Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Abstract

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A 2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/ acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a "sideways sliding" mechanism to explain how a conserved membrane-embedded ␣-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.

Using Spatial Analysis of ANR1 Gene Transcription Rates for Detecting Nitrate Irregularities in Cherry Tomatoes (Solanum lycopersicum var. cerasiforme) Organic Greenhouses

“…The analysis is based on sampling flag leaves which are characterized by rapid changes in molecular expression [18]. In the framework of the analysis, the cells walls of the flag leaves are broken [19], the mRNAs of the carrier proteins are processed [20], and the mRNA amounts of the examined genes is calculated (in our case, those related to the plant nutrition).…”

The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A2 attack

“…LplT promotes energy-independent trans-bilayer migration of each of the major LPL metabolites in Gram-negative bacteria (i.e. lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), and lyso-cardiolipin) from the periplasmic to the cytoplasmic leaflet of the IM (23). Aas is a peripheral membrane protein on the cytoplasmic side of the IM (24), positioned to convert transported LPLs to their respective parent diacyl-PL species (PE or PG) or a triacyl form of cardiolipin (CL) (23).…”

“…lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), and lyso-cardiolipin) from the periplasmic to the cytoplasmic leaflet of the IM (23). Aas is a peripheral membrane protein on the cytoplasmic side of the IM (24), positioned to convert transported LPLs to their respective parent diacyl-PL species (PE or PG) or a triacyl form of cardiolipin (CL) (23). The potentially membrane-disruptive properties of LPLs raised the possibility that the LplT/Aas protein system plays a housekeeping role to minimize accumulation of LPLs generated de novo as byproducts of normal bacterial envelope modification processes, as in the biogenesis of OM lipoproteins (25,26).…”

“…The potentially membrane-disruptive properties of LPLs raised the possibility that the LplT/Aas protein system plays a housekeeping role to minimize accumulation of LPLs generated de novo as byproducts of normal bacterial envelope modification processes, as in the biogenesis of OM lipoproteins (25,26). Deletion of lplT or aas gene, however, resulted in only a modest elevation of LPL in E. coli and no change in bacterial growth or viability (22,23,26). In contrast, OM-depleted spheroplasts generated from E. coli cells that lacked LplT or Aas function exhibited increased sensitivity to venom-derived sPLA 2 (23), suggesting that LplT/Aas provide a "self-defense" mechanism to counteract the membranedisruptive effects of LPLs produced under hoststressed conditions that include activation of sPLA 2 action.…”

“…Deletion of lplT or aas gene, however, resulted in only a modest elevation of LPL in E. coli and no change in bacterial growth or viability (22,23,26). In contrast, OM-depleted spheroplasts generated from E. coli cells that lacked LplT or Aas function exhibited increased sensitivity to venom-derived sPLA 2 (23), suggesting that LplT/Aas provide a "self-defense" mechanism to counteract the membranedisruptive effects of LPLs produced under hoststressed conditions that include activation of sPLA 2 action.…”