Acidic phospholipid deficiency results in an inhibition of chromosomal replication and cell-cycle specific arrest in Escherichia coli
Fingland, Nicholas Kyle
Beyond the simplistic function as an environmental barrier, lipids have a variety of functions. The wealth of knowledge available on Escherichia coli (E. coli), and its small subset of lipid species, makes it an ideal organism to study lipid function. In E. coli, acidic phospholipids (aPLs) have been suggested to be uniquely important for chromosomal replication among phospholipids, since growth-arrest caused by depletion of aPLs is rescued by bypassing normal mechanisms for initiating replication.In E. coli coordinated activation and deactivation of DnaA promotes proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC), assuring occurrence once per cell-cycle. In vitro, aPLs reactivate DnaA, and in vivo aPL depletion results in growth-arrest. Growth is restored by the expression of a mutant DnaA, DnaA(L366K), or through oriC-independent initiation, suggesting aPLs participate in DnaA- oriC-dependent replication initiation. Although compelling, a gap remained linking these studies with aPLs influencing the frequency of initiation in vivo. Moreover, aPLs have also been suggested to influence cell division; therefore, their role in the overall cell-cycle was unclear. I hypothesized that aPLs are required for initiation-dependent cell-cycle progression in E. coli. The role of aPLs in cell-cycle progression and DNA replication was assessed in E. coli with inducible control of aPL production using flow cytometry, chromosomal labeling, and quantitative polymerase chain reaction (qPCR).We found when aPLs were depleted, replication was inhibited with a concomitant reduction of chromosomal content and cell mass prior to growth-arrest, showing aPLs are required for normal initiation frequency and that inhibition contributed towards growth-arrest. The observed biosynthetic shutdown was independent of the stringent response, which elicits a similar phenotype during inhibition of fatty acid synthesis. Restoration of aPLs resulted in a resumption of DNA replication prior to division, indicating a cell-cycle-specific growth-arrest had occurred. Independently, each technique showed a deficiency in aPLs prolonged chromosomal replication. Expression of DnaA(L366K) lowers the DNA-to-cell mass ratio regardless of aPL cellular content, suggesting initiation activity of DnaA(L366K) is independent of aPL-reactivation. This work not only demonstrates that aPLs are required for oriC-dependent replication initiation and cell-cycle-specific progression, but likely also DNA elongation.
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