Mutations in DnaA protein suppress the growth arrest of acidic phospholipid-deficient Escherichia coli cells

Cell growth arrests when the concentrations of anionic phospholipids drop b elow a critical level in Escherichia coil, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been intro duced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions. E xpression of DnaA proteins possessing certain small deletions or substitute d amino acids restored growth to cells deficient in acidic phospholipids, w hereas expression of wild-type DnaA did not. The mutations include substitu tions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA. Marker frequency analysi s indicated that initiation of replication occurs at or near oriC in acidic phospholipid-deficient cells rescued by the expression of DnaA having a po int mutation in the membrane-binding domain, DnaA(L366K). Flow cytometry re vealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta 363-367) reduced the frequency with which replication was initia ted and disturbed the synchrony of initiations.