Outer Membrane Lipid Research Articles

Vibrio cholerae Sheds Its Coat to Make Itself Comfortable in the Gut

Invertebrates molt, furry mammals shed, and human skin exfoliates. In this issue of Cell Host & Microbe, Zingl etal. describe a virulence mechanism in which the bacterial pathogen Vibrio cholerae jettisons outer membrane proteins and lipids in vesicles as it enters the mammalian intestine.

From the GRAM-Negative Bacterial Extracellular Space to Periplasmic Space with EPR: Exploring the Escherichia Coli Vitamin B12 Transporter, BtuB, in Whole Cells

Outer membrane (OM) biogenesis processes are pivotal for Gram-negative bacterial survival. These sophisticated and well-coordinated pathways are involved in synthesis, maturation and translocation of OM lipids and OM proteins such as lipopolysaccharides (LPS), lipoproteins (LP), and outer membrane proteins (OMPs). Assembly of LPS and OMPs within the OM can impact the structure and function of OMPs within bacteria. Additionally, function of some OMPs mediated by inner membrane (IM) and periplasmic proteins. For example, the active transport of TonB dependent transporters (TBDTs) in the OM is coupled with IM proton motive force (PMF) via TonB complex in the IM. Thereby, hindering the studies of TBDTs’ transport process in isolated systems compared to their native environment, whole cells. Recently, we demonstrated an effective approach to study a TBDT, BtuB, in Escherichia coli via EPR spectroscopy (CW EPR and DEER). This approach has recently been further modified to provide more physiological conditions and is utilized to explore the dynamics of BtuB loops and core regions exposed to the extracellular space. Based on DEER analysis, we were able to detect new substrate-dependent conformational states between sites T188 (loop) and V90 (core) which are associated with the previously characterized molecular switch, R14-D3161. Additionally, we have successfully overcome the periplasmic reduction potential and spin-labeled a range of sites within the periplasmic space, which permitted potential exploration of the transport process. In the isolated systems Ton box, a folded BtuB core region in the periplasmic side, undergoes substrate induced unfolding, but we have observed novel unfolded states of apo BtuB Ton box BtuB in whole cells. [1] Lukasik, S. M., David Ho, K. W., and Cafiso, D. S. (2007), Journal of molecular biology 370, 807-811.

Cryo-EM Structure of a Bacterial Lipid Transporter YebT

The outer membrane (OM) of Gram-negative bacteria is asymmetric, with lipopolysaccharides (LPSs) on the outer surface and phospholipids (PLs) on the inner surface. This unique organization of OM makes Gram-negative bacteria resistant to many toxic chemicals. How this asymmetric distribution of lipids is maintained has been studied for decades with previous reports of an Mla (Maintenance of OM Lipid Asymmetry) system to be involved. Furthermore, the OM of Gram-negative bacteria is about 20 nm away from inner membrane (IM) where the lipids are synthesized. Therefore, how nascent lipids travel across the periplasmic space and arrive at the inner surface of OM is another interesting question. YebT is a homologue of MlaD in the Mla pathway, but its role in lipid distribution of the OM and IM is largely unknown. Here we report the first high-resolution (~3.0 Å) cryo-EM structure of full-length E. coli YebT in a substrate-bound state. Our structure with details of lipid interaction indicates that YebT is a lipid transporter spanning between IM and OM. We also demonstrate the symmetry mismatch in YebT and the existence of many other conformations of YebT revealing the intrinsic dynamics of this lipid channel. And a brief discussion on possible mechanisms of lipid transport is also included.

Self-identity barcodes encoded by six expansive polymorphic toxin families discriminate kin in myxobacteria

Myxobacteria are an example of how single-cell individuals can transition into multicellular life by an aggregation strategy. For these and all organisms that consist of social groups of cells, discrimination against, and exclusion of, nonself is critical. In myxobacteria, TraA is a polymorphic cell surface receptor that identifies kin by homotypic binding, and in so doing exchanges outer membrane (OM) proteins and lipids between cells with compatible receptors. However, TraA variability alone is not sufficient to discriminate against all cells, as traA allele diversity is not necessarily high among local strains. To increase discrimination ability, myxobacteria include polymorphic OM lipoprotein toxins called SitA in their delivered cargo, which poison recipient cells that lack the cognate, allele-specific SitI immunity protein. We previously characterized 3 SitAI toxin/immunity pairs that belong to 2 families. Here, we discover 4 additional SitA families. Each family is unique in sequence, but share the characteristic features of SitA: OM-associated toxins delivered by TraA. We demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strikingly numerous and diverse, with most genomes possessing >30 and up to 83 distinct sitAI loci. Interestingly, all SitA protein families are serially transferred between cells, allowing a SitA inhibitor cell to poison multiple targets, including cells that never made direct contact. The expansive suites of sitAI loci thus serve as identify barcodes to exquisitely discriminate against nonself to ensure populations are genetically homogenous to conduct cooperative behaviors.

The MmpL3 interactome reveals a complex crosstalk between cell envelope biosynthesis and cell elongation and division in mycobacteria

Integral membrane transporters of the Mycobacterial Membrane Protein Large (MmpL) family and their interactome play important roles in the synthesis and export of mycobacterial outer membrane lipids. Despite the current interest in the mycolic acid transporter, MmpL3, from the perspective of drug discovery, the nature and biological significance of its interactome remain largely unknown. We here report on a genome-wide screening by two-hybrid system for MmpL3 binding partners. While a surprisingly low number of proteins involved in mycolic acid biosynthesis was found to interact with MmpL3, numerous enzymes and transporters participating in the biogenesis of peptidoglycan, arabinogalactan and lipoglycans, and the cell division regulatory protein, CrgA, were identified among the hits. Surface plasmon resonance and co-immunoprecipitation independently confirmed physical interactions for three proteins in vitro and/or in vivo. Results are in line with the focal localization of MmpL3 at the poles and septum of actively-growing bacilli where the synthesis of all major constituents of the cell wall core are known to occur, and are further suggestive of a role for MmpL3 in the coordination of new cell wall deposition during cell septation and elongation. This novel aspect of the physiology of MmpL3 may contribute to the extreme vulnerability and high therapeutic potential of this transporter.

A Vibrio cholerae BolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity.

BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen's morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaGV. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaGV. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant's morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG's genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.IMPORTANCE BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

Bacterial lipocalins protect bacteria from antibiotic-induced membrane lipid peroxidation

Burkholderia cenocepacia, an opportunistic bacterium causing chronic respiratory infections in patients with cystic fibrosis, produces the bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures antibiotics outside bacterial cells, providing a novel extracellular mechanism of antimicrobial resistance. Sublethal concentrations of bactericidal antibiotics (e.g. polymyxin B, norfloxacin) and superoxide ion stress inducers (e.g. paraquat) stimulate transcription of bcnA, suggesting BcnA may also play a role in oxidative stress responses. bcnA gene homologues can be genetically linked to an upstream gene encoding a predicted membrane cytochrome b-561 protein, which we have named bcoA (bcnA cytochrome oxidase-associated gene). RT-PCR showed that both bcnA and bcoA are co-transcribed in B. cenocepacia. We hypothesize that BcnA and BcoA may be components of an unrecognized pathway to protect B. cenocepacia and other bacteria from membrane lipid-derived peroxidation damage. Here, we show that bcnA and bcoA deletion mutants display enhanced membrane lipid peroxidation and fail to survive under conditions that stimulate peroxidative stress (e.g. cold stress and salt stress). Compared to wild type, the levels of the lipid peroxidation biomarker malondialdehyde are also increased in the mutants upon exposure to near-lethal concentrations of polymyxin B and norfloxacin. No differences in bacterial survival were detected in parental and mutants under anaerobiosis. These findings indicate that BcnA and BcoA participate in a previously unrecognised peroxidation detoxification pathway that likely protects the outer membrane lipid bilayer under extreme stress conditions such as antibiotic killing.

