Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions. Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild-type DnaA did not. The mutations include substitutions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA. Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid- deficient cells rescued by the expression of DnaA having a point mutation in the membrane-binding domain, DnaA(L366K). Flow cytometry revealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta363-367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.