Bacterial outer membrane constriction

The outer membrane of Gram‐negative bacteria is a crucial permeability barrier allowing the cells to survive a myriad of toxic compounds, including many antibiotics. This innate form of antibiotic resistance is compounded by the evolution of more active mechanisms of resistance such as efflux pumps, reducing the already limited number of clinically relevant treatments for Gram‐negative pathogens. During cell division Gram‐negative bacteria must coordinate constriction of the outer membrane in conjunction with other crucial layers of the cell envelope, the peptidoglycan cell wall and the inner membrane. Coordination is crucial in maintaining structural integrity of the envelope, and represents a highly vulnerable time for the cell as any failure can be fatal, if not least disadvantageous. However, the molecular mechanisms of cell division and how the biogenesis of the three layers is synchronised during constriction remain largely unknown. Perturbations of the outer membrane have been shown to increase the effectiveness of antibiotics in vitro, and so with improved understanding of this process we may be able to exploit this vulnerability and improve the effectiveness of antibiotic treatments. In this review the current knowledge of how Gram‐negative bacteria facilitate constriction of their outer membranes during cell division will be discussed.     

Membrane Vesicle Formation as a Multiple-Stress Response Mechanism Enhances Pseudomonas putida DOT-T1E Cell Surface Hydrophobicity and Biofilm Formation

Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.     

Atomistic Scale Effects of Lipopolysaccharide Modifications on Bacterial Outer Membrane Defenses

Lipopolysaccharides (LPS) are a main constituent of the outer membrane of Gram-negative bacteria. Salmonella enterica, like many other bacterial species, are able to chemically modify the structure of their LPS molecules through the PhoPQ pathway as a defense mechanism against the host immune response. These modifications make the outer membrane more resistant to antimicrobial peptides (AMPs), large lipophilic drugs, and cation depletion, and are crucial for survival within a host organism. It is believed that these LPS modifications prevent the penetration of large molecules and AMPs through a strengthening of lateral interactions between neighboring LPS molecules. Here, we performed a series of long-timescale molecular dynamics simulations to study how each of three key S. enterica lipid A modifications affect bilayer properties, with a focus on membrane structural characteristics, lateral interactions, and the divalent cation bridging network. Our results discern the unique impact each modification has on strengthening the bacterial outer membrane through effects such as increased hydrogen bonding and tighter lipid packing. Additionally, one of the modifications studied shifts Ca²⁺ from the lipid A region, replacing it as a major cross-linking agent between adjacent lipids and potentially making bacteria less susceptible to AMPs that competitively displace cations from the membrane surface. These results further improve our understanding of outer membrane chemical properties and help elucidate how outer membrane modification systems, such as PhoPQ in S. enterica, are able to alter bacterial virulence.     

High‐resolution architecture of the outer membrane of the Gram‐negative bacteria Roseobacter denitrificans

The outer membrane of Gram‐negative bacteria protects the cell against bactericidal substances. Passage of nutrients and waste is assured by outer membrane porins, beta‐barrel transmembrane channels. While atomic structures of several porins have been solved, so far little is known on the supramolecular structure of the outer membrane. Here we present the first high‐resolution view of a bacterial outer membrane gently purified maintaining remnants of peptidoglycan on the perisplasmic surface. Atomic force microscope images of outer membrane fragments of the size of ～50% of the bacterial envelope revealed that outer membrane porins are by far more densely packed than previously assumed. Indeed the outer membrane is a molecular sieve rather than a membrane. Porins cover ～70% of the membrane surface and form locally regular lattices. The potential role of exposed aromatic residues in the formation of the supramolecular assembly is discussed. Finally, we present first structural data of the outer membrane porin from the marine Gram‐negative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.     

Checks and balances in membrane phospholipid class and acyl chain homeostasis,the yeast perspective

Glycerophospholipids are the most abundant membrane lipid constituents in most eukaryotic cells. As a consequence, phospholipid class and acyl chain homeostasis are crucial for maintaining optimal physical properties of membranes that in turn are crucial for membrane function. The topic of this review is our current understanding of membrane phospholipid homeostasis in the reference eukaryote Saccharomyces cerevisiae. After introducing the physical parameters of the membrane that are kept in optimal range, the properties of the major membrane phospholipids and their contributions to membrane structure and dynamics are summarized. Phospholipid metabolism and known mechanisms of regulation are discussed, including potential sensors for monitoring membrane physical properties. Special attention is paid to processes that maintain the phospholipid class specific molecular species profiles, and to the interplay between phospholipid class and acyl chain composition when yeast membrane lipid homeostasis is challenged. Based on the reviewed studies, molecular species selectivity of the lipid metabolic enzymes, and mass action in acyl-CoA metabolism are put forward as important intrinsic contributors to membrane lipid homeostasis.     

Structural biology of membrane-intrinsic beta-barrel enzymes: sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel beta-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three beta-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipidColon, two colonslipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane beta-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two beta-barrel enzymes of unknown structure; namely, the Salmonella enterica 3'-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O(2) to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how beta-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger beta-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

细菌溶血磷脂的生物合成及生物学功能

溶血磷脂(lysophospholipids, LPLs)是细胞膜中的一类脂质代谢中间产物，主要由磷脂分子被水解后生成。LPL的生物学功能与其前体磷脂有明显的区别。在真核细胞中，LPL是一种参与多种胞内生物信号调控的重要活性分子，但在细菌中，LPL的生物学功能还未被充分揭示。LPL通常是细菌细胞膜中的次要组分，在环境压力条件下其含量可显著升高。除了参与细胞膜磷脂代谢，LPL被认为在细菌环境适应性及致病性中发挥重要作用。其在细胞膜中的累积可以显著提高细菌在环境压力下的存活及增殖效率，同时还是细菌感染过程中重要的信号分子。近期有研究表明，LPL可能是细菌新发现的潜在毒力因子。本文因此将结合最新研究数据，对不同种类LPL的从头合成通路以及LPL在细菌抵御环境压力和细菌-宿主互作等方面所发挥的生物学功能进行综述，为对细菌致病机制和防治细菌感染的相关研究提供新的思路和参考借鉴。     

The outer membrane protein OmpW forms an eight-stranded beta-barrel with a hydrophobic channel

Escherichia coli OmpW belongs to a family of small outer membrane proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. To gain insight into the function of these proteins A we have determined the crystal structure of E. coli OmpW to 2.7-A resolution. The structure shows that OmpW forms an 8-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound n-dodecyl-N,N-dimethylamine-N-oxide detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of n-dodecyl-N,N-dimethylamine-N-oxide. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial outer membrane.     

Antigenic changes in lipopolysaccharide I of Rhizobium leguminosarum bv. viciae in root nodules of Vicia sativa subsp. nigra occur during release from infection threads.

Three different monoclonal antibodies raised against the O antigen-containing lipopolysaccharide (LPS I) of free-living cells were used in an immunocytochemical study to follow the fate of LPS I on the outer membrane of Rhizobium leguminosarum bv. viciae 248 during the nodulation of Vicia sativa subsp. nigra. After immunogold labeling, the LPS I epitopes were detected on the outer membrane of bacteria present in infection threads throughout the nodule. Epitopes were not detectable on bacteria released from the infection thread. The data show that the LPS I epitopes present on rhizobia in infection droplets disappear shortly before or during endocytosis of the bacteria into the host plant cell cytoplasm. The abruptness of the change suggests an active degradation or modification of LPS I epitopes rather than only a repression of their synthesis.     

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel β-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three β-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipid∷lipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane β-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two β-barrel enzymes of unknown structure; namely, the Salmonella enterica 3′-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O₂ to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how β-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger β-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

E. coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

细菌sRNA对环境胁迫的响应机制

细菌小RNA (Small RNAs,sRNAs)是一类长度大约在40?400个核酸之间,不编码蛋白质的RNA,在细菌适应环境方面起重要的调节作用。当环境中温度、营养、外膜蛋白、pH、铁等条件改变时,sRNA常常通过连接双组分信号转导系统和调节蛋白,来传递压力信号并调节应激响应,其作用方式一般是通过碱基互补配对的方式与靶mRNA结合,从而调控靶mRNA的翻译和稳定性;或直接与靶标蛋白质结合,调节靶标蛋白质的生物活性。本文总结了细菌在多种环境压力下,sRNA的调控响应机制。     

E.?coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

The lipid A palmitoyltransferase PagP: molecular mechanisms and role in bacterial pathogenesis

Palmitoylated lipid A can both protect pathogenic bacteria from host immune defences and attenuate the activation of those same defences through the TLR4 signal transduction pathway. A palmitate chain from a phospholipid is incorporated into lipid A by an outer membrane enzyme PagP, which is an 8-stranded antiparallel beta-barrel preceded by an amino-terminal amphipathic alpha-helix. The PagP barrel axis is tilted by 25 degrees with respect to the membrane normal. An interior hydrophobic pocket in the outer leaflet-exposed half of the molecule functions as a hydrocarbon ruler that allows the enzyme to distinguish palmitate from other acyl chains found in phospholipids. Internalization of a phospholipid palmitoyl group within the barrel appears to occur by lateral diffusion from the outer leaflet through non-hydrogen-bonded regions between beta-strands. The MsbA-dependent trafficking of lipids from the inner membrane to the outer membrane outer leaflet is necessary for lipid A palmitoylation in vivo. The mechanisms by which bacteria regulate pagP gene expression strikingly reflect the corresponding pathogenic lifestyle of the bacterium. Variations on PagP structure and function can be illustrated with the known homologues from Gram-negative bacteria, which include pathogens of humans and other mammals in addition to pathogens of insects and plants. The PagP enzyme is potentially a target for the development of anti-infective agents, a probe of outer membrane lipid asymmetry, and a tool for the synthesis of lipid A-based vaccine adjuvants and endotoxin antagonists.     

