title: Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli.
abstract: In Escherichia coli, coordinated activation and deactivation of DnaA allows for proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC) and assures initiation occurs once per cell cycle. In vitro, acidic phospholipids reactivate DnaA, and in vivo depletion of acidic phospholipids, results in growth arrest. Growth can be restored by the expression of a mutant form of DnaA, DnaA(L366K), or by oriC-independent DNA synthesis, suggesting acidic phospholipids are required for DnaA- and oriC-dependent replication. We observe here that when acidic phospholipids were depleted, replication was inhibited with a concomitant reduction of chromosomal content and cell mass prior to growth arrest. This global shutdown of biosynthetic activity was independent of the stringent response. Restoration of acidic phospholipid synthesis resulted in a resumption of DNA replication prior to restored growth, indicating a possible cell-cycle-specific growth arrest had occurred with the earlier loss of acidic phospholipids. Flow cytometry, thymidine uptake, and quantitative polymerase chain reaction data suggest that a deficiency in acidic phospholipids prolonged the time required to replicate the chromosome. We also observed that regardless of the cellular content of acidic phospholipids, expression of mutant DnaA(L366K) altered the DNA content-to-cell mass ratio.
authors.affiliation: Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC 20057, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Fingland
authors.forename: Ingvild
authors.initials: I
authors.lastname: Flåtten
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Downey
authors.forename: Solveig
authors.initials: S
authors.lastname: Fossum-Raunehaug
authors.forename: Kirsten
authors.initials: K
authors.lastname: Skarstad
authors.forename: Elliott
authors.initials: E
authors.lastname: Crooke
mesh_terms: D059447:Cell Cycle Checkpoints / Q000235:genetics / Q000502:physiology; D002876:Chromosomes, Bacterial; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry; D010743:Phospholipids / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018741:Replication Origin / Q000235:genetics / Q000502:physiology
chemical_list: D004269:DNA, Bacterial; D010743:Phospholipids