Lipid trafficking to the outer membrane of Gram-negative bacteria

The envelope of Gram-negative bacteria is composed of two distinct lipid membranes: an inner membrane and outer membrane. The outer membrane is an asymmetric bilayer with an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharide. Most of the steps of lipid synthesis occur within the cytoplasmic compartment of the cell. Lipids must then be transported across the inner membrane and delivered to the outer membrane. These topological features combined with the ability to apply the tools of biochemistry and genetics make the Gram-negative envelope a fascinating model for the study of lipid trafficking. In addition, as lipopolysaccharide is essential for growth of most strains and is a potent inducer of the mammalian innate immune response via activation of Toll-like receptors, Gram-negative lipid transport is also a promising target for the development of novel antibacterial and anti-inflammatory compounds. This review focuses on recent developments in our understanding of lipid transport across the inner membrane and to the outer membrane of Gram-negative bacteria. © 2006 Blackwell Publishing Ltd

Density gradient enrichment of Escherichia coli conditional msbA mutants

Insight into the mechanism of lipid transport to the outer membrane of gram-negative bacteria has been hampered by the lack of an effective genetic screen for defective mutants. This work demonstrates an enrichment of conditional mutants defective in lipopolysaccharide export by Ludox density gradient centrifugation and selection for detergent resistance. New temperature-sensitive mutants with lipid export defects were isolated with single missense mutations in msbA. The results demonstrate the power of this approach for the study of lipid export in Escherichia coli. Copyright © 2007, American Society for Microbiology. All Rights Reserved

Escherichia coli YqjA, a member of the conserved DedA/Tvp38 membrane protein family, is a putative osmosensing transporter required for growth at alkaline pH

© 2015, American Society for Microbiology. The ability to persist and grow under alkaline conditions is an important characteristic of many bacteria. In order to survive at alkaline pH, Escherichia coli must maintain a stable cytoplasmic pH of about 7.6. Membrane cation/proton antiporters play a major role in alkaline pH homeostasis by catalyzing active inward proton transport. The DedA/Tvp38 family is a highly conserved membrane protein family of unknown function present in most sequenced genomes. YqjA and YghB are members of the E. coli DedA family with 62% amino acid identity and partially redundant functions. We have shown that E. coli with ΔyqjA and ΔyghB mutations cannot properly maintain the proton motive force (PMF) and is compromised in PMF-dependent drug efflux and other PMF-dependent functions. Furthermore, the functions of YqjA and YghB are dependent upon membrane-embedded acidic amino acids, a hallmark of several families of proton-dependent transporters. Here, we show that the ΔyqjA mutant (but not ΔyghB) cannot grow under alkaline conditions (ranging from pH 8.5 to 9.5), unlike the parent E. coli. Overexpression of yqjA restores growth at alkaline pH, but only when more than ~100mM sodium or potassium is present in the growth medium. Increasing the osmotic pressure by the addition of sucrose enhances the ability of YqjA to support growth under alkaline conditions in the presence of low salt concentrations, consistent with YqjA functioning as an osmosensor. We suggest that YqjA possesses proton-dependent transport activity that is stimulated by osmolarity and that it plays a significant role in the survival of E. coli at alkaline pH

Members of the conserved DedA family are likely membrane transporters and are required for drug resistance in Escherichia coli

Bacterial resistance to antibiotics and biocides is an increasing public health problem. Genes encoding integral membrane proteins belonging to the DedA family are present in most bacterial genomes, including Escherichia coli. An E. coli strain lacking partially redundant DedA family genes yqjA and yghB (strain BC202) displays temperature sensitivity and cell division defects. These phenotypes can be corrected by overexpression of mdfA, anNa+-K+/H+ antiporter of the major facilitator superfamily. We show that BC202 is hypersensitive to several biocides and cationic compounds that are known substrates of several multidrug resistance transporters, including MdfA, EmrE, and AcrB. The introduction of deletions of genes encoding these drug transporters into BC202 results in additional sensitivity. Expression of wild-type yghB or yqjA can restore drug resistance, but this is eliminated upon mutation of two membrane-embedded acidic amino acids (E39 or D51 in either protein). This dependence upon membrane-embedded acidic amino acids is a hallmark of proton-dependent antiporters. Overexpression of mdfA in BC202 or artificially restoring proton motive force (PMF) restores wild-type resistance to substrates of MdfA as well as other drug resistance transporters such as EmrE and AcrAB. These results suggest that YqjA and YghB may be membrane transporters required for PMF-dependent drug efflux in E. coli. Copyright © 2014, American Society for Microbiology. All Rights Reserved

Multiple deletions reveal the essentiality of the DedA membrane protein family in Escherichia coli