Prediction of the Closed Conformation and Insights into the Mechanism of the Membrane Enzyme LpxR

Covalent modification of outer membrane lipids of Gram-negative bacteria can impact the ability of the bacterium to develop resistance to antibiotics as well as modulating the immune response of the host. The enzyme LpxR from Salmonella typhimurium is known to deacylate lipopolysaccharide molecules of the outer membrane, however the mechanism of action is unknown. Here we employ molecular dynamics and Monte Carlo simulations to study the conformational dynamics and substrate binding of LpxR in representative outer membrane models and also detergent micelles. We examine the roles of conserved residues and provide an understanding of how LpxR binds its substrate. Our simulations predict that the catalytic H122 must be Nɛ-protonated for a single water molecule to occupy the space between it and the scissile bond, with a free binding energy of −8.5 kcal mol−1. Furthermore, simulations of the protein within a micelle enable us to predict the structure of the putative ‘closed’ protein. Our results highlight the need for including dynamics, a representative environment and the consideration of multiple tautomeric and rotameric states of key residues in mechanistic studies; static structures alone do not tell the full story.

Dewetting-induced formation and mechanical properties of synthetic bacterial outer membrane models (GUVs) with controlled inner-leaflet lipid composition.

The double-membrane cellular envelope of Gram-negative bacteria enables them to endure harsh environments and represents a barrier to many clinically available antibiotics. The outer membrane (OM) is exposed to the environment and is the first point of contact involved in bacterial processes such as signaling, pathogenesis, and motility. As in the cytoplasmic membrane, the OM in Gram-negative bacteria has a phospholipid-rich inner leaflet and an outer leaflet that is predominantly composed of lipopolysaccharide (LPS). We report on a microfluidic technique for fabricating monodisperse asymmetric giant unilamellar vesicles (GUVs) possessing the Gram-negative bacterial OM lipid composition. Our continuous microfluidic fabrication technique generates 50-150 μm diameter water-in-oil-in-water double emulsions at high-throughput. The water-oil and oil-water interfaces facilitate the self-assembly of phospholipid and LPS molecules to create the inner and outer leaflets of the lipid bilayer, respectively. The double emulsions have ultrathin oil shells, which minimizes the amount of residual organic solvent that remains trapped between the leaflets of the GUV membrane. An extraction process by ethanol and micropipette aspiration of the ultrathin oil shells triggers an adhesive interaction between the two lipid monolayers assembled on the water-oil and oil-water interfaces (i.e., dewetting transition), forcing them to contact and form a lipid bilayer membrane. The effect of different inner-leaflet lipid compositions on the emulsion/vesicle stability and the dewetting transition is investigated. We also report on the values for bending and area expansion moduli of synthetic asymmetric model membranes with lipid composition/architecture that is physiologically relevant to the OM in Pseudomonas aeruginosa bacteria.

Characterization of Interactions and Phospholipid Transfer between Substrate Binding Proteins of the OmpC-Mla System

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts. This barrier function depends critically on the asymmetric lipid distribution across the bilayer, with lipopolysaccharides (LPS) facing outside and phospholipids (PLs) facing inside. In Escherichia coli, the OmpC-Mla system is believed to maintain OM lipid asymmetry by removing surface exposed PLs and shuttling them back to the inner membrane (IM). How proteins in the pathway interact to mediate PL transport across the periplasm is not known. Evidence for direct transfer of PLs between these proteins is also lacking. In this study, we mapped the interaction surfaces between the two PL-binding proteins, MlaC and MlaD, using site-specific in vivo photo-cross-linking, and obtained a physical picture for how these proteins may transfer PLs. Furthermore, we demonstrated using purified proteins that MlaD spontaneously transfers PLs to MlaC, suggesting that the latter has a higher affinity for PLs. Our work provides insights into the mechanism of bacterial intermembrane lipid transport important for the maintenance of OM lipid asymmetry.

Prediction of the Closed Conformation and Insights into the Mechanism of the Membrane Enzyme LpxR

Covalent modification of outer membrane lipids of Gram-negative bacteria can impact the ability of the bacterium to develop resistance to antibiotics as well as modulating the immune response of the host. The enzyme LpxR from Salmonella typhimurium is known to deacylate lipopolysaccharide molecules of the outer membrane; however, the mechanism of action is unknown. Here, we employ molecular dynamics and Monte Carlo simulations to study the conformational dynamics and substrate binding of LpxR in representative outer membrane models as well as detergent micelles. We examine the roles of conserved residues and provide an understanding of how LpxR binds its substrate. Our simulations predict that the catalytic H122 must be Nε-protonated for a single water molecule to occupy the space between it and the scissile bond, with a free binding energy of −8.5 kcal mol−1. Furthermore, simulations of the protein within a micelle enable us to predict the structure of the putative “closed” protein. Our results highlight the need for including dynamics, a representative environment, and the consideration of multiple tautomeric and rotameric states of key residues in mechanistic studies; static structures alone do not tell the full story.