Leptospira: a spirochaete with a hybrid outer membrane

Leptospira is a genus of spirochaetes that includes organisms with a variety of lifestyles ranging from aquatic saprophytes to invasive pathogens. Adaptation to a wide variety of environmental conditions has required leptospires to acquire a large genome and a complex outer membrane with features that are unique among bacteria. The most abundant surface‐exposed outer membrane proteins are lipoproteins that are integrated into the lipid bilayer by amino‐terminal fatty acids. In contrast to many spirochaetes, the leptospiral outer membrane also includes lipopolysaccharide and many homologues of well‐known beta‐barrel transmembrane outer membrane proteins. Research on leptospiral transmembrane outer membrane proteins has lagged behind studies of lipoproteins because of their aberrant behaviour by Triton X‐114 detergent fractionation. For this reason, transmembrane outer membrane proteins are best characterized by assessing membrane integration and surface exposure. Not surprisingly, some outer membrane proteins that mediate host–pathogen interactions are strongly regulated by conditions found in mammalian host tissues. For example, the leptospiral immunoglobulin‐like (Lig) repeat proteins are dramatically induced by osmolarity and mediate interactions with host extracellular matrix proteins. Development of molecular genetic tools are making it possible to finally understand the roles of these and other outer membrane proteins in mechanisms of leptospiral pathogenesis.     

Structure,biosynthesis and function of unusual lipids A from nodule-inducing and N2-fixing bacteria

This review focuses on the chemistry and structures of (Brady)rhizobium lipids A, indispensable parts of lipopolysaccharides. These lipids contain unusual (ω-1) hydroxylated very long chain fatty acids, which are synthesized by a very limited group of bacteria, besides rhizobia. The significance and requirement of the very long chain fatty acids for outer membrane stability as well as the genetics of the synthesis pathway are discussed. The biological role of these fatty acids for bacterial life in extremely different environments (soil and intracellular space within nodules) is also considered.     

Transport of outer membrane lipids in mycobacteria

The complex organization of the mycobacterial cell wall poses unique challenges for the study of its assembly. Although mycobacteria are classified evolutionarily as Gram-positive bacteria, their cell wall architecture more closely resembles that of Gram-negative organisms. They possess not only an inner cytoplasmic membrane, but also a bilayer outer membrane that encloses an aqueous periplasm and includes diverse lipids that are required for the survival and virulence of pathogenic species. Questions surrounding how mycobacterial outer membrane lipids are transported from where they are made in the cytoplasm to where they function at the cell exterior are thus similar, and similarly compelling, to those that have driven the study of Gram-negative outer membrane transport pathways. However, little is understood about these processes in mycobacteria. Here we contextualize these questions by comparing our current knowledge of mycobacteria with better-defined systems in other organisms. Based on this analysis, we propose possible models and highlight continuing challenges to improving our understanding of outer membrane assembly in these medically and environmentally important bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Modeling of mechanosensitive channel gating in response to wall shear stress

The accurate biological function of mechanosensitive (MS) channels is crucial for maintaining the viability of living cells. For instance, in vascular endothelial cells, calcium influx from the extracellular environment into cytoplasm is regulated by stretch-activated channels. However, the mechanism by which cells sense force remains unclear. For this study, we hypothesized that gating of ion channels is simply regulated by the direct mechanical stress induced in a membrane. We modeled a membrane channel using crystallographic data of the bacteria Mycobacterium tuberculosis (Tb-MscL) because MscL homologs are integral membrane proteins with sequence similarity to most known ion channels. Molecular dynamics (MD) simulations were performed to elucidate the gating mechanism of the channel protein in response to the fluid shear stress. Results suggest that the stretched membrane drives the interfacial part of the protein–membrane complex to expand and maintains the stability of the constricted part of the transmembrane pore. Moreover, structural similarities between Tb-MscL and the family of ligand-gated ion channels suggest that the conformational change of this model in response to fluid shear stress is useful for modeling the gating mechanism in a broad class of gated channels.     

Structure and mechanism of the bacterial lipid ABC transporter,MlaFEDB

The cell envelope of Gram-negative bacteria is composed of an inner membrane, outer membane, and an intervening periplasmic space. How the outer membrane lipids are trafficked and assembled there, and how the asymmetry of the outer membrane is maintained is an area of intense research. The Mla system has been implicated in the maintenance of lipid asymmetry in the outer membrane, and is generally thought to drive the removal of mislocalized phospholipids from the outer membrane and their retrograde transport to the inner membrane. At the heart of the Mla pathway is a structurally unique ABC transporter complex in the inner membrane, called MlaFEDB. Recently, an explosion of cryo-EM studies has begun to shed light on the structure and lipid translocation mechanism of MlaFEDB, with many parallels to other ABC transporter families, including human ABCA and ABCG, as well as bacterial lipopolysaccharide and O-antigen transporters. Here we synthesize information from all available structures, and propose a model for lipid trafficking across the cell envelope by MlaFEDB.