Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli

• Published in: MicrobiologyOpen (Weinheim) Vol. 1; no. 4; pp. 450 - 466
• Main Authors: Fingland, Nicholas, Flåtten, Ingvild, Downey, Christopher D., Fossum‐Raunehaug, Solveig, Skarstad, Kirsten, Crooke, Elliott
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-12-2012
• Subjects: Anionic phospholipids; Cell Cycle Checkpoints - genetics; Cell Cycle Checkpoints - physiology; chromosomal replication; Chromosomes, Bacterial; DNA Replication; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DnaA; E. coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Flow Cytometry; oriC; Original Research; Phospholipids - metabolism; Point Mutation; Polymerase Chain Reaction; Replication Origin - genetics; Replication Origin - physiology
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.46

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.
Depletion of acidic phospholipids in Escherichia coli leads to inhibited chromosomal replication and a decrease in cell mass, followed by growth arrest. Restoration of acidic phospholipid synthesis results 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.