by high concentrations of NaCl.

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 agarose‐gel electrophoresis in the presence of the DNA intercalating agent chloroquine at a low concentration (2 μg mL −1 ). 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 μg mL −1 ). 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).…”

Conserved hydrophobic amino acid residues in the N-terminal region of DnaA protein are involved in DnaA–DnaA interaction

“…Therefore, the activities of DnaA for adenine-nucleotide-binding, acidic phospholipid-binding, and ATPase are important in the regulatory mechanism of DNA replication. Although DnaA also changes DNA supercoiling by sequence-independent DNA binding activity [15][16][17], its role in DNA replication is unknown. Since DnaA has multiple activities, determination of the functional domain (identification of essential amino acid residues) for each activity is important in order to fully understand the function and structure of DnaA.…”