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.

Crosstalk between DnaA Protein, the Initiator of Escherichia coli Chromosomal Replication, and Acidic Phospholipids Present in Bacterial Membranes

“…In prokaryotes, the role of acidic phospholipids also appears to be linked to chromosomal and cell division-related events [35] including the initiation of chromosomal DNA replication [36–39]. In vivo evidence links proper cellular levels of PG and CL with continued cell growth [40,41] and normal chromosomal replication [36–39], in that reduced levels of acidic phospholipids, arising from repressed expression of pgsA , result in arrested-growth and inhibited chromosomal replication in otherwise wild-type E. coli .…”

“…In vivo evidence links proper cellular levels of PG and CL with continued cell growth [40,41] and normal chromosomal replication [36–39], in that reduced levels of acidic phospholipids, arising from repressed expression of pgsA , result in arrested-growth and inhibited chromosomal replication in otherwise wild-type E. coli .…”

“…Acidic phospholipids have the ability to promote the exchange of the tightly bound allosteric effectors ADP and ATP (see sections 3), and studies have shown that acidic phospholipids can inhibit the formation of the replicatively active nucleoprotein complex at the origin of chromosomal replication ( oriC ) in E. coli [49,50] (see section 7). The molar ratios of membrane phospholipids appears to change as cells pass from exponential growth into stationary phase [51,52] and recent work shows that depletion of cellular acidic phospholipids leads to under initiation of replication from oriC during the cell-cycle [39]. …”

ExbB Cytoplasmic Loop Deletions Cause Immediate, Proton Motive Force-Independent Growth Arrest

“…Alternative candidates include inhibition of key cellular processes, such as DNA replication, protein synthesis, cell membrane, and peptidoglycan synthesis. When acidic phospholipids are depleted, DnaA arrests DNA synthesis; however, that depletion requires hours (54). Inhibition of protein synthesis seems unlikely given the very low steady-state levels of the ⌬10 proteins that lead to growth arrest.…”

The initiator protein DnaA, chromosomal origin oriC and their interaction to generate origin recognition complex and pre-replication complex like nucleoprotein structures in Escherichia coli

“…In vitro acidic phospholipids such as phosphotidylglycerol (PG) and cardiolipin (CL) present in a fluid bilayer promote the conversion of inactive ADP-DnaA to replicatively proficient ATP-DnaA 22,23 . In vivo evidence links proper cellular levels of acidic phospholipids such as PG and CL, with continued cell growth 24,25 and normal chromosomal replication in E. coli 26 , whereas reduced levels of acidic phospholipids, result in arrested-growth 24,25 and inhibited chromosomal replication in otherwise wild-type E. coli 26 . High resolution X-ray crystal structures of truncated ADP-DnaA 14 and ATP-DnaA 27 protein (domains III-IV) from thermophilic bacteria Aquifex aeolicus, have provided insight into the conformational differences between the two nucleotide forms of DnaA protein and explain how ATPDnaA protein can accommodate and stabilise DnaA protomer interaction 14,27 .…”