Crosstalk between Prokaryotic Replication Initiation and Acidic Phospholipids

Abstract

The role of cellular membranes in DNA replication has been speculated on for over five decades. Biochemical studies show that Escherichia coli acidic phospholipids influence the chromosomal replication activity of the initiator protein DnaA. In vivo studies also suggest crosstalk between inner membrane acidic phospholipids and replication initiation machinery. However, the manner in which acidic phospholipids affect chromosomal replication is still unclear. Separately, acidic phospholipids are also involved in the maturation of the major outer membrane lipoprotein (Lpp). The depletion of acidic phospholipids adversely affects the maturation of Lpp, which leads to the accumulation of immature intermediates at the inner membrane, inhibiting growth. In this study, we investigate how Lpp(C21G), which accumulates as a growth arresting Lpp intermediate in cells with normal levels of acidic phospholipids, might poison E. coli chromosomal replication. We find that the ectopic expression of DnaA(L366K) or deletion of fis (encoding Factor for Inversion Stimulation) is able to rescue growth in cells with membranes that otherwise would be perturbed by accumulated Lpp(C21G). The DnaA(L366K)-mediated growth rescue occurs by reduced expression of Lpp(C21G) via a σE-dependent small regulator RNA (sRNA), MicL-S.In contrast, growth rescue via fis deletion is only partially dependent on this MicL-S/ Lpp(C21G) pathway; the deletion of fis also rescues Lpp(C21G) growth arrest in cells lacking physiological levels of phosphatidylglycerol (PG) and cardiolipin (CL), independently of MicL-S. Lipidomic analysis of cells unable to synthesize PG and CL and also lacking Fis shows elevated levels of phosphatidic acid, phosphatidylethanolamine, diacylglycerol, and lysophosphatidylethanolamine levels. Our results suggest a close link between the physiological state of the bacterial cell membrane and DnaA- and Fis-dependent growth.