Outer Membrane Phospholipase A Research Articles

Allosteric modulation of integral protein activity by differential stress in asymmetric membranes.

The activity of integral membrane proteins is tightly coupled to the properties of the surrounding lipid matrix. In particular, transbilayer asymmetry, a hallmark of all plasma membranes, might be exploited to control membrane-protein activity. Here, we hypothesized that the membrane-embedded enzyme outer membrane phospholipase A (OmpLA) is susceptible to the lateral pressure differences that build up between such asymmetric membrane leaflets. Upon reconstituting OmpLA into synthetic, chemically well-defined phospholipid bilayers exhibiting different lateral pressure profiles, we indeed observed a substantial decrease in the enzyme's hydrolytic activity with increasing membrane asymmetry. No such effects were observed in symmetric mixtures of the same lipids. To quantitatively rationalize how the differential stress in asymmetric lipid bilayers inhibits OmpLA, we developed a simple allosteric model within the lateral pressure framework. Thus, we find that membrane asymmetry can serve as the dominant factor in controlling membrane-protein activity, even in the absence of specific, chemical cues or other physical membrane determinants such as hydrophobic mismatch.

Inactivation of the Mla system and outer-membrane phospholipase A results in disrupted outer-membrane lipid asymmetry and hypervesiculation in Bordetella pertussis

Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. With the goal of improving the production of outer-membrane vesicles (OMVs), we studied here the mechanisms that are involved in maintaining lipid asymmetry in the outer membrane of this organism. We identified homologues of the phospholipid (PL)-transport systems Mla and Pqi and of outer-membrane phospholipase A (OMPLA). Inactivation of mlaF, encoding the ATPase of the Mla system, together with pldA, which encodes OMPLA, resulted in an accumulation of PLs at the cell surface as demonstrated by the binding of a phosphatidylethanolamine-specific fluorescent probe to intact cells of this strain. The corresponding single mutations did hardly or not affect binding of the probe. These results are consistent with a retrograde transport directionality of the Mla system in B. pertussis and indicate that PLs accumulating at the cell surface in the mlaF mutant are degraded by OMPLA. Consequently, the mlaF mutant showed a conditional growth defect due to the production of free fatty acids by OMPLA, which could be compensated by inactivation of OMPLA or by sequestration of the produced fatty acids with starch. The mlaF pldA double mutant showed markedly increased OMV production, and representative antigens were detected in these OMVs as in wild-type OMVs. Further phenotypic characterization showed that the barrier function of the outer membrane of the mlaF pldA mutant was compromised as manifested by increased susceptibility to SDS and to several antibiotics. Moreover, inactivation of mlaF alone or together with pldA resulted in increased biofilm formation, which was, however, not directly related to increased vesiculation as the addition of purified OMVs to the wild-type strain decreased biofilm formation. We conclude that the absence of MlaF together with OMPLA results in PL accumulation in the outer leaflet of the outer membrane, and the increased vesiculation of the mutant could be useful in the development of novel, OMV-based pertussis vaccines.

Control of Outer Membrane Phospholipase a (OmpLA) Functions on an Artificial Lipid Bilayer Membrane

Giant lipid vesicles consisted of a lipid bilayer membrane are used to investigate the function of several membrane proteins in a living cell. The membrane protein-reconstituted giant lipid vesicles, which emulated cellular functions, was created. However, to occur the complex functions into the giant lipid vesicles, the generation of giant lipid vesicles containing a diverse set of membrane proteins at sufficient concentrations is difficult.In this study, we observe dual functions (small molecule transportation and budding of small vesicle) on an artificial lipid bilayer using Outer membrane phospholipase A (OmpLA) expressed in E.Coli outer membranes. OmpLA has not only enzyme activity but also the function of molecular transportation. In the presence of calcium ions, OmpLA catalyzes phospholipids to lysolipid and fatty acid. By measuring ion currents of OmpLA nanopores using a patch-clamp method of the artificial lipid bilayer, the reconstitution of OmpLA into artificial lipid bilayers was promoted by adding anionic and cationic lipids. To the best of our knowledge, we have revealed for the first time that the lipid composition controlled OmpLA orientation into the lipid bilayer because hydrophilic region of OmpLA has asymmetric distribution of charge. In addition, we observed the transportation of small molecules through OmpLA pore on giant lipid vesicles. Finally, by control of OmpLA orientation, we showed a budding of small vesicles from giant lipid vesicles by changing a curvature of the lipid membranes due to OmpLA enzyme activity. The giant lipid vesicles containing OmpLA will be contributed to regulate cell functions and recognize the living cells by changing the external environment of the artificial cell model.

PMAP-36 reduces the innate immune response induced by Bordetella bronchiseptica-derived outer membrane vesicles

Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines.

Structures of Unfolded Outer Membrane Proteins in Complex with Chaperones

The viability of Gram-negative bacteria is ensured by the presence of outer membrane proteins (OMPs) acting as channels, porins, lipases and receptors. They are considered as interesting drug targets and thus obtaining a molecular perspective about various stages of their biogenesis is of great interest. However, the biogenesis of OMPs is biologically puzzling because it is not driven by ATP. We lack a detailed understanding of the various steps involved in this process. Unlike other proteins, OMPs adopt an active conformation only on reaching the outer membrane and thus have to be transferred from the cytoplasm through the periplasm in a folding competent state. Chaperones like Seventeen kilodalton protein (Skp) and Survival Factor A (SurA) protect these nascent hydrophobic proteins against aggregation. Yet, it remains elusive which conformations and interaction dynamics occur in the chaperone and unstructured OMP interplay. Using single molecule Förster Resonance Energy Transfer (smFRET), we investigate the structure of unfolded OMPs in chaperone-bound and unbound states. We find that both OMPs adopt a rather compact conformation in a low denaturant buffer. Remarkably, when bound with periplasmic chaperones, we observe that this compact state is dissolved by the chaperones differently for both OMPs. The small 8 beta-stranded OmpX shows heterogeneous conformations with sub-millisecond dynamics, while the 12 beta-stranded Outer membrane phospholipase A (OmpLA) appears to be pulled and pushed upon by the chaperones in expanded conformations. In both the cases, the chaperone-OMP interactions were remarkably long-lasting for hours, even below the dissociation constant. Our findings hint towards mechanisms by which OMPs are arranged in a folding competent state before reaching the outer membrane and shed light on the elusive mechanism of chaperone-OMP interaction in periplasm.

Slow Interconversion in a Conformationally Heterogeneous Unfolded-State Ensemble of Outer Membrane Phospholipase A

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of Outer Membrane Phospholipase A (OmpLA) to provide a detailed view of its structural dynamics from nanoseconds to milliseconds. We find that, even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states as compared with a random-coil polypeptide may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.

Slow Interconversion in a Heterogeneous Unfolded-State Ensemble of Outer-Membrane Phospholipase A

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of outer-membrane phospholipase A (OmpLA) to provide a detailed view of its structural dynamics on timescales from nanoseconds to milliseconds. We find that even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain-reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states, as compared with a random-coil polypeptide, may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.

Outer membrane phospholipase A\u2019s roles in Helicobacter pylori acid adaptation

BackgroundThe pH of the human gastric mucosa varies around 2.5 so that only bacteria with strong acidic stress tolerance can colonize it. The ulcer causing Helicobacter pylori thrives in the gastric mucosa. We analyse the roles of the key outer membrane protein OMPLA in its roles in acid tolerance.ResultsThe homology model of Helicobacter pylori outer membrane phospholipase A (OMPLA) reveals a twelve stranded β-barrel with a pore that allows molecules to pass with a diameter up to 4 Å. Structure based multiple sequence alignments revealed the functional roles of many amino acids, and led to the suggestion that OMPLA has multiple functions. Besides its role as phospholipase it lets urea enter and ammonium exit the periplasm. Combined with an extensive literature study, our work leads to a comprehensive model for H. pylori’s acid tolerance. This model is based on the conversion of urea into ammonium, and it includes multiple roles for OMPLA and involves two hitherto little studied membrane channels in the OMPLA operon.ConclusionThe three-dimensional model of OMPLA predicts a transmembrane pore that can aid H. pylori’s acid tolerance through urea influx and ammonium efflux. After urea passes through OMPLA into the periplasm, it passes through the pH-gated inner membrane channel UreI into the cytoplasm where urease hydrolyses it into NH3 and CO2. Most of the NH3 becomes NH4+ that is likely to need an inner membrane channel to reach the periplasm. Two genes that are co-regulated with OMPLA in gastric Helicobacter operons could aid this transport. The NH4+ that might leave the cell through the OMPLA pore has been implicated in H. pylor’s pathogenesis.

Real-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy

Phospholipases are abundant in various types of cells and compartments, where they play key roles in physiological processes as diverse as digestion, cell proliferation, and neural activation. In Gram-negative bacteria, outer membrane phospholipase A (OmpLA) is involved in outer-membrane lipid homeostasis and bacterial virulence. Although the enzymatic activity of OmpLA can be probed with an assay relying on an artificial monoacyl thioester substrate, only little is known about its activity on diacyl phospholipids. Here, we used high-speed atomic force microscopy (HS-AFM) to directly image enzymatic phospholipid degradation by OmpLA in real time. In the absence of Ca2+, reconstituted OmpLA diffused within a phospholipid bilayer without revealing any signs of phospholipase activity. Upon the addition of Ca2+, OmpLA was activated and degraded the membrane with a turnover of ~2 phospholipid molecules per second and per OmpLA dimer until most of the membrane phospholipids were hydrolyzed and the protein became tightly packed.

The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri.

Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

Selective Pressure for Rapid Membrane Integration Constrains the Sequence of Bacterial Outer Membrane Proteins

The Moon-Fleming hydrophobicity scale suggests that the small thermodynamic penalty for inserting a basic residue into the lipid bilayer of the bacterial outer membrane (OM) does not account for the extreme evolutionary selection against arginine residues on the lipid-facing surfaces of OM proteins (OMPs). To explain this phenomenon, we investigated the consequences of introducing lipid-facing arginine residues into the β-barrel of two E. coli OMPs, an autotransporter (EspP) and outer membrane phospholipase A (OmpLA). We found that lipid-facing arginine residues do not affect the targeting of EspP to the Bam complex, an essential hetero-oligomer that catalyzes the membrane integration of OMPs, but markedly slow the membrane integration of the β-barrel domain. OmpLA mutants that contain lipid-facing arