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Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria

  • Jim E. Horne
    Affiliations
    Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
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  • David J. Brockwell
    Affiliations
    Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
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  • Sheena E. Radford
    Correspondence
    For correspondence: Sheena E. Radford
    Affiliations
    Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom
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Open AccessPublished:June 04, 2020DOI:https://doi.org/10.1074/jbc.REV120.011473
      β-Barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonization and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP-folding landscape and discuss the factors that guide folding in vitro and in vivo. We particularly focus on the composition, architecture, and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo. Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance it is an attractive target. To bring down this vital OMP-supported barrier to antibiotics, we must first understand how bacterial cells build it.
      Proteins that span lipid bilayers come in two types, either α-helical or β-barrels. Whereas the cytosolic inner membranes (IMs) of bacteria and the plasma membrane of eukaryotes are comprised only of α-helical membrane proteins, β-barrel outer membrane proteins (OMPs) are found exclusively in the outer membranes (OMs) of diderm bacteria as well as in bacterially derived eukaryotic organelles, such as mitochondria and chloroplasts. The “OMPome” (the complement of OMPs encoded for by a genome) of Escherichia coli consists of a large number of proteins ranging in barrel size from 8 to 26 β-strands and includes monomers, small assemblies (dimers, trimers etc.), and oligomeric structures that can form up to 60-stranded pores (Fig. 1). Some OMPs comprise only the integral membrane β-barrel structure, whereas others have soluble domains in the periplasm or on the extracellular surface of the OM. Some OMPs have low copy number or can be absent in the OM under “standard” growth conditions (e.g. the E. coli porin OmpN) (
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      Figure thumbnail gr1
      Figure 1Structures of transmembrane proteins found in the OM of E. coli K-12 MG1655. A list of all known and predicted transmembrane proteins in the OM of E. coli K-12 strain MG1655 was manually curated, creating the “OMP-ome.” The Protein Data Bank was then searched for solved structures of these proteins or close homologues. Where no high-resolution solved 3D structures were available, homology models were generated using the I-TASSER server (RRID:SCR_014627) (
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      • Zhang Y.
      I-TASSER server: new development for protein structure and function predictions.
      ). For two proteins, NfrA (the N4 bacteriophage receptor), and FlgH (the flagellar L-ring protein), no homology models could be generated. Predictions for YaiO, YcgI, YdbH, and YhjY generated deformed or broken barrels (possibly due to a lack of homology to existing structures), but their predictions are displayed to indicate their approximate structure. Extracellular domains of autotransporters have only been shown where accurate models could be built or crystal structures were available. OMPs are grouped here by the number of β-strands and then by protein family. The non-OMP subunits of the BAM complex are labeled below the central BamA subunit. Protein names are in red if they represent pseudogenes (inactivated by mutation in this strain) and blue if they are encoded on the F plasmid. The color of the box surrounding the protein names represents the number of β-strands in the β-barrel. Light Orange, 8; red, 10; light blue, 12; violet, 14; pink, 16; purple, 18; light green, 22; dark green, 24; brown, 26; black, oligomeric split β-barrel; gray, α-helical transmembrane region. Structures were aligned with each other by their β-barrel domains and rendered individually in PyMOL 2.X (Schrödinger, LLC). A list of the proteins with their associated family and PDB code can be found in Table S1.
      Table 1Summary of BAM-dependent and BAM-independent OMPs in the OM of different bacteria
      OMP(s)FamilyNo. of β-strandsOrganismReference
      BAM catalysis–involved
          OmpA, OmpX, OmpT, OmPLA, OmpGVaried small barrels8–14E. coli
      These studies were all performed in vitro.
      Refs.
      • Burgess N.K.
      • Dao T.P.
      • Stanley A.M.
      • Fleming K.G.
      β-Barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro.
      • Gessmann D.
      • Chung Y.H.
      • Danoff E.J.
      • Plummer A.M.
      • Sandlin C.W.
      • Zaccai N.R.
      • Fleming K.G.
      Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA.
      ,
      • Iadanza M.G.
      • Higgins A.J.
      • Schiffrin B.
      • Calabrese A.N.
      • Brockwell D.J.
      • Ashcroft A.E.
      • Radford S.E.
      • Ranson N.A.
      Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM.
      ,
      • Hagan C.L.
      • Kim S.
      • Kahne D.
      Reconstitution of outer membrane protein assembly from purified components.
      ,
      • Hagan C.L.
      • Westwood D.B.
      • Kahne D.
      Bam lipoproteins assemble BamA in vitro.
      ,
      • Patel G.J.
      • Kleinschmidt J.H.
      The lipid bilayer-inserted membrane protein BamA of Escherichia coli facilitates insertion and folding of outer membrane protein A from its complex with Skp.
      • Hussain S.
      • Bernstein H.D.
      The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition.
          VariousAutotransporters12E. coliRefs.
      • Hussain S.
      • Bernstein H.D.
      The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition.
      • Doyle M.T.
      • Bernstein H.D.
      Bacterial outer membrane proteins assemble via asymmetric interactions with the BamA β-barrel.
      ,
      • Ieva R.
      • Bernstein H.D.
      Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane.
      ,
      • Sauri A.
      • Soprova Z.
      • Wickström D.
      • de Gier J.-W.
      • Van der Schors R.C.
      • Smit A.B.
      • Jong W.S.P.
      • Luirink J.
      The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease.
      ,
      • Bodelón G.
      • Marín E.
      • Fernández L.A.
      Role of periplasmic chaperones and BamA (YaeT/Omp85) in folding and secretion of intimin from enteropathogenic Escherichia coli strains.
      ,
      • Norell D.
      • Heuck A.
      • Tran-Thi T.-A.
      • Götzke H.
      • Jacob-Dubuisson F.
      • Clausen T.
      • Daley D.O.
      • Braun V.
      • Müller M.
      • Fan E.
      Versatile in vitro system to study translocation and functional integration of bacterial outer membrane proteins.
      • Roman-Hernandez G.
      • Peterson J.H.
      • Bernstein H.D.
      Reconstitution of bacterial autotransporter assembly using purified components.
          OprDOuter membrane porin18P. aeruginosaRef.
      • Klein K.
      • Sonnabend M.S.
      • Frank L.
      • Leibiger K.
      • Franz-Wachtel M.
      • Macek B.
      • Trunk T.
      • Leo J.C.
      • Autenrieth I.B.
      • Schütz M.
      • Bohn E.
      Deprivation of the periplasmic chaperone SurA reduces virulence and restores antibiotic susceptibility of multidrug-resistant Pseudomonas aeruginosa.
          LamBSugar porin18E. coliRefs.
      • Ruiz N.
      • Falcone B.
      • Kahne D.
      • Silhavy T.J.
      Chemical conditionality: a genetic strategy to probe organelle assembly.
      ,
      • Wu T.
      • Malinverni J.
      • Ruiz N.
      • Kim S.
      • Silhavy T.J.
      • Kahne D.
      Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli.
          VariousTonB-dependent receptors22Caulobacter crescentusRefs.
      • Rassam P.
      • Copeland N.A.
      • Birkholz O.
      • Tóth C.
      • Chavent M.
      • Duncan A.L.
      • Cross S.J.
      • Housden N.G.
      • Kaminska R.
      • Seger U.
      • Quinn D.M.
      • Garrod T.J.
      • Sansom M.S.P.
      • Piehler J.
      • Baumann C.G.
      • et al.
      Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.
      ,
      • Ryan K.R.
      • Taylor J.A.
      • Bowers L.M.
      The BAM complex subunit BamE (SmpA) is required for membrane integrity, stalk growth and normal levels of outer membrane β-barrel proteins in Caulobacter crescentus.
          TolCOuter membrane factor3 × 4 (12)E. coliRef.
      • Werner J.
      • Misra R.
      YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent outer membrane proteins of Escherichia coli.
          FimD
      These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      Fimbrial usher24E. coliRef.
      • Palomino C.
      • Marín E.
      • Fernández L.Á.
      The fimbrial usher FimD follows the SurA-BamB pathway for its assembly in the outer membrane of Escherichia coli.
          LptDLPS assembly26E. coliRefs.
      • Lee J.
      • Xue M.
      • Wzorek J.S.
      • Wu T.
      • Grabowicz M.
      • Gronenberg L.S.
      • Sutterlin H.A.
      • Davis R.M.
      • Ruiz N.
      • Silhavy T.J.
      • Kahne D.E.
      Characterization of a stalled complex on the β-barrel assembly machine.
      ,
      • Ruiz N.
      • Falcone B.
      • Kahne D.
      • Silhavy T.J.
      Chemical conditionality: a genetic strategy to probe organelle assembly.
      ,
      • Lee J.
      • Sutterlin H.A.
      • Wzorek J.S.
      • Mandler M.D.
      • Hagan C.L.
      • Grabowicz M.
      • Tomasek D.
      • May M.D.
      • Hart E.M.
      • Silhavy T.J.
      • Kahne D.
      Substrate binding to BamD triggers a conformational change in BamA to control membrane insertion.
      • Lee J.
      • Tomasek D.
      • Santos T.M.
      • May M.D.
      • Meuskens I.
      • Kahne D.
      Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA.
      • Chimalakonda G.
      • Ruiz N.
      • Chng S.-S.
      • Garner R.A.
      • Kahne D.
      • Silhavy T.J.
      Lipoprotein LptE is required for the assembly of LptD by the β-barrel assembly machine in the outer membrane of Escherichia coli.
          PilQ
      These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      Type IV pilus secretin14 × 4 (56)Neisseria meningitidisRefs.
      • Voulhoux R.
      • Bos M.P.
      • Geurtsen J.
      • Mols M.
      • Tommassen J.
      Role of a highly conserved bacterial protein in outer membrane protein assembly.
      ,
      • Volokhina E.B.
      • Beckers F.
      • Tommassen J.
      • Bos M.P.
      The β-barrel outer membrane protein assembly complex of Neisseria meningitidis.
      BAM-independent folding
          PulD
      These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      , XcpQ
      These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      , GspD
      These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      T2SS secretin15 × 4 (60)Klebsiella oxytoca, P. aeruginosa, E. coliRefs.
      • Collin S.
      • Guilvout I.
      • Chami M.
      • Pugsley A.P.
      YaeT-independent multimerization and outer membrane association of secretin PulD.
      • Hoang H.H.
      • Nickerson N.N.
      • Lee V.T.
      • Kazimirova A.
      • Chami M.
      • Pugsley A.P.
      • Lory S.
      Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.
      ,
      • Huysmans G.H.M.
      • Guilvout I.
      • Chami M.
      • Nickerson N.N.
      • Pugsley A.P.
      Lipids assist the membrane insertion of a BAM-independent outer membrane protein.
      • Dunstan R.A.
      • Hay I.D.
      • Wilksch J.J.
      • Schittenhelm R.B.
      • Purcell A.W.
      • Clark J.
      • Costin A.
      • Ramm G.
      • Strugnell R.A.
      • Lithgow T.
      Assembly of the secretion pores GspD, Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA.
          pIV
      These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      Phage secretin?Phage f1Refs.
      • Kazmierczak B.I.
      • Mielke D.L.
      • Russel M.
      • Model P.
      pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer.
      ,
      • Nickerson N.N.
      • Abby S.S.
      • Rocha E.P.C.
      • Chami M.
      • Pugsley A.P.
      A single amino acid substitution changes the self-assembly status of a Type IV piliation secretin.
          CsgG
      These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      ,
      Also contains an N-terminal lipid anchor.
      Curli secretion9 × 4 (36)E. coliRef.
      • Dunstan R.A.
      • Hay I.D.
      • Wilksch J.J.
      • Schittenhelm R.B.
      • Purcell A.W.
      • Clark J.
      • Costin A.
      • Ramm G.
      • Strugnell R.A.
      • Lithgow T.
      Assembly of the secretion pores GspD, Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA.
      a These studies were all performed in vitro.
      b These proteins are often assembled as part of larger protein machineries or export/import pathways and may also include their own targeting and assembly factors.
      c Also contains an N-terminal lipid anchor.
      This review aims to provide a holistic view of our current understanding of the process of OMP biogenesis, including 1) the composition and physical and chemical properties of the OM in vivo; 2) current knowledge of the determinants of OMP folding through in vitro studies; and 3) how OMP folding depends on parameters such as the lipid composition, physical environment, and the presence/absence of BAM. Although information is drawn from different organisms, we focus on OMPs and the OM of E. coli because of the position of this bacterium as the de facto model organism for studying these processes.

      Another brick in the wall: Building the OM

      To understand OMP folding and biogenesis, it is first important to review our current understanding of the composition and architecture of the environment in which this process takes place: the complex and crowded bacterial OM.

      Lipid types found in the OM

      The OM of Gram-negative bacteria is unusual in that it is a highly asymmetric lipid bilayer, comprising an inner leaflet enriched in phospholipids and an outer leaflet containing lipopolysaccharide (LPS) (Fig. 2) (
      • Henderson J.C.
      • Zimmerman S.M.
      • Crofts A.A.
      • Boll J.M.
      • Kuhns L.G.
      • Herrera C.M.
      • Trent M.S.
      The power of asymmetry: architecture and assembly of the Gram-negative outer membrane lipid bilayer.
      ). This is in contrast to the IM in Gram-negative and Gram-positive bacteria, which mostly contain phospholipids mixed between both leaflets. In Gram-negative bacteria, phospholipids in the OM generally have the canonical structure expected of a phospholipid, containing two hydrophobic acyl chains, with different length and degree of saturation. These are connected via an ester linkage to a headgroup that can be zwitterionic, or positively or negatively charged (Fig. 2). Cardiolipin (CL) is also found in the OM and has the appearance of a phospholipid dimer (Fig. 2). LPS is a much bulkier molecule, made up of a variable number of acyl chains (between 4 and 8, depending on the species) (
      • Kim S.
      • Patel D.S.
      • Park S.
      • Slusky J.
      • Klauda J.B.
      • Widmalm G.
      • Im W.
      Bilayer properties of lipid A from various Gram-negative bacteria.
      ). The acyl chain can vary in length both within each molecule and between species (e.g. C10:0–C14:0 in Bordetella pertussis, C12:0–C14:0 in E. coli, C14:0–C21:0 in Chlamydia trachomatis, and C14:0–C28:0 in Agrobacterium tumefaciens) (
      • Kosma P.
      Chlamydial lipopolysaccharide.
      ,
      • Raetz C.R.H.
      • Whitfield C.
      Lipopolysaccharide endotoxins.
      ,
      • Silipo A.
      • De Castro C.
      • Lanzetta R.
      • Molinaro A.
      • Parrilli M.
      Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction.
      ,
      • Albitar-Nehme S.
      • Basheer S.M.
      • Njamkepo E.
      • Brisson J.-R.
      • Guiso N.
      • Caroff M.
      Comparison of lipopolysaccharide structures of Bordetella pertussis clinical isolates from pre- and post-vaccine era.
      ). Furthermore, the acyl chains of LPS are usually shorter than those of the average phospholipid and are almost always saturated (
      • Zähringer U.
      • Lindner B.
      • Rietschel E.T.
      Molecular structure of lipid A, the endotoxic center of bacterial lipopolysaccharides.
      ) (Fig. 2). The acyl chains in LPS are connected to a disaccharide diphosphate headgroup, which in turn is connected to a conserved “core” region made up of chained sugar groups, and then finally a highly variable sugar-containing O-antigen region (Fig. 2) (
      • Miller S.I.
      • Ernst R.K.
      • Bader M.W.
      LPS, TLR4 and infectious disease diversity.
      ,
      • Steimle A.
      • Autenrieth I.B.
      • Frick J.-S.
      Structure and function: lipid A modifications in commensals and pathogens.
      ). Together, these sugar regions convey a large net negative charge to the outer surface of bacteria (
      • Steimle A.
      • Autenrieth I.B.
      • Frick J.-S.
      Structure and function: lipid A modifications in commensals and pathogens.
      ).
      Figure thumbnail gr2
      Figure 2Common lipid types found in bacterial outer membranes and/or used in in vitro studies of OMP folding. Top, schematic of the generic structure of phospholipids and LPS. Bacterial lipids can be conceptualized as having two “domains”: a polar headgroup and a hydrophobic acyl tail region. In phospholipids, the acyl tails are connected by an ester linkage to a phosphate group and a variable headgroup region. PC and PE are zwitterionic, whereas PG carries a net negative charge. Note that PC lipids are not commonly found in bacterial membranes but are often used for OMP folding-studies in vitro due to their net neutral charge and propensity to form bilayers. Cardiolipin comprises two acyl tail regions connected by phosphate groups via a glycerol linkage and carries a net double negative charge. LPS is found exclusively in the OM of Gram-negative bacteria and varies considerably between species in both the number and length of acyl tails in the lipid A region and the sugar composition in the polysaccharide region (shown below). Here the most common structure of lipid A-Kdo2 for E. coli K-12 LPS is shown in full. Bottom, the architecture of a generic LPS is shown. The lipid A and core region are consistent with LPS found in E. coli K-12; however, this strain does not naturally produce an O-antigen, whereas many environmental and clinical strains do. Strains lacking the O-antigen region are said to contain “rough” LPS, and this can further be divided into subtypes dependent on truncations in the core region. The most extreme of these that is still viable at 37 °C under laboratory growth conditions is “deep rough” LPS, containing only lipid A-Kdo2. The O-antigen region is highly variable within species and can contain as many as 40 glycan repeats. Kdo, keto-deoxyoctulosonate; LDmanHep, l-glycero-d-manno-heptose; Glc, glucose; Gal, galactose; P, phosphate group; PEtN, phosphorylethanolamine.

