SummaryDnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperaturesensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42ЊC (non-permissive temperature) and at 37ЊC (semi-permissive temperature), but not at 28ЊC (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28ЊC), showed a higher level of unsaturation of fatty acids at 28ЊC. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

We report in this paper that the amino acid residues Ile-26 and Leu-40 of the DnaA protein are essential for the DNA replication activity in vitro. Lines of evidence to support this conclusion are as follows. Variants of the DnaA protein containing either an Ile-26-Ser or Leu-40-Ser replacement were unable to support oriC DNA replication in vitro. Though the mutant DnaA proteins retained the capability to bind oriC DNA, they were unable to open the duplex DNA at oriC. Based on these and other results, we conclude that the N-terminal region of the DnaA protein is involved in the oligomerization of this protein, an essential step for the duplex opening activity at oriC.

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.

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.

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.