The architecture of the OmpC–MlaA complex sheds light on the maintenance of outer membrane lipid asymmetry in Escherichia coli

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. In Escherichia coli, the complex comprising osmoporin OmpC and the OM lipoprotein MlaA is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How this complex performs this function is unknown. Here, we defined the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. Using in vivo photo-cross-linking and molecular dynamics simulations, we established that MlaA interacts extensively with OmpC and is located entirely within the lipid bilayer. In addition, MlaA forms a hydrophilic channel, likely enabling PL translocation across the OM. We further showed that flexibility in a hairpin loop adjacent to the channel is critical in modulating MlaA activity. Finally, we demonstrated that OmpC plays a functional role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antibacterial drug development.

Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways

Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.

Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli

A distinctive feature of the Gram‐negative bacterial cell envelope is the presence of an asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, thus allowing survival of bacteria in harsh environments. In Escherichia coli, the OmpC‐Mla system is responsible for maintenance of OM lipid asymmetry. Osmoporin OmpC and the OM lipoprotein MlaA form a complex proposed to remove PLs from the outer leaflet of the OM. How this complex is organized in the OM to perform this function is not known. In this report, we define the molecular architecture of the OmpC‐MlaA complex to gain insights into its function in PL transport. We show that MlaA sits entirely within the OM lipid bilayer, and interacts with the OmpC trimer at its dimeric interfaces. Molecular dynamics simulations reveal a membrane‐spanning hydrophilic channel within MlaA, suggesting a path for PL translocation across the OM. We demonstrate that a hydrophobic hairpin loop adjacent to this putative channel is critical for modulating the activity of MlaA; restricting flexibility of this structure significantly perturbs the function of the complex. Finally, we establish that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work provides a glimpse into the molecular mechanism of how the OmpC‐MlaA complex may extract PLs from the outer leaflet of the OM, and highlights key features that could be exploited in the development of future antimicrobial drugs.Support or Funding InformationNational University of Singapore Graduate School for Integrative Sciences and Engineering scholarship (to J.Y.).Singapore Ministry of Education Academic Research Fund Tier 3 grant (MOE2012‐T3‐1‐008) grant to (P.J.B.).Singapore Ministry of Education Academic Research Fund Tier 1 and Tier 2 (MOE2013‐T2‐1‐148) grants (to S.‐S.C.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.

The increasing incidence of multidrug-resistant Acinetobacter baumannii (MDRAb) infections worldwide has necessitated the development of novel antibiotics. Human defensin 5 (HD5) is an endogenous peptide with a complex architecture and antibacterial activity against MDRAb In the present study, we attempted to simplify the structure of HD5 by removing disulfide bonds. We found that the Cys2-4 bond was most indispensable for HD5 to inactivate MDRAb, although the antibacterial activity of the derivative was significantly attenuated. We then replaced the noncationic and nonhydrophobic residues with electropositive Arg to increase the antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both showed that HD5d5 was much more effective than HD5 at eliminating MDRAb Further investigations revealed that HD5d5 efficiently bound to outer membrane lipid A and penetrated membranes, leading to bacterial collapse and peptide translocation. Compared to HD5, more HD5d5 molecules were located in the cytoplasm of MDRAb, and HD5d5 was more efficient at reducing the activities of superoxide dismutase and catalase, causing the accumulation of reactive oxygen species that are detrimental to microbes. In addition, HD5 failed to suppress the pathogenic outer membrane protein A of Acinetobacter baumannii (AbOmpA) at concentrations up to 50 μg/ml, whereas HD5d5 strongly bound to AbOmpA and exhibited a dramatic toxin-neutralizing ability, thus expanding the repertoire of drugs that is available to treat MDRAb infections.

Identification of novel lipid modifications and intermembrane dynamics in Corynebacterium glutamicum using high-resolution mass spectrometry [S

The complex cell envelopes of Corynebacterineae contribute to the virulence of pathogenic species (such as Mycobacterium tuberculosis and Corynebacterium diphtheriae) and capacity of nonpathogenic species (such as Corynebacterium glutamicum) to grow in diverse niches. The Corynebacterineae cell envelope comprises an asymmetric outer membrane that overlays the arabinogalactan-peptidoglycan complex and the inner cell membrane. Dissection of the lipid composition of the inner and outer membrane fractions is important for understanding the biogenesis of this multilaminate wall structure. Here, we have undertaken the first high-resolution analysis of C. glutamicum inner and outer membrane lipids. We identified 28 lipid (sub)classes (>233 molecular species), including new subclasses of acylated/acetylated trehalose mono/dicorynomycolic acids, using high-resolution LC/MS/MS coupled with mass spectral library searches in MS-DIAL. All lipid subclasses exhibited polarized distributions across the inner and outer membrane fractions generated by differential solvent extraction. Strikingly, deletion of the TmaT protein, which is required for transport of trehalose corynomycolates across the inner membrane, led to the accumulation of triacylglycerols in the inner membrane and to suppressed synthesis of phosphatidylglycerol and alanylated lipids. These analyses indicate unanticipated connectivity in the synthesis and/or transport of different lipid classes in C. glutamicum.

Membrane-spanning electron transfer proteins from electrogenic bacteria: Production and investigation.

Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions can proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering, and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.

Inactivation of the arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of proteus vulgaris

Colistin has become a last-line antibiotic for the treatment of multidrug-resistant bacterial infections; however, resistance to colistin has emerged in recent years. Some bacteria, such as Proteus and Serratia spp., are intrinsically resistant to colistin although the exact mechanism of resistance is unknown. Here we identified the molecular support for intrinsic colistin resistance in Proteus spp. by comparative genomic, transcriptomic and proteomic analyses of colistin-susceptible (CSUR P1868_S) and colistin-resistant (CSUR P1867_R) strains of an atypical Proteus vulgaris. A significant difference in outer membrane glycoside structures in both strains that was corroborated by MALDI-TOF/MS analysis was found, which showed an absence of 4-amino-4-deoxy-l-arabinose (L-Ara4N) in the outer membrane lipid A moiety of the susceptible strain. Comparative genomic analysis with other resistant strains of P. vulgaris available in a local database found a mutation in the arnBCADTEF operon of the susceptible strain. Transcriptomic analysis of genes belonging to the arnBCADTEF operon showed a significant decrease in mRNA expression level of these genes in the susceptible strain, supporting addition of L-Ara4N in the outer membrane lipid A moiety as an explanation for colistin resistance. Insertion of the arnD gene that was suggested to be altered in the susceptible strain by in silico analysis led to a 16-fold increase of colistin MIC in the susceptible strain, confirming its role in colistin resistance in this species. Here we show that constitutive activation of the arn operon and addition of L-Ara4N is the main molecular mechanism of colistin resistance in P. vulgaris.

Global landscape of cell envelope protein complexes in Escherichia coli.

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the β-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

Dynamics of OmpF Trimer Formation in the Bacterial Outer Membrane of Escherichia coli.

The self-assembly of outer membrane protein F (OmpF) in the outer membrane of Escherichia coli Gram-negative bacteria was studied using multiscale molecular dynamics simulations. To accommodate the long time scale required for protein assembly, coarse-grained parametrization of E. coli outer membrane lipids was first developed. The OmpF monomers formed stable dimers at specific protein-protein interactions sites irrespective of the lipid membrane environment. The dimer intermediate was asymmetric but provided a template to form a symmetric trimer. Superposition analysis of the self-assembled trimer with the X-ray crystal structure of the trimer available in the protein data bank showed excellent agreement with global root-mean-square deviation of less than 2.2 Å. The free energy change associated with dimer formation was -26 ± 1 kcal mol-1, and for a dimer to bind to a monomer and to form a trimer yielded -56 ± 4 kcal mol-1. Based on thermodynamic data, an alternate path to trimer formation via interaction of two dimers is also presented.