      Essentiality of specific lipids

      E. coli is remarkably tolerant of modifications in its lipid biosynthesis pathways, with viable strains including bacteria in which synthesis of phosphatidylethanolamine (PE) (
      • DeChavigny A.
      • Heacock P.N.
      • Dowhan W.
      Sequence and inactivation of the pss gene of Escherichia coli: phosphatidylethanolamine may not be essential for cell viability.
      ,
      • Rowlett V.W.
      • Mallampalli V.K.P.S.
      • Karlstaedt A.
      • Dowhan W.
      • Taegtmeyer H.
      • Margolin W.
      • Vitrac H.
      Impact of membrane phospholipid alterations in Escherichia coli on cellular function and bacterial stress adaptation.
      ), phosphatidylglycerol (PG) and CL (
      • Kikuchi S.
      • Shibuya I.
      • Matsumoto K.
      Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin.
      ,
      • Matsumoto K.
      Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids.
      ), or CL alone (
      • Rowlett V.W.
      • Mallampalli V.K.P.S.
      • Karlstaedt A.
      • Dowhan W.
      • Taegtmeyer H.
      • Margolin W.
      • Vitrac H.
      Impact of membrane phospholipid alterations in Escherichia coli on cellular function and bacterial stress adaptation.
      ,
      • Tan B.K.
      • Bogdanov M.
      • Zhao J.
      • Dowhan W.
      • Raetz C.R.H.
      • Guan Z.
      Discovery of a cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates.
      ) is eliminated (see Fig. 2 for the structure of common lipid types); phosphatidylcholine (PC) synthesis is induced synthetically (E. coli lacks PC in its IM or OM, although this phospholipid is present in some bacterial membranes) (
      • Chen F.
      • Zhao Q.
      • Cai X.
      • Lv L.
      • Lin W.
      • Yu X.
      • Li C.
      • Li Y.
      • Xiong M.
      • Wang X.-G.
      Phosphatidylcholine in membrane of Escherichia coli changes bacterial antigenicity.
      ,
      • Geiger O.
      • López-Lara I.M.
      • Sohlenkamp C.
      Phosphatidylcholine biosynthesis and function in bacteria.
      ); gluco- or galactolipids are utilized (
      • Wikström M.
      • Kelly A.A.
      • Georgiev A.
      • Eriksson H.M.
      • Klement M.R.
      • Bogdanov M.
      • Dowhan W.
      • Wieslander A.
      Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function.
      ); or even archaeal lipids are incorporated into the membrane (
      • Caforio A.
      • Siliakus M.F.
      • Exterkate M.
      • Jain S.
      • Jumde V.R.
      • Andringa R.L.H.
      • Kengen S.W.M.
      • Minnaard A.J.
      • Driessen A.J.M.
      • van der Oost J.
      Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane.
      ). Although these strains are able to survive under laboratory conditions, their growth and virulence are affected (in some cases severely), stress responses are up-regulated, and defects of varying acuteness are seen in the structure and permeability of the cell envelope (
      • Rowlett V.W.
      • Mallampalli V.K.P.S.
      • Karlstaedt A.
      • Dowhan W.
      • Taegtmeyer H.
      • Margolin W.
      • Vitrac H.
      Impact of membrane phospholipid alterations in Escherichia coli on cellular function and bacterial stress adaptation.
      ,
      • Rossi R.M.
      • Yum L.
      • Agaisse H.
      • Payne S.M.
      Cardiolipin synthesis and outer membrane localization are required for Shigella flexneri virulence.
      ). The effect of such changes in lipid composition in the OM on OMP biogenesis has not been investigated in detail for all of these strains. However, in PE-deficient strains, OmpF folding is impaired in a titratable manner, with complete lack of PE reducing folding yields from ∼100% in WT to <15% (
      • Rowlett V.W.
      • Mallampalli V.K.P.S.
      • Karlstaedt A.
      • Dowhan W.
      • Taegtmeyer H.
      • Margolin W.
      • Vitrac H.
      Impact of membrane phospholipid alterations in Escherichia coli on cellular function and bacterial stress adaptation.
      ). Lack of CL causes less severe defects but still reduces OmpF folding yields to ∼25% (
      • Rowlett V.W.
      • Mallampalli V.K.P.S.
      • Karlstaedt A.
      • Dowhan W.
      • Taegtmeyer H.
      • Margolin W.
      • Vitrac H.
      Impact of membrane phospholipid alterations in Escherichia coli on cellular function and bacterial stress adaptation.
      ) and has also been shown to cause mislocalization of the OMP IcsA, which normally resides at the cell pole in Shigella flexneri (
      • Rossi R.M.
      • Yum L.
      • Agaisse H.
      • Payne S.M.
      Cardiolipin synthesis and outer membrane localization are required for Shigella flexneri virulence.
      ). Interestingly, in E. coli, lack of CL causes severe distention/detachment of the OM from the IM at the cell poles, and CL and PG have been observed to accumulate at cell poles and division sites (
      • Oliver P.M.
      • Crooks J.A.
      • Leidl M.
      • Yoon E.J.
      • Saghatelian A.
      • Weibel D.B.
      Localization of anionic phospholipids in Escherichia coli cells.
      ), suggesting a role for CL in maintaining cell shape and integrity at sites of negative curvature (
      • Koppelman C.M.
      • Den Blaauwen T.
      • Duursma M.C.
      • Heeren R.M.
      • Nanninga N.
      Escherichia coli minicell membranes are enriched in cardiolipin.
      ). PG null only mutants have not been described, as CL utilizes PG for its biosynthesis. However, the creation of viable strains absent in PG synthesis also requires mutations in the major E. coli OM lipoprotein Lpp (Braun's lipoprotein), suggesting that lack of PG causes lethality primarily though lethal accumulation of Lpp at the IM (
      • Matsumoto K.
      Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids.
      ,
      • Suzuki M.
      • Hara H.
      • Matsumoto K.
      Envelope disorder of Escherichia coli cells lacking phosphatidylglycerol.
      ). The first step in lipoprotein maturation after translocation into the periplasm involves the transfer of a diacyl moiety from PG (
      • Nakayama H.
      • Kurokawa K.
      • Lee B.L.
      Lipoproteins in bacteria: structures and biosynthetic pathways.
      ), and its absence presumably stalls maturation at this point. However, the OM lipoproteins LptE and BamD are essential in E. coli, so the viability of these lpp mutant strains suggests that alternate maturation pathways or sources of diacylglycerol must exist (
      • Grabowicz M.
      • Silhavy T.J.
      Redefining the essential trafficking pathway for outer membrane lipoproteins.
      ,
      • Grabowicz M.
      Lipoprotein transport: greasing the machines of outer membrane biogenesis: re-examining lipoprotein transport mechanisms among diverse Gram-negative bacteria while exploring new discoveries and questions.
      ). As BamA and LptD are essential OMPs, the fact that bacteria can still grow and divide in these strains (albeit poorly) suggests that other lipids can moonlight for the loss of PE, PG, or CL or that there is no absolute need for a particular phospholipid type as a minimum requirement for OMP biogenesis in E. coli. Nonetheless, the severe defects observed in these strains show that outside the laboratory, all of these components are needed for bacterial viability. This highlights that whereas a stable bilayer is the minimum requirement to fold an OMP, to understand how OMP biogenesis occurs in biologically relevant environments, consideration of the complexity of the OM environment is crucial.

      Organization of lipid types within the OM

      The E. coli OM contains PE, PG, CL, and LPS (Fig. 2). These lipid types are divided asymmetrically between the inner and outer leaflets of the OM, with the outer leaflet containing almost 100% LPS and the inner leaflet containing ∼80% PE, ∼15% PG, and ∼5% CL (Figure 2, Figure 3). By contrast, the IM also contains ∼5% CL with a lower ratio of PE/PG of ∼70%/25% (
      • McMorran L.M.
      • Brockwell D.J.
      • Radford S.E.
      Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro in vivo: what have we learned to date?.
      ). Although it is physically possible for phospholipids to flip from the inner leaflet to the outer leaflet, this process is likely to be intrinsically slow (occurring on the order of hours or longer in vesicles in vitro) (
      • Bai J.
      • Pagano R.E.
      Measurement of spontaneous transfer and transbilayer movement of BODIPY-labeled lipids in lipid vesicles.
      ,
      • Nakano M.
      • Fukuda M.
      • Kudo T.
      • Matsuzaki N.
      • Azuma T.
      • Sekine K.
      • Endo H.
      • Handa T.
      Flip-flop of phospholipids in vesicles: kinetic analysis with time-resolved small-angle neutron scattering.
      ). However, this process may be accelerated under conditions of OM/bilayer stress, such as exposure to antimicrobial peptides or detergents, in strains with truncated LPS (see Fig. 2), or after loss of OMPs (
      • Nikaido H.
      Molecular basis of bacterial outer membrane permeability revisited.
      ,
      • Vaara M.
      • Vaara T.
      Polycations as outer membrane-disorganizing agents.
      ,
      • Paul S.
      • Chaudhuri K.
      • Chatterjee A.N.
      • Das J.
      Presence of exposed phospholipids in the outer membrane of Vibrio cholerae.
      ,
      • Contreras F.-X.
      • Sánchez-Magraner L.
      • Alonso A.
      • Goñi F.M.
      Transbilayer (flip-flop) lipid motion and lipid scrambling in membranes.
      ). This process is therefore associated with increased permeability of the OM. Regardless of such events, the asymmetry of the OM is actively maintained in E. coli by the maintenance of lipid asymmetry (Mla) system, which removes errant phospholipids specifically from the outer leaflet of the OM to maintain its barrier function (
      • Malinverni J.C.
      • Silhavy T.J.
      An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane.
      ,
      • Abellón-Ruiz J.
      • Kaptan S.S.
      • Baslé A.
      • Claudi B.
      • Bumann D.
      • Kleinekathöfer U.
      • van den Berg B.
      Structural basis for maintenance of bacterial outer membrane lipid asymmetry.
      ,
      • Powers M.J.
      • Trent M.S.
      Intermembrane transport: glycerophospholipid homeostasis of the Gram-negative cell envelope.
      ).
      Figure thumbnail gr3
      Figure 3Model depicting the structural organization of the E. coli OM. A schematic displays the degree of crowding in the OM. A, view of an imagined OM showing the dense packing of different size OMPs in monomers, dimers, and trimers interspersed with LPS in the outer leaflet (top) and phospholipids in the inner leaflet (bottom). Phospholipids are represented as dark gray circles with a diameter proportional to the headgroup size of PE/PG and LPS as light gray circles with a diameter proportional to the size of lipid A. Different OMPs are represented as idealized circles with diameters proportional to their number of strands. Blue outline, abundant 8-stranded; orange outline, rare 8-stranded; black, 16-stranded porin trimers; red, other 16-stranded; yellow, 22-stranded; green, 26-stranded. The overall LPR in this schematic is ∼9:1, with ∼2 LPS and ∼7 phospholipids per OMP, consistent with estimates for the LPR of the E. coli OM. B, left, high-resolution AFM image of OM extracts from Roseobacter dentrificans imaged from the periplasmic side showing a dense lattice of porin trimers. Right, atomic model of the packing of porin trimers derived from the AFM data. Reproduced with permission from Jarosławski et al. (
      • Jarosławski S.
      • Duquesne K.
      • Sturgis J.N.
      • Scheuring S.
      High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans.
      ). This research was originally published in Molecular Microbiology. Jarosławski, S., Duquesne, K., Sturgis, J. N., and Scheuring, S. High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans. Molecular Microbiology 2009; 74:1211–1222. © Wiley–Blackwell. C, view of an imagined OM with the same LPR as in A but assuming a more extreme clustering of most OMPs. Only the inner leaflet is shown. Despite having the same LPR values, the buried surface area of the clustered OMPs frees up more lipid to form larger bulk lipid domains.

      The lipid acyl chain composition is diverse

      The acyl chain composition of lipids in the OM of E. coli is more variable than that of their headgroups. The acyl chain composition of the OM depends on the growth conditions, with acyl chains varying in length from C12 to C18, as well as in the degree of saturation or the presence of cyclopropyl modifications (Fig. 2) (
      • Ingram L.O.
      Changes in lipid composition of Escherichia coli resulting from growth with organic solvents and with food additives.
      ,
      • McGarrity J.T.
      • Armstrong J.B.
      The effect of temperature and other growth conditions on the fatty acid composition of Escherichia coli.
      ,
      • Ingram L.O.
      Regulation of fatty acid composition in Escherichia coli: a proposed common mechanism for changes induced by ethanol, chaotropic agents, and a reduction of growth temperature.
      ,
      • Arneborg N.
      • Salskov-Iversen A.
      • Mathiasen T.
      The effect of growth rate and other growth conditions on the lipid composition of Escherichia coli.
      ,
      • Gidden J.
      • Denson J.
      • Liyanage R.
      • Ivey D.M.
      • Lay J.O.
      Lipid compositions in Escherichia coli Bacillus subtilis during growth as determined by MALDI-TOF and TOF/TOF mass spectrometry.
      ,
      • Jeucken A.
      • Molenaar M.R.
      • van de Lest C.H.A.
      • Jansen J.W.A.
      • Helms J.B.
      • Brouwers J.F.
      A comprehensive functional characterization of Escherichia coli lipid genes.
      ). Lipidomics has provided insights into the range of acyl groups found in E. coli membranes. Early experiments in E. coli K1062 reported the presence of C12:0, C14:0, C16:0, C16:1, C18:1, and cyclo-C19:0 acyl chains in phospholipids from the IM and OM (
      • Overath P.
      • Brenner M.
      • Gulik-Krzywicki T.
      • Shechter E.
      • Letellier L.
      Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli.
      ). Examining total lipid content in E. coli K-12 strain LM3118 grown at 37 °C and harvested in stationary phase showed that the acyl chains of PE and PG lipids were comprised primarily of C12:0, C14:0, C16:0, C16:1, C18:0, and C18:1 (with C16:0 being about 3 times more abundant than the other acyl chains), with a lesser contribution from C15:0, cyclo-C17:0, and cyclo-C19:0 (
      • Oursel D.
      • Loutelier-Bourhis C.
      • Orange N.
      • Chevalier S.
      • Norris V.
      • Lange C.M.
      Identification and relative quantification of fatty acids in Escherichia coli membranes by gas chromatography/mass spectrometry.
      ,
      • Oursel D.
      • Loutelier-Bourhis C.
      • Orange N.
      • Chevalier S.
      • Norris V.
      • Lange C.M.
      Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization.
      ). These acyl chains are combined to form a large variety of phospholipid types, most containing at least one unsaturated acyl bond or cyclo-propyl group, although diC16:0PE, diC16:0PG, C16:0C14:0PG, C16:0C12:0PE, diC14:0PE, and diC12:0PE lipids were also observed. Despite cyclo-propyl acyl chain–containing lipids being relatively understudied, the most common lipid species detected under these conditions was C16:0/cyclo-C17:0 (Fig. 2). Cyclo-propyl–containing lipids are produced in large quantities in stationary phase cultures from the conversion of double bonds in unsaturated chains to cyclo-propyl groups (
      • Wang A.‐Y.
      • Cronan J.E.
      The growth phase-dependent synthesis of cyclopropane fatty acids in Escherichia coli is the result of an RpoS(KatF)-dependent promoter plus enzyme instability.
      ,
      • Grogan D.W.
      • Cronan J.E.
      Cyclopropane ring formation in membrane lipids of bacteria.
      ). Although the physiological functions of these modifications are unclear, they appear to be related to protection of the bacteria against a variety of adverse environmental conditions (
      • Grogan D.W.
      • Cronan J.E.
      Cyclopropane ring formation in membrane lipids of bacteria.
      ), including acid shock (
      • Chang Y.Y.
      • Cronan J.E.
      Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli.
      ,
      • Kim B.H.
      • Kim S.
      • Kim H.G.
      • Lee J.
      • Lee I.S.
      • Park Y.K.
      The formation of cyclopropane fatty acids in Salmonella enterica serovar Typhimurium.
      ), osmotic shock (
      • Asakura H.
      • Ekawa T.
      • Sugimoto N.
      • Momose Y.
      • Kawamoto K.
      • Makino S.-I.
      • Igimi S.
      • Yamamoto S.
      Membrane topology of Salmonella invasion protein SipB confers osmotolerance.
      ), high alcohol concentrations (
      • Grandvalet C.
      • Assad-García J.S.
      • Chu-Ky S.
      • Tollot M.
      • Guzzo J.
      • Gresti J.
      • Tourdot-Maréchal R.
      Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation.
      ), or high temperature (
      • Annous B.A.
      • Kozempel M.F.
      • Kurantz M.J.
      Changes in membrane fatty acid composition of Pediococcus sp. strain NRRL B-2354 in response to growth conditions and its effect on thermal resistance.
      ). Furthermore, OMP folding in E. coli occurs primarily during exponential growth (
      • Rassam P.
      • Copeland N.A.
      • Birkholz O.
      • Tóth C.
      • Chavent M.
      • Duncan A.L.
      • Cross S.J.
      • Housden N.G.
      • Kaminska R.
      • Seger U.
      • Quinn D.M.
      • Garrod T.J.
      • Sansom M.S.P.
      • Piehler J.
      • Baumann C.G.
      • et al.
      Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.
      ,
      • Ursell T.S.
      • Trepagnier E.H.
      • Huang K.C.
      • Theriot J.A.
      Analysis of surface protein expression reveals the growth pattern of the Gram-negative outer membrane.
      ), while expression is down-regulated (
      • Johansen J.
      • Rasmussen A.A.
      • Overgaard M.
      • Valentin-Hansen P.
      Conserved small non-coding RNAs that belong to the sigmaE regulon: role in down-regulation of outer membrane proteins.
      ,
      • Guillier M.
      • Gottesman S.
      • Storz G.
      Modulating the outer membrane with small RNAs.
      ) and OMPs are lost from the OM (
      • Allen R.J.
      • Scott G.K.
      Biosynthesis and turnover of outer-membrane proteins in Escherichia coli ML308-225.
      ) when bacteria enter stationary phase. Thus, the relevance of this lipid modification for OMP biogenesis may be minor under favorable growth conditions.
      Few studies have examined whether significant differences or biases exist between the acyl chain composition of phospholipids in the IM and those of the OM of Gram-negative bacteria. Some have reported an enrichment of shorter acyl chains (C12:0 and C14:0) (
      • Overath P.
      • Brenner M.
      • Gulik-Krzywicki T.
      • Shechter E.
      • Letellier L.
      Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli.
      ), saturated fatty acids (
      • Lugtenberg E.J.J.
      • Peters R.
      Distribution of lipids in cytoplasmic and outer membranes of Escherichia coli K12.
      ), lyso-PE lipids (
      • White D.A.
      • Lennarz W.J.
      • Schnaitman C.A.
      Distribution of lipids in the wall and cytoplasmic membrane subfractions of the cell envelope of Escherichia coli.
      ), and C16:0 acyl chains in the OM and a depletion of polyunsaturated acyl chains (
      • Ishinaga M.
      • Kanamoto R.
      • Kito M.
      Distribution of phospholipid molecular species in outer and cytoplasmic membrane of Escherichia coli.
      ). These biases, however, generally vary with growth conditions, and it is unclear to what extent this simply reflects the presence of the lipid A component of the outer leaflet. Regulatory systems that alter the acyl chain composition of lipids specific to the OM are known for LPS, including enzymes that alter the acyl chains attached to lipid A to modulate the endotoxicity of this lipid type during growth in a host (
      • Henderson J.C.
      • Zimmerman S.M.
      • Crofts A.A.
      • Boll J.M.
      • Kuhns L.G.
      • Herrera C.M.
      • Trent M.S.
      The power of asymmetry: architecture and assembly of the Gram-negative outer membrane lipid bilayer.
      ). Modulation of the acyl chain content of lipid A has also been shown to occur in E. coli when under selective pressure from an external insult by the addition of a bactericidal BamA-specific antibody. This suggests a direct link between modulation of lipid content and a selective pressure to efficiently fold OMPs (
      • Storek K.M.
      • Auerbach M.R.
      • Shi H.
      • Garcia N.K.
      • Sun D.
      • Nickerson N.N.
      • Vij R.
      • Lin Z.
      • Chiang N.
      • Schneider K.
      • Wecksler A.T.
      • Skippington E.
      • Nakamura G.
      • Seshasayee D.
      • Koerber J.T.
      • et al.
      Monoclonal antibody targeting the β-barrel assembly machine of Escherichia coli is bactericidal.
      ). However, it is not clear whether this change in lipid A reflects a need to aid the function of an essential BAM client (e.g. LptD), is related to conformational changes in BamA, or is simply a response to a defective permeability barrier.
      This diversity of acyl chain types gives E. coli a wide range of lipids with which it can tailor the biophysical properties of its membranes both globally and locally. This variety may allow it to deal with local minor deformations of the membrane, due to either random thermal fluctuations or the presence of membrane-bound or embedded proteins, as acyl chains can diffuse laterally and occupy the most energetically favorable position, dependent on the match between their own physicochemical properties (length and saturation) and those of the membrane environment.

      A crowded environment

      Although the familiar fluid-mosaic model of membranes found in most textbooks depicts a biological membrane with just a few proteins floating in a “sea” of lipid, the OM is markedly different, containing instead a much higher fraction of protein by weight, with lipid/protein ratios (LPRs) (w/w) estimated to be between 0.14 and 0.36 (
      • Overath P.
      • Brenner M.
      • Gulik-Krzywicki T.
      • Shechter E.
      • Letellier L.
      Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli.
      ,
      • Jarosławski S.
      • Duquesne K.
      • Sturgis J.N.
      • Scheuring S.
      High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans.
      ), corresponding to only 2–4 LPS and 4–10 phospholipid molecules per OMP (
      • Lessen H.J.
      • Fleming P.J.
      • Fleming K.G.
      • Sodt A.J.
      Building blocks of the outer membrane: calculating a general elastic energy model for β-barrel membrane proteins.
      ). Estimates based on biochemical studies suggest that as much as 50% of the surface area of the OM may be occupied by OMPs (
      • Lugtenberg B.
      • Van Alphen L.
      Molecular architecture and functioning of the outer membrane of Escherichia coli and other Gram-negative bacteria.
      ), whereas AFM studies (
      • Jarosławski S.
      • Duquesne K.
      • Sturgis J.N.
      • Scheuring S.
      High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans.
      ) (Fig. 3B), extrapolation from the copy numbers of OMPs measured by proteomics (
      • Li G.-W.
      • Burkhardt D.
      • Gross C.
      • Weissman J.S.
      Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources.
      ,
      • Soufi B.
      • Krug K.
      • Harst A.
      • Macek B.
      Characterization of the E. coli proteome and its modifications during growth and ethanol stress.
      ), and the above measurements of the LPR suggest that this value may be even higher. For example, a copy number of 100,000 for OmpA would imply that ∼6–20% of the surface area of E. coli (dependent on the size of the bacterium) would be occupied by this protein alone. Hence, the OM could be considered more like a protein-rich layer solubilized in a relatively small amount of lipid (Fig. 3, A and B). Despite the low LPR of the OM, the diffusion rates of OMPs in the OM of E. coli are similar to those of inner membrane proteins (IMPs) but are, on average, slower (diffusion coefficients of 0.006–0.15 μm2/s for OMPs versus 0.001–0.4 μm2/s for IMPs) (
      • Rassam P.
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      • Kaminska R.
      • Seger U.
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      • Garrod T.J.
      • Sansom M.S.P.
      • Piehler J.
      • Baumann C.G.
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      Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.
      ,
      • Oddershede L.
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      • Grego S.
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      The motion of a single molecule, the λ-receptor, in the bacterial outer membrane.
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      • Kumar M.
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      Mobility of cytoplasmic, membrane, and DNA-binding proteins in Escherichia coli.
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      • Ritchie K.
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      Single-molecule imaging in live bacteria cells.
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      • Oh D.
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      Dynamics of the serine chemoreceptor in the Escherichia coli inner membrane: a high-speed single-molecule tracking study.
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      • Oswald F.
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      • Lill H.
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      MreB-dependent organization of the E. coli cytoplasmic membrane controls membrane protein diffusion.
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      • Gibbs K.A.
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      Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB.
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      • Spector J.
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      • Lill Y.
      • Sharma O.
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      Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex.
      ,
      • Rothenberg E.
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      Single-virus tracking reveals a spatial receptor-dependent search mechanism.
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      Protein-protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins.
      ,
      • Deich J.
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      Visualization of the movement of single histidine kinase molecules in live Caulobacter cells.
      ,
      • Mullineaux C.W.
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      • Ray N.
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      Diffusion of green fluorescent protein in three cell environments in Escherichia coli.
      ,
      • Leake M.C.
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      • Bai F.
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      Stoichiometry and turnover in single, functioning membrane protein complexes.
      ,
      • Leake M.C.
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      Variable stoichiometry of the TatA component of the twin-arginine protein transport system observed by in vivo single-molecule imaging.
      ) (Fig. 4A). For comparison, the length elongation rate of E. coli alone is ∼0.006 μm/s (
      • Szczepaniak J.
      • Holmes P.
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      The lipoprotein Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism.
      ), whereas the diffusion coefficients of LPS in the OM of Salmonella typhimurium are ∼0.00005 and 0.02 μm2 s−1 (for O-antigen–containing and truncated “deep rough” LPS, respectively (Fig. 2)) (
      • Mühlradt P.F.
      • Menzel J.
      • Golecki J.R.
      • Speth V.
      Lateral mobility and surface density of lipopolysaccharide in the outer membrane of Salmonella typhimurium.
      ,
      • Schindler M.
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      Lateral diffusion of lipopolysaccharide in the outer membrane of Salmonella typhimurium.
      ), lipid probes in the IM of E. coli ∼0.8-1.5 μm2/s (
      • Oswald F.
      • Varadarajan A.
      • Lill H.
      • Peterman E.J.G.
      • Bollen Y.J.M.
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      ,
      • Nenninger A.
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      • Robson A.
      • Lenn T.
      • Xue Q.
      • Leake M.C.
      • Mullineaux C.W.
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      • Mika J.T.
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      • Brooks N.J.
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      • Kuimova M.K.
      Measuring the viscosity of the Escherichia coli plasma membrane using molecular rotors.
      ), and the periplasm, cytoplasm, and buffer ∼3, 0.4-9, and ∼87 μm2/s, respectively (
      • Kumar M.
      • Mommer M.S.
      • Sourjik V.
      Mobility of cytoplasmic, membrane, and DNA-binding proteins in Escherichia coli.
      ,
      • Mullineaux C.W.
      • Nenninger A.
      • Ray N.
      • Robinson C.
      Diffusion of green fluorescent protein in three cell environments in Escherichia coli.
      ,
      • Elowitz M.B.
      • Surette M.G.
      • Wolf P.E.
      • Stock J.B.
      • Leibler S.
      Protein mobility in the cytoplasm of Escherichia coli.
      ,
      • Cluzel P.
      • Surette M.
      • Leibler S.
      An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells.
      ,
      • Konopka M.C.
      • Shkel I.A.
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      Crowding and confinement effects on protein diffusion in vivo.
      ,
      • Potma E.O.
      • de Boeij W.P.
      • Bosgraaf L.
      • Roelofs J.
      • van Haastert P.J.
      • Wiersma D.A.
      Reduced protein diffusion rate by cytoskeleton in vegetative and polarized dictyostelium cells.
      ,
      • Terry B.R.
      • Matthews E.K.
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      Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy.
      ) (Fig. 4B). What particularly distinguishes OMPs from IMPs is their restricted diffusion areas, with diffusion being confined within clusters in the OM, compared with free diffusion of most IMPs in the IM (
      • Rassam P.
      • Copeland N.A.
      • Birkholz O.
      • Tóth C.
      • Chavent M.
      • Duncan A.L.
      • Cross S.J.
      • Housden N.G.
      • Kaminska R.
      • Seger U.
      • Quinn D.M.
      • Garrod T.J.
      • Sansom M.S.P.
      • Piehler J.
      • Baumann C.G.
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      Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.
      ,
      • Kleanthous C.
      • Rassam P.
      • Baumann C.G.
      Protein-protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins.
      ,
      • Gunasinghe S.D.
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      • Lithgow T.
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      ,
      • Lill Y.
      • Jordan L.D.
      • Smallwood C.R.
      • Newton S.M.
      • Lill M.A.
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      ). These observations can be explained by the propensities of OMPs to form clusters (
      • Rassam P.
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      • Birkholz O.
      • Tóth C.
      • Chavent M.
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      Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.
      ,
      • Ursell T.S.
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      • Huang K.C.
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      Analysis of surface protein expression reveals the growth pattern of the Gram-negative outer membrane.
      ,
      • Jarosławski S.
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      • Gunasinghe S.D.
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      • Lill Y.
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      • Smit J.
      • Nikaido H.
      Outer membrane of Gram-negative bacteria. XVIII. Electron microscopic studies on porin insertion sites and growth of cell surface of Salmonella typhimurium.
      ) and/or to interact strongly with LPS or components of the cell envelope (
      • Arunmanee W.
      • Pathania M.
      • Solovyova A.S.
      • Le Brun A.P.
      • Ridley H.
      • Baslé A.
      • van den Berg B.
      • Lakey J.H.
      Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis.
      ,
      • Verhoeven G.S.
      • Dogterom M.
      • den Blaauwen T.
      Absence of long-range diffusion of OmpA in E. coli is not caused by its peptidoglycan binding domain.
      ). OMPs that have a lower tendency to cluster and/or interact with cell envelope components less strongly may exhibit higher diffusion coefficients but will ultimately become “corralled” within OMP-LPS domains. On this point, an abundance of clustered OMPs with low mobility may make the OM locally rigid. Indeed, molecular dynamics (MD) simulations have shown that membranes containing 8–12-stranded OMPs are much stiffer than membranes containing only DMPC (diC14:0PC) (
      • Lessen H.J.
      • Fleming P.J.
      • Fleming K.G.
      • Sodt A.J.
      Building blocks of the outer membrane: calculating a general elastic energy model for β-barrel membrane proteins.
      ). However, simulations investigating larger length scales have shown that crowding a bilayer with some OMPs (i.e. BtuB), but not others (i.e. OmpF), can reduce the global bending rigidity of a POPE (C16:0C18:1PE)/POPC (C16:0C18:1PC) membrane (
      • Fowler P.W.
      • Hélie J.
      • Duncan A.
      • Chavent M.
      • Koldsø H.
      • Sansom M.S.P.
      Membrane stiffness is modified by integral membrane proteins.
      )—an effect that would explain how a cell with a protein-rich OM could still maintain its shape. An interesting alternative possibility to explain the potentially incompatible concepts of OMP folding and a protein-dense, lipid-poor OM would be to consider the OM as an inhomogeneous mixture of protein and lipid (Fig. 3C). The view of OMP monomers or trimers, well-solubilized with lipid, would leave little bulk lipid available for nascent OMPs to fold. However, by clustering OMPs together into regions resembling two-dimensional crystals (i.e. forming regions highly enriched with OMPs and little to no lipid—a local LPR closer to 1:1 (mol/mol)), sufficient lipid-rich regions would be created to enable OMP folding and insertion (Fig. 3, B and C). Regardless of which model is correct, this locally stiff, crowded, and confined environment, with a relative paucity of free lipid, poses a challenging environment into which OMPs must fold.
      Figure thumbnail gr4
      Figure 4Comparison of physical properties of bacterial membranes. A, box plots showing the range of diffusion coefficients reported for OMPs and IMPs (see “A crowded environment”). Boxes show interquartile range calculated by the Tukey method with the median indicated as a boldface horizontal line. Whiskers show the minimum and maximum values. B, comparison of the diffusion coefficients of membrane proteins with other components of bacteria. Whiskers are only shown for components that have three or more values reported in the literature. All values are reported from in vivo studies. LPS, diffusion of LPS molecules in S. typhimurium. Lipid, diffusion rate of a fluorescent lipid reporter probe in E. coli membranes. Peri, diffusion of soluble protein in the E. coli periplasm. Cyto, diffusion of soluble protein in the E. coli cytoplasm. C, viscosities of different membrane environments as measured by the use of fluorescent BODIPY C10 lipid reporter probes. E. coli data are from Mika et al. (
      • Mika J.T.
      • Thompson A.J.
      • Dent M.R.
      • Brooks N.J.
      • Michiels J.
      • Hofkens J.
      • Kuimova M.K.
      Measuring the viscosity of the Escherichia coli plasma membrane using molecular rotors.
      ), and synthetic phospholipid data are from Wu et al. (
      • Wu Y.
      • Stefl M.
      • Olzyńska A.
      • Hof M.
      • Yahioglu G.
      • Yip P.
      • Casey D.R.
      • Ces O.
      • Humpolíčková J.
      • Kuimova M.K.
      Molecular rheometry: direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging.
      ). BODIPY C10 specifically incorporates into the IM of E. coli, and removal of the OM minimally affects the measured viscosity. Synthetic phospholipid 200 nm LUVs were comprised of DLPC, DMPC, POPC, or DOPC.

      More than a mix of lipid types

      The physical properties of lipid membranes

      Lipid bilayers can be characterized by a number of physical, mechanical, and chemical parameters, including stored curvature elastic stress (lateral pressure), melting temperature, the bulk lipid phase, the presence of lipid rafts, membrane viscosity, and headgroup charge (
      • Heberle F.A.
      • Feigenson G.W.
      Phase separation in lipid membranes.
      ,
      • Bigay J.
      • Antonny B.
      Curvature, lipid packing, and electrostatics of membrane organelles: defining cellular territories in determining specificity.
      ,
      • Budin I.
      • de Rond T.
      • Chen Y.
      • Chan L.J.G.
      • Petzold C.J.
      • Keasling J.D.
      Viscous control of cellular respiration by membrane lipid composition.
      ) (Fig. 5). Many of these properties are interrelated and can be modulated by altering the acyl chain composition and/or the phospholipid headgroup and by altering the relative amounts of phospholipid, CL, and LPS (
      • Jackson M.B.
      • Cronan J.E.
      An estimate of the minimum amount of fluid lipid required for the growth of Escherichia coli.
      ,
      • Janoff A.S.
      • Haug A.
      • McGroarty E.J.
      Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.
      ,
      • Nakayama H.
      • Mitsui T.
      • Nishihara M.
      • Kito M.
      Relation between growth temperature of E. coli and phase transition temperatures of its cytoplasmic and outer membranes.
      ,
      • Janoff A.S.
      • Gupte S.
      • McGroarty E.J.
      Correlation between temperature range of growth and structural transitions in membranes and lipids of Escherichia coli K12.
      ,
      • Souzu H.
      Escherichia coli B membrane stability related to cell growth phase. Measurement of temperature dependent physical state change of the membrane over a wide range.
      ). Stored curvature elastic stress makes membranes more rigid and less elastic in terms of their ability to deform or bend. This property can be introduced by the presence of nonbilayer-forming lipids in otherwise bilayer-forming membranes (Fig. 5). For example, PE lipids create negative curvature, whereas PG and PC lipids have zero or low spontaneous curvature, which allows them to readily form bilayers (
      • van den Brink-van der Laan E.
      • Killian J.A.
      • de Kruijff B.
      Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.
      ). Doping bilayers containing PC or PG with PE or CL generates a tension in packing of the different lipid types, creating a crowding, or pressure, near the center of the bilayer where each leaflet meets (Fig. 5), which is further altered by the length of the acyl chains (with shorter chains reducing this elastic stress) (
      • van den Brink-van der Laan E.
      • Killian J.A.
      • de Kruijff B.
      Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.
      ).
      Figure thumbnail gr5
      Figure 5Physical and mechanical properties of a lipid bilayer. A, top, the phase of a lipid bilayer depends on the temperature, with the lipids being in an ordered (gel) phase below the Tm and in a (liquid) disordered phase above the Tm. At the transition temperature, frustration between packing of regions of gel and liquid phase causes defects to occur at these boundaries. Bottom, a typical differential scanning calorimetry curve illustrating the thermal response of a DMPC (diC14:0PC) bilayer with the regions of each phase colored as above. B, the hydrophobic thickness of a membrane depends on the lipid acyl chain length. However, when an OMP becomes embedded in a lipid bilayer, the membrane responds by trying to “match” the hydrophobic thickness of the bilayer to that of the protein to minimize the energetic penalty of exposing polar lipid headgroups to a hydrophobic OMP surface or hydrophobic acyl tails to polar OMP loops. C, mixtures of lipids can separate, forming “rafts” or domains dependent on the physical conditions and lipid type. CL has a high propensity for negative curvature and has been shown to be enriched at cell poles and division sites where the membrane constricts. CL has also been observed to bind to membrane protein complexes such as the BAM complex (
      • Chorev D.S.
      • Baker L.A.
      • Wu D.
      • Beilsten-Edmands V.
      • Rouse S.L.
      • Zeev-Ben-Mordehai T.
      • Jiko C.
      • Samsudin F.
      • Gerle C.
      • Khalid S.
      • Stewart A.G.
      • Matthews S.J.
      • Grünewald K.
      • Robinson C.V.
      Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry.
      ) and cluster under patches of LPS in MD simulations (
      • Shearer J.
      • Khalid S.
      Communication between the leaflets of asymmetric membranes revealed from coarse-grain molecular dynamics simulations.
      ,
      • Shearer J.
      • Jefferies D.
      • Khalid S.
      Outer membrane proteins OmpA, FhuA, OmpF, EstA, BtuB, and OmpX have unique lipopolysaccharide fingerprints.
      ), suggesting that it may help stabilize bilayer packing defects (which might be induced by LPS) and stabilize regions of large hydrophobic mismatch (e.g. around embedded proteins such as BamA). D, schematic describing stored curvature elastic stress and how this depends on lipid type. Adapted from Booth and Curnow (
      • Booth P.J.
      • Curnow P.
      Folding scene investigation: membrane proteins.
      ). This research was originally published in Current Opinion in Structural Biology. Booth, P. J., and Curnow, P. Folding scene investigation: membrane proteins. Current Opinion in Structural Biology 2009; 19:8–13. © Elsevier. Attractive and repulsive interactions driven by the packing of lipids create a pressure differential along the normal of the membrane that must be overcome to deform or alter lipid packing. Incorporation of lipids that have a tendency toward negative curvature (due to the relative size of the headgroup versus the acyl chain (e.g. PE lipids)) into a bilayer formed from lipids with a neutral or low curvature tendency (e.g. PC lipids) generates a stress force within the bilayer due to the opposing tendencies for bilayer formation of these lipids.
      The lipid phase of a membrane is also dependent on its lipid composition and on the temperature (Fig. 5). Bilayers exist primarily in one of two major states, a solid “gel” phase in which the acyl chains are tightly packed and the mobility of lipid molecules is low, and a “liquid” phase, where lipid mobility is higher (
      • Kranenburg M.
      • Smit B.
      Phase behavior of model lipid bilayers.
      ,
      • Brown D.A.
      • London E.
      Structure and origin of ordered lipid domains in biological membranes.
      ,
      • M'Baye G.
      • Mély Y.
      • Duportail G.
      • Klymchenko A.S.
      Liquid ordered and gel phases of lipid bilayers: fluorescent probes reveal close fluidity but different hydration.
      ). Furthermore, analogous to the familiar phase change of ice to water, lipids in a gel-phase membrane can “melt” to become the liquid phase at a temperature characteristic of the particular lipid type—called the transition temperature, Tm. Lipid mixtures can also adopt a “liquid-ordered” phase (with the classical pure liquid phase described as “liquid-disordered”). This liquid-ordered phase contains lipids that are highly mobile but have well-ordered acyl chains and is often associated with the formation of lipid rafts in cholesterol-containing membranes in eukaryotes (
      • Quinn P.J.
      • Wolf C.
      The liquid-ordered phase in membranes.
      ,
      • Mouritsen O.G.
      The liquid-ordered state comes of age.
      ,
      • Sodt A.J.
      • Sandar M.L.
      • Gawrisch K.
      • Pastor R.W.
      • Lyman E.
      The molecular structure of the liquid-ordered phase of lipid bilayers.
      ). Although sterol lipids are rare in bacteria, CL may play a similar role in increasing lipid order, and there is evidence that CL can participate in the formation of rafts/domains in membranes in vitro and in vivo (Fig. 5) (although it is likely this mechanism is distinct from that of cholesterol) (
      • Boscia A.L.
      • Treece B.W.
      • Mohammadyani D.
      • Klein-Seetharaman J.
      • Braun A.R.
      • Wassenaar T.A.
      • Klösgen B.
      • Tristram-Nagle S.
      X-ray structure, thermodynamics, elastic properties and MD simulations of cardiolipin/dimyristoylphosphatidylcholine mixed membranes.
      ,
      • Bramkamp M.
      • Lopez D.
      Exploring the existence of lipid rafts in bacteria.
      ). In silico, CL has been observed to form clusters under patches of LPS in simulations of bacterial OMs in a role that may compensate for packing defects in the outer leaflet of the OM (
      • Shearer J.
      • Khalid S.
      Communication between the leaflets of asymmetric membranes revealed from coarse-grain molecular dynamics simulations.
      ,
      • Shearer J.
      • Jefferies D.
      • Khalid S.
      Outer membrane proteins OmpA, FhuA, OmpF, EstA, BtuB, and OmpX have unique lipopolysaccharide fingerprints.
      ).

      The physical properties of native lipid extracts

      The OM differs from the IM in its highly asymmetric structure, large fraction of proteins by weight, permeability to small molecules (<600 Da), and lack of energization across it (
      • Vergalli J.
      • Bodrenko I.V.
      • Masi M.
      • Moynié L.
      • Acosta-Gutiérrez S.
      • Naismith J.H.
      • Davin-Regli A.
      • Ceccarelli M.
      • van den Berg B.
      • Winterhalter M.
      • Pagès J.-M.
      Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria.
      ,
      • Henderson J.C.
      • Zimmerman S.M.
      • Crofts A.A.
      • Boll J.M.
      • Kuhns L.G.
      • Herrera C.M.
      • Trent M.S.
      The power of asymmetry: architecture and assembly of the Gram-negative outer membrane lipid bilayer.
      ,
      • Overath P.
      • Brenner M.
      • Gulik-Krzywicki T.
      • Shechter E.
      • Letellier L.
      Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli.
      ,
      • Jarosławski S.
      • Duquesne K.
      • Sturgis J.N.
      • Scheuring S.
      High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans.
      ,
      • Thanassi D.G.
      • Stathopoulos C.
      • Karkal A.
      • Li H.
      Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (review).
      ). However, relatively little is known about how these unique features of the OM affect its mechanical properties and how this differs from the IM. E. coli is known to alter the lipid content of its membranes, particularly the length and degree of saturation of acyl chains, in response to changes in growth temperature. This process, termed “homeoviscous adaptation” (
      • Sinensky M.
      Homeoviscous adaptation—a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli.
      ), suggests that bacteria actively maintain their membranes at a constant level of “fluidity,” or in a particular phase, that enables them to adjust to different environments. For example, whereas total lipid extract from E. coli K-12 W3110 showed approximately the same headgroup content (with a minor monotonic increase in CL and decrease in PG) when grown at 30, 37, 42, or 45 °C (
      • Velázquez J.B.
      • Fernández M.S.
      GPS, the slope of Laurdan generalized polarization spectra, in the study of phospholipid lateral organization and Escherichia coli lipid phases.
      ), the ratio of saturated over unsaturated acyl chains increased with temperature. This suggests a change to a more “rigid” mixture of phospholipids at higher growth temperatures to balance the increased fluidity caused by the input of thermal energy.
      Membrane lipid properties can be probed using fluorescent reporter dyes that partition into membranes (either globally or into specific lipid phases) and alter their excitation or emission properties according to the local lipid environment. Hence, these dyes can be used as reporters of membrane viscosity, degree of hydration, phase, and mobility (
      • Demchenko A.P.
      • Mély Y.
      • Duportail G.
      • Klymchenko A.S.
      Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes.
      ). The fluorophore laurdan partitions into membranes through its acyl tail, whereas its naphthalene-based headgroup resides in the interfacial region of bilayers, where its fluorescence excitation and emission spectra are sensitive to the degree of hydration of the bilayer, allowing it to report on the phase and order of lipids in a bilayer (
      • Leung S.S.W.
      • Brewer J.
      • Bagatolli L.A.
      • Thewalt J.L.
      Measuring molecular order for lipid membrane phase studies: linear relationship between Laurdan generalized polarization and deuterium NMR order parameter.
      ). Using laurdan, a Tm of E. coli total lipid extract of <14 °C was determined for bacteria grown at 30 °C or 37 °C, whereas the Tm was higher (Tm ∼20-22 °C) for bacteria grown at 42 °C and elevated again (Tm ∼27 °C) for bacteria grown at 45 °C (
      • Velázquez J.B.
      • Fernández M.S.
      GPS, the slope of Laurdan generalized polarization spectra, in the study of phospholipid lateral organization and Escherichia coli lipid phases.
      ). This shows that the lipids in E. coli are natively in the liquid phase, and their Tm is as much as 20 °C below the growth temperature. However, when the OM and IM are considered separately, it becomes clear that “global” lipid properties do not accurately capture the differences between these two different membranes. Deuterium NMR studies, used to measure the order of acyl chains, found that lipids in an OM preparation of E. coli L51 were less fluid than those from the IM and that the Tm was ∼7 °C higher than the IM (
      • Davis J.H.
      • Nichol C.P.
      • Weeks G.
      • Bloom M.
      Study of the cytoplasmic and outer membranes of Escherichia coli by deuterium magnetic resonance.
      ,
      • Nichol C.P.
      • Davis J.H.
      • Weeks G.
      • Bloom M.
      Quantitative study of the fluidity of Escherichia coli membranes using deuterium magnetic resonance.
      ). Electron spin resonance experiments on IMs and OMs of E. coli W1485 doped with a spin-labeled stearic acid probe found the Tm of the OM (26 °C) to be ∼13 °C higher than the IM (13 °C) when the bacteria were grown at 37 °C (
      • Janoff A.S.
      • Haug A.
      • McGroarty E.J.
      Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.
      ). Fluorescence polarization studies using IM and OM extracts from E. coli B doped with parinaric acid also found that the phase transition of the OM was ∼15 °C higher than the IM, initiating its phase transition at 40 °C (
      • Souzu H.
      Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase.
      ).

      E. coli membranes in situ

      Few studies on the properties of bacterial membrane lipid order (or phase) in vivo have been reported to date, but current data suggest that the organization of the OM is more complicated than that derived using lipid extracts, as described above. Differential scanning calorimetry studies on whole cells of E. coli W945 grown at 20 or 37 °C observed two reversible transitions, one well below the growth temperature, which was suggested to correspond to the IM, and another slightly above the growth temperature, which was assigned to the OM (
      • Melchior D.L.
      • Steim J.M.
      Thermotropic transitions in biomembranes.
      ). Experiments on fixed cells using laurdan and the dye 1,3-diphenyl-1,3,5-hexatriene as a probe of local viscosity (
      • Demchenko A.P.
      • Mély Y.
      • Duportail G.
      • Klymchenko A.S.
      Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes.
      ) have shown that E. coli membranes are predominantly in the liquid phase. These experiments also revealed that these membranes are heterogeneous and contain at least two distinct phases, one more liquid and one less so, possibly indicating the presence of distinct lipid domains (
      • Vanounou S.
      • Pines D.
      • Pines E.
      • Parola A.H.
      • Fishov I.
      Coexistence of domains with distinct order and polarity in fluid bacterial membranes.