Membrane Stress Caused by Unprocessed Outer Membrane Lipoprotein Intermediate Pro-Lpp Affects DnaA and Fis-Dependent Growth.

Digvijay Patil,Dan Xun,Markus Schueritz,Shivani Bansal,Amrita Cheema,Rahul Saxena,Elliott Crooke

doi:10.3389/fmicb.2021.677812

Digvijay Patil, Dan Xun + Show 5 more

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https://doi.org/10.3389/fmicb.2021.677812

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Abstract

In Escherichia coli, repression of phosphatidylglycerol synthase A gene (pgsA) lowers the levels of membrane acidic phospholipids, particularly phosphatidylglycerol (PG), causing growth-arrested phenotype. The interrupted synthesis of PG is known to be associated with concomitant reduction of chromosomal content and cell mass, in addition to accumulation of unprocessed outer membrane lipoprotein intermediate, pro-Lpp, at the inner membrane. However, whether a linkage exists between the two altered-membrane outcomes remains unknown. Previously, it has been shown that pgsA+ cells overexpressing mutant Lpp(C21G) protein have growth defects similar to those caused by the unprocessed pro-Lpp intermediate in cells lacking PG. Here, we found that the ectopic expression of DnaA(L366K) or deletion of fis (encoding Factor for Inversion Stimulation) permits growth of cells that otherwise would be arrested for growth due to accumulated Lpp(C21G). The DnaA(L366K)-mediated restoration of growth occurs by reduced expression of Lpp(C21G) via a σE-dependent small-regulatory RNA (sRNA), MicL-S. In contrast, restoration of growth via fis deletion is only partially dependent on the MicL-S pathway; deletion of fis also rescues Lpp(C21G) growth arrest in cells lacking physiological levels of PG and cardiolipin (CL), independently of MicL-S. Our results suggest a close link between the physiological state of the bacterial cell membrane and DnaA- and Fis-dependent growth.

Highlights

In Escherichia coli, DnaA protein initiates chromosomal DNA replication once, and only once, per cell cycle (Bramhill and Kornberg, 1988; Marszalek and Kaguni, 1994; Baker and Bell, 1998)
We examine the possible role of the σE-MicL/Lpp protective loop in the ability of DnaA(L366K) to facilitate growth of cells carrying mutant Lpp(C21G) or lacking the ability to synthesize the acidic phospholipids
Wondering whether there is a link of some fashion between the two growth-arrest phenotypes led us to assess the capacity of DnaA(L366K) to restore growth in bacterial cells exogenously overproducing Lpp(C21G)

Summary

Introduction

In Escherichia coli, DnaA protein initiates chromosomal DNA replication once, and only once, per cell cycle (Bramhill and Kornberg, 1988; Marszalek and Kaguni, 1994; Baker and Bell, 1998). DnaA shows a high affinity for both ATP (KD = 0.03 μM) and ADP (KD = 0.1 μM) (Sekimizu et al, 1987). Biochemical studies established the role of acidic phospholipids in the in vitro rejuvenation of inactive ADP-DnaA to active ATP-DnaA Limited proteolysis of DnaA in the presence of acidic phospholipids to generate functional fragments revealed a specific region of DnaA involved in membrane interaction (Garner and Crooke, 1996). Cytolocalization studies involving an allelic replacement of the chromosomal copy of dnaA with a gene encoding GFP-DnaA demonstrated a discrete, longitudinal helical arrangement of GFP-DnaA along the cell periphery (Boeneman et al, 2009), supporting that DnaA is located along the inner leaflet of the cytoplasmic membrane

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