The DedA family is a highly conserved, ancient family of membrane proteins with representatives in most sequenced genomes. A characteristic of prokaryotic DedA family genes is extensive gene duplication, with most bacterial genomes carrying two or more homologues. The Escherichia coli genome carries eight DedA genes, each individually nonessential. We previously described an E. coli mutant (BC202; ΔyghB:: kanR, ΔyqjA:: tetR) with in-frame deletions of two DedA genes encoding proteins with 61% amino acid identity. BC202 fails to complete cell division or grow at elevated temperatures. Here, we report that restoration of normal growth and cell division of BC202 is possible by overexpression of a subset of the eight E. coli DedA genes (yabI, yohD, yqjA and yghB) but not others (dedA, ydjX, ydjZ and yqaA), suggesting the existence of two functional groups within the family. We have constructed individual E. coli strains in which all eight DedA genes are deleted in a nonpolar manner, and growth is supported by a single DedA family gene under control of an inducible promoter. Strain BAL801 (with growth supported by cloned dedA) and BAL802 (with growth supported by cloned yqjA) exhibit slow growth that is absolutely dependent upon the presence of the arabinose inducer. Growth in the presence of glucose results in cell death. These results indicate that while not individually essential, the E. coli DedA family proteins are collectively essential. These observations suggest important functions for the E. coli DedA protein family. © 2012 SGM

ATPase activity of the MsbA lipid flippase of Escherichia coli

Escherichia coli MsbA, the proposed inner membrane lipid flippase, is an essential ATP-binding cassette transporter protein with homology to mammalian multidrug resistance proteins. Depletion or loss of function of MsbA results in the accumulation of lipopolysaccharide and phospholipids in the inner membrane of E. coli. MsbA modified with an N-terminal hexahistidine tag was overexpressed, solubilized with a nonionic detergent, and purified by nickel affinity chromatography to ∼95% purity. The ATPase activity of the purified protein was stimulated by phospholipids. When reconstituted into liposomes prepared from E. coli phospholipids, MsbA displayed an apparent Km of 878 μM and a Vmax of 37 nmol/min/mg for ATP hydrolysis in the presence of 10 mM Mg2+. Preincubation of MsbA-containing liposomes with 3-deoxy-D-mannooctulosonic acid (Kdo)2-lipid A increased the ATPase activity 4-5-fold, with half-maximal stimulation seen at 21 μM Kdo2-lipid A. Addition of Kdo2-lipid A increased the Vmax to 154 nmol/min/mg and decreased the Km to 379 μM. Stimulation was only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IVA. MsbA containing the A270T substitution, which renders cells temperature-sensitive for growth and lipid export, displayed ATPase activity similar to that of the wild type protein at 30°C but was significantly reduced at 42°C. These results provide the first in vitro evidence that MsbA is a lipid-activated ATPase and that hexaacylated lipid A is an especially potent activator

Loss of outer membrane proteins without inhibition of lipid export in an Escherichia coli YaeT mutant

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with TV. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 °C. After 30 min at 44 °C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 °C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Cpx-dependent expression of YqjA requires cations at elevated pH

© FEMS 2017. All rights reserved. Under alkaline pH conditions, Escherichia coli must maintain a stable cytoplasmic pH of about 7.6 that is acidic relative to the environment. Bacteria employ various mechanisms to survive alkaline pH; however, membrane cation/H+ antiporters play a primary role by facilitating inward transport of protons. Escherichia coli YqjA belongs to the DedA/Tvp38 membrane protein family and, along with its paralog YghB, is required for growth at 42°C, proper cell division and antibiotic resistance. YqjA is required for viability at alkaline pH, requiring cations sodium or potassium to support growth under these conditions, suggesting it may be a transporter. We measured yqjA expression at different pHs and cation concentrations using a yqjA promoter-lacZ fusion. We found that yqjA promoter activity was highest at alkaline pH. Increased activity of the yqjA promoter required both the transcriptional regulator CpxR, in agreement with previous results, and sodium or potassium salts at alkaline pH. Extracellular cations are also required for activity of cpxP and degP promoters at alkaline pH, suggesting this is a general property of the Cpx regulon. To our knowledge, this is the first demonstration of cation-dependent expression of Cpx-regulated genes at alkaline pH

Multiple envelope stress response pathways are activated in an Escherichia coli strain with mutations in two members of the deda membrane protein family

We have reported that simultaneous deletion of two Escherichia coli genes, yqjA and yghB, encoding related and conserved inner membrane proteins belonging to the DedA protein family results in a number of intriguing phenotypes, including temperature sensitivity at 42°C, altered membrane lipid composition, and cell division defects. We sought to characterize these and other phenotypes in an effort to establish a function for this protein family in E. coli. Here, using reporter assays, we show that the major envelope stress response pathways Cpx, Psp, Bae, and Rcs are activated in strain BC202 (W3110; yqjA yghB) at the permissive growth temperature of 30°C. We previously demonstrated that 10mMMg2+, 400mMNaCl, and overexpression of tatABC are capable of restoring normal growth to BC202 at elevated growth temperatures. Deletion of the cpxR gene from BC202 results in the loss of the ability of these supplements to restore growth at 42°C. Additionally, we report that the membrane potential of BC202 is significantly reduced and that cell division and growth can be restored either by expression of the multidrug transporter MdfA from a multicopy plasmid or by growth at pH 6.0. Together, these results suggest that the DedA family proteins YqjA and YghB are required for general envelope maintenance and homeostasis of the proton motive force under a variety of growth conditions. © 2013, American Society for Microbiology

MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymysin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells