Asymmetric Outer Membrane Research Articles

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli

Gram-negative bacteria can survive in harsh environments in part because the asymmetric outer membrane (OM) hinders the entry of toxic compounds. Lipid asymmetry is established by having phospholipids (PLs) confined to the inner leaflet of the membrane and lipopolysaccharides (LPS) to the outer leaflet. Perturbation of OM lipid asymmetry, characterized by PL accumulation in the outer leaflet, disrupts proper LPS packing and increases membrane permeability. The multi-component Mla system prevents PL accumulation in the outer leaflet of the OM via an unknown mechanism. Here, we demonstrate that in Escherichia coli, the Mla system maintains OM lipid asymmetry with the help of osmoporin OmpC. We show that the OM lipoprotein MlaA interacts specifically with OmpC and OmpF. This interaction is sufficient to localize MlaA lacking its lipid anchor to the OM. Removing OmpC, but not OmpF, causes accumulation of PLs in the outer leaflet of the OM in stationary phase, as was previously observed for MlaA. We establish that OmpC is an additional component of the Mla system; the OmpC-MlaA complex may function to remove PLs directly from the outer leaflet to maintain OM lipid asymmetry. Our work reveals a novel function for the general diffusion channel OmpC in lipid transport.

Charge and aggregation pattern govern the interaction of plasticins with LPS monolayers mimicking the external leaflet of the outer membrane of Gram-negative bacteria

Bacterial resistance to antibiotics has become today a major public health issue. In the development of new anti-infectious therapies, antimicrobial peptides appear as promising candidates. However, their mechanisms of action against bacterial membranes are still poorly understood. We describe for the first time the interaction and penetration of plasticins into lipid monolayers and bilayers modeling the two leaflets of the asymmetrical outer membrane of Gram-negative bacteria. The lipid composition of these monolayers mimics that of each leaflet: mixtures of LPS Re 595 mutant and wild type S-form from Salmonella enterica for the external leaflet, and SOPE/SOPG/cardiolipin (80/15/5) for the inner one. The analysis of the interfacial behavior of native (PTCDA1) and modified (PTCDA1-KF) antimicrobial plasticins showed that PTCDA1-KF exhibited better surface properties than its unmodified counterpart. Both peptides could penetrate into the model monolayers at concentrations higher than 0.1μM. The penetration was particularly enhanced for PTCDA1-KF into the mixed LPS monolayer, due to attractive electrostatic interactions. Grazing X-ray diffraction and atomic force microscopy studies revealed the changes in LPS monolayers organization upon peptide insertion. The interaction of plasticins with liposomes was also monitored by light scattering and circular dichroism techniques. Only the cationic plasticin achieved full disaggregation and structuration in α helices, whereas the native one remained aggregated and unstructured. The main steps of the penetration mechanism of the two plasticins into lipid models of the external leaflet of the outer membrane of Gram-negative bacteria have been established.

Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids.

Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl β-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. Here, we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl β-diol substrate analogues. By applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and that a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in the regulation of DIM biosynthesis.

The transcriptomic response of Acinetobacter baumannii to colistin and doripenem alone and in combination in an in vitro pharmacokinetics/pharmacodynamics model.

Colistin remains a last-line treatment for MDR Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against MDR strains. In order to understand the bacterial responses to these antibiotics, we analysed the transcriptome of A. baumannii following exposure to each. RNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics (PK)/pharmacodynamics model to mimic the PK of both antibiotics in patients. After treatment with colistin (continuous infusion at 2 mg/L), >400 differentially regulated genes were identified, including many associated with outer membrane biogenesis, fatty acid metabolism and phospholipid trafficking. No genes were differentially expressed following treatment with doripenem (Cmax 25 mg/L, t1/2 1.5 h) for 15 min, but 45 genes were identified as differentially expressed after 1 h of growth under this condition. Treatment of A. baumannii with both colistin and doripenem together for 1 h resulted in >450 genes being identified as differentially expressed. More than 70% of these gene expression changes were also observed following colistin treatment alone. These data suggest that colistin causes gross damage to the outer membrane, facilitates lipid exchange between the inner and outer membrane and alters the normal asymmetric outer membrane composition. The transcriptional response to colistin was highly similar to that observed for an LPS-deficient strain, indicating that many of the observed changes are responses to outer membrane instability resulting from LPS loss.

Elucidating biosynthesis of the outer membrane lipid phthiocerol dimycocerosate by Mycobacterium tuberculosis PapA5 (768.13)

Mycobacterium tuberculosis (Mtb) has a complex, asymmetric outer membrane consisting of mycolic acids and other unique lipids. Phthiocerol dimycocerosate (PDIM) is a wax ester lipid found in the outer membrane, at the interface between the bacterium and host, where it has been implicated in Mtb virulence. My work focuses on gaining a better understanding of how PDIM biosynthesis is regulated in Mtb by studying PapA5, an acyltransferase responsible for acylating phthiocerol with mycocerosic acid. We show using a series of synthetic substrates that PapA5 has a preference for longer alkyl chains. We demonstrate under novel biphasic reaction conditions that PapA5 is capable of catalyzing two successive acylation reactions, consistent with its proposed role in modifying both hydroxyls on the phthiocerol moiety. The introduction of bulky amino acid side chains in the putative substrate‐binding pocket of PapA5 yielded partial or complete inhibition of product formation. These data help confirm the location of the substrate‐binding channels. Furthermore, the introduction of a salt bridge or a disulfide bond to constrain a hypothesized flexible helix led to partial inhibition of product. These data support a role for conformational flexibility in ligand binding and/or product release. Finally, in vitro phosphorylation of PapA5 altered product yield, demonstrating a possible mechanism of regulation for PDIM biosynthesis.Grant Funding Source: Supported by a Stony Wold‐Herbert Fund award to JCS

Probing the Cell Growth-Dependent Spatial Distribution and Dynamics of Proteins Inserted in the Bacterial Outer Membrane

The Gram-negative cell envelope incorporates two lipid bilayers: an inner membrane (IM) with a protein and phospholipid composition characteristic of a typical cell membrane and an atypical, asymmetric outer membrane (OM). Protein insertion and time-dependent re-distribution in the IM is fairly well understood, but these processes remain poorly characterised in the OM. The pattern of insertion for an endogenous OM porin over-produced in Escherichia coli and its rearrangement during cell growth has recently been visualised. In this published work, distinct fluorescent puncta coincident with newly inserted proteins appeared randomly in the OM as discrete bursts, and they did not re-distribute appreciably during cell growth. We build on this previous work by addressing whether protein production levels characteristic of normal exponential cell growth alter the protein insertion process in E. coli JM83. To specifically label low-abundance outer membrane receptors (BtuB and Cir), and avoid the problems of over-expression, we have utilised organic dye-labelled colicin molecules (colicins E9 and Ia) engineered to retain high-affinity receptor binding (Kd ≈ pM) and prevent OM translocation. This allowed us to follow insertion of BtuB and Cir in live cells and their re-distribution during cell growth and binary fission using both fluorescence recovery after photo-bleaching (FRAP) confocal microscopy and single-molecule total internal reflection fluorescence (TIRF) microscopy. Single particle tracking and FRAP microscopy of colicin-receptor complexes revealed that BtuB and Cir are sequestered to randomly positioned patches in the OM, which persist during cell growth, and through multiple rounds of binary fission. Work is underway to correlate the random sites of receptor protein insertion with the periplasmic/OM molecular machinery necessary to mediate this process.

Understanding Colicin N Import into Gram Negative Bacterial Cells using Small Angle Neutron Scattering

Escherichia coli (E. coli) are gram-negative bacteria commonly found in human guts but which can cause food poisoning or severe systemic infections such as meningitis. Unlike other bacteria, gram-negative cells have an additional asymmetric outer membrane (OM) which consists of tightly-packed lipopolysaccharides in the outer leaflet of membrane and phospholipids on the inside. The OM is a selective permeability barrier and protects E. coli from antibiotics and bile acids. The occurrence of antibiotic-resistant bacteria is becoming increasingly serious and owing to the tightly-packed OM gram-negative bacteria can show even higher antibiotic resistance. In the search for novel effective antibiotics, Colicin N (ColN) is a promising model. ColN is a bacterial toxin produced and secreted by E. coli in time of stress. It translocates across the OM of target cells and kills via a voltage dependent channel in the inner membrane. ColN exploits outer membrane protein F (OmpF) as both a receptor and translocator. The pore-forming domain binds to the outside of OmpF (1, 2). Here we study the OmpF/ColN translocation complex in the presence of non-ionic detergents, where binding is driven by the translocation domain. The structure of complexes was determined by small angle neutron scattering using the SANS2D beam line and utilizing a contrast variation strategy with selective deuteration of proteins and Octyl Glucoside (OG). Mixing of three OG forms achieved exact contrast matches with the solvent. This enabled a low resolution structure of the translocon to be derived from ab initio modeling and revealed that the translocation and receptor-binding domains of ColN bind to the middle of the OmpF trimer.1. Baboolal, T. G. et al. Structure16, 371, (2008).2. Clifton, L. A. et al. J. Biol. Chem.287, 337,(2012).

Inhibition of Lipopolysaccharide Transport to the Outer Membrane in Pseudomonas aeruginosa by Peptidomimetic Antibiotics

The asymmetric outer membrane (OM) of Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet and phospholipid in the inner leaflet. During OM biogenesis, LPS is transported from the periplasm into the outer leaflet by a complex comprising the OM proteins LptD and LptE. Recently, a new family of macrocyclic peptidomimetic antibiotics that interact with LptD of the opportunistic human pathogen Pseudomonas aeruginosa was discovered. Here we provide evidence that the peptidomimetics inhibit the LPS transport function of LptD. One approach to monitor LPS transport involved studies of lipid A modifications. Some modifications occur only in the inner membrane while others occur only in the OM, and thus provide markers for LPS transport within the bacterial envelope. We prepared a conditional lptD mutant of P. aeruginosa PAO1 that allowed control of lptD expression from the rhamnose promoter. With this mutant, the effects caused by the antibiotic on the wild-type strain were compared with those caused by depleting LptD in the mutant strain. When LptD was depleted in the mutant, electron microscopy revealed accumulation of membrane-like material within cells and OM blebbing; this mirrored similar effects in the wild-type strain caused by the antibiotic. Moreover, the bacterium responded to the antibiotic, and to depletion of LptD, by introducing the same lipid A modifications, consistent with inhibition by the antibiotic of LptD-mediated LPS transport. This conclusion was further supported by monitoring the radiolabelling of LPS from [¹⁴C]acetate, and by fractionation of IM and OM components. Overall, the results provide support for a mechanism of action for the peptidomimetic antibiotics that involves inhibition of LPS transport to the cell surface.

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs), and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella, and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation, and isomerization pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, the pathways required for the biosynthesis of the OM and the regulatory circuits that control them have evolved to suit the lifestyle of each organism.

LpxI: A Novel Phosphosaccharolipid Hydrolase

Gram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids, while the outer leaflet contains mainly lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. LPS is anchored to the outer membrane by lipid A, a unique glucosamine-based saccharolipid. Lipid A biosynthesis is required for bacterial viability and pathogenesis. While most lipid A biosynthetic genes are present in a single copy, one gene, lpxH, encoding a membrane-associated specific UDP-diacylglucosamine hydrolase, is absent in ∼30% of Gram-negative bacteria. We hypothesized that a transformational analogue of lpxH must exist in these organisms. We identified this gene, designated lpxI, in Caulobacter crescentus, and confirmed its ability to cover for a deficiency of lpxH in Eschserichia coli. LpxI lacks homology to any other known enzyme. We over-expressed LpxI, purified it, and obtained protein crystals. We solved the X-ray crystal structure of this peripheral membrane enzyme using a single-wavelength anomalous dispersion (SAD) dataset collected on Se-Met derivatized protein. The structural data reveal two domains, each having a novel fold. Unexpectedly, “apo” LpxI co-purified and co-crystallized stoichiometrically with its product, diacylglucosamine-1-phosphate (lipid X), the saccharolipid precursor of lipid A. We then identified and determined the 2.5 A X-ray crystal structure of an inactive point mutant of Caulobacter crescentus LpxI, which co-purified in a stoichiometric ratio with its substrate, UDP-2,3-diacyglucosamine. The conformation of substrate-liganded LpxI differs significantly from the product -liganded wild-type enzyme. Taken together with analytical unltracentrifugation data, this observation suggests that large-scale domain re-arrangement occurs during LpxI substrate binding and/or catalysis. These data provide an interesting example of lipid-protein interaction, and set the stage for additional structural and mechanistic work.

Protecting Bacterial Cells from Rupture: A Multiscale Simulation Study of Braun's Lipoprotein

The cell envelope of Gram-negative bacteria is composed of two membranes. The inner membrane has a relatively simple phospholipid composition, whilst the asymmetric outer membrane is a complex mixture of lipopolysaccharide (LPS) and phospholipids. The region between the two membranes, know as the periplasm, is largely composed of peptidoglycan that maintains the cell shape and protects its from rupture. Braun's lipoprotein (BLP), a coiled-coiled trimeric protein located within the periplasm, has long been known to covalently bind to the peptidoglycan and to anchor the peptidoglycan to the inner leaflet of the outer membrane. Recently, however, it has been shown that in addition to this structural role, BLP is able to adopt a trans-membrane orientation across the outer membrane.In this present study we have performed a series of molecular dynamics simulations of BLP using both atomistic and coarse-grained approaches. An atomistic model of the peptidoglycan has been developed and incorporated with our complex model of the E. coli outer membrane that contains LPS in the outer leaflet and mixtures of phospholipids in the inner leaflet. Multiple simulations of BLP, including the peptidoglycan bound and free forms, plus BLP in a trans-membrane orientation have been performed. These simulations have enabled us to explore the conformational dynamics, oligomerisation and environmental interactions of BLP in these different orientations.

Assembly of Outer Membrane β-Barrel Proteins: the Bam Complex.

The major class of integral proteins found in the outer membrane (OM) of E. coli and Salmonella adopt a β-barrel conformation (OMPs). OMPs are synthesized in the cytoplasm with a typical signal sequence at the amino terminus, which directs them to the secretion machinery (SecYEG) located in the inner membrane for translocation to the periplasm. Chaperones such as SurA, or DegP and Skp, escort these proteins across the aqueous periplasm protecting them from aggregation. The chaperones then deliver OMPs to a highly conserved outer membrane assembly site termed the Bam complex. In E. coli, the Bam complex is composed of an essential OMP, BamA, and four associated OM lipoproteins, BamBCDE, one of which, BamD, is also essential. Here we provide an overview of what we know about the process of OMP assembly and outline the various hypotheses that have been proposed to explain how proteins might be integrated into the asymmetric OM lipid bilayer in an environment that lacks obvious energy sources. In addition, we describe the envelope stress responses that ensure the fidelity of OM biogenesis and how factors, such as phage and certain toxins, have coopted this essential machine to gain entry into the cell.

Concentration‐dependent oligomerization and oligomeric arrangement of LptA

Gram-negative bacteria such as Escherichia coli have an inner membrane and an asymmetric outer membrane (OM) that together protect the cytoplasm and act as a highly selective permeability barrier. Lipopolysaccharide (LPS) is the major component of the outer leaflet of the OM and is essential for the survival of nearly all Gram-negative bacteria. Recent advances in understanding the proteins involved in the transport of LPS across the periplasm and into the outer leaflet of the OM include the identification of seven proteins suggested to comprise the LPS transport (Lpt) system. Crystal structures of the periplasmic Lpt protein LptA have recently been reported and show that LptA forms oligomers in either an end-to-end arrangement or a side-by-side dimer. It is not known if LptA oligomers bridge the periplasm to form a large, connected protein complex or if monomeric LptA acts as a periplasmic shuttle to transport LPS across the periplasm. Therefore, the studies presented here focus specifically on the LptA protein and its oligomeric arrangement and concentration dependence in solution using experimental data from several biophysical approaches, including laser light scattering, crosslinking, and double electron electron resonance spectroscopy. The results of these complementary techniques clearly show that LptA readily associates into stable, end-to-end, rod-shaped oligomers even at relatively low local protein concentrations and that LptA forms a continuous array of higher order oligomeric end-to-end structures as a function of increasing protein concentration.

Melittin vs E.coli: Insights from Molecular Dynamics Simulations

The interactions of antimicrobial peptides with eukaryotic cell membranes have been well-studied by experimental and computational methods. However, the molecular-level interactions of these peptides with Gram-negative bacteria such as E.coli are more difficult to study, largely due to the complex nature of the asymmetric outer membrane of Gram-negative bacteria. While cell lysis is thought to proceed via destruction of the inner membrane, it is the outer membrane that is the first barrier encountered by antimicrobial peptides as they attempt to reach their target site.In the present work, we employ atomistic molecular dynamics simulations to study the interaction of melittin, the major component of honey bee toxin, with detailed models of the E.coli outer membrane. Experimental data (ref), has indicated that upon interaction, melittin induces reorganisation of the lipopolysaccharide (LPS) component of the bacterial outer membrane. We have simulated multiple copies of melittin in lipid bilayers composed of varying depths of LPS in the outer leaflet, and realistic mixtures of phospholipids in the inner leaflet. Our simulations reveal details of the melittin-outer membrane recognition process and the structural stability and dynamics of melittin as it interacts with the outer membrane. Furthermore, our simulations provide specific, molecular-level details of the interaction of melittin with the E.coli outer membrane that may aid the future design of novel antimicrobial agents.

The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel

The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane β-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD β-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the β-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.

Discovery, characterization, and structural determination of a novel UDP‐2,3‐diacylglucosamine hydrolase

Gram‐negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids, while the outer leaflet contains mainly lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram‐negative bacteria from antibiotics and other environmental stressors. LPS is anchored to the outer membrane by Lipid A, a unique glucosamine‐based saccharolipid. Lipid A biosynthesis is required for viability and pathogenesis. While most lipid A biosynthetic genes are present in a single copy, one gene, lpxH, encoding a membrane‐associated specific UDP‐diacylglucosamine hydrolase, is absent in ~30% of Gram‐negative bacteria. We hypothesized that a transformational analogue of lpxH must exist in these organisms. We identified this gene, designated lpxI, in Caulobacter crescentus, and confirmed its ability to cover for a deficiency of lpxH in Eschserichia coli. LpxI lacks homology to any other known enzyme. We over‐expressed LpxI, purified it, and obtained protein crystals. We solved the x‐ray crystal structure of this peripheral membrane enzyme to a resolution of 2.7 angstroms using a single‐wavelength anomalous dispersion (SAD) dataset collected on Se‐Met derivatized protein. The structural data reveal two domains, each having a novel fold. Unexpectedly, “apo” LpxI co‐purified and co‐crystallized in a 1:1 stoichiometric ratio with its product, lipid X. These data provide an interesting example of lipid‐protein interaction, and set the stage for additional structural and mechanistic work. Support was provided by NIH grant GM5150 to C.R.H.R., Specialized Center grant of the Protein Structure Initiative U54 GM074929‐01 to R.M.S., and NSF GRF grant 2005029353 to L.E.M.

A novel biological synthesis and purification of lipid A precursors

Gram‐negative bacteria possess an asymmetric outer membrane in which the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram‐negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine‐based saccharolipid that is further modified with core and O‐antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. In macrophages, lipid A has been found to stimulate TLR4 (toll like receptor 4), which in turn activates the inflammatory cascade. Given lipid A's role in both pathogen viability and virulence, the genetic engineering of Gram‐negative bacteria to produce non‐canonical lipid A may play an important role in the development of attenuated vaccine strains. Here, we engineer E. coli to produce a non‐canonical lipid A precursor, 2,3‐diamino‐2,3‐dideoxy‐D‐glucopyranose lipid X. We then purify this compound from bacterial homogenate, verify its structure, and evaluate the specificity of various orthologues of LpxB, the bacterial lipid A disaccharide synthase, toward this non‐canonical substrate. This study presents the first biological synthesis and purification of this lipid A precursor. Funding: NIH 5R01GM0531310‐15 to CR and NSF GRF2005029353 to LM.

Effect of Cations on the Structure of Bilayers Formed by Lipopolysaccharides Isolated from Pseudomonas aeruginosa PAO1

The asymmetric outer membrane of Gram-negative bacteria contains lipopolysaccharides (LPSs) which contribute significantly to the bacterium's surface properties and play a crucial role in regulating membrane permeability. We report on neutron diffraction studies performed on aligned, self-assembled bilayers of Na-, Ca-, and Mg-salt forms of LPS isolated from Pseudomonas aeruginosa PAO1. From the one-dimensional neutron scattering length density profiles we find that water penetrates Ca2+-LPS bilayers to a lesser extent than either Na+- or Mg2+-LPS bilayers. This differential water penetration could have implications as to how small molecules permeate the outer membrane of Gram-negative bacteria and, possibly, how nonlamellar phases are formed.

Identification of a Novel Protein with a Role in Lipoarabinomannan Biosynthesis in Mycobacteria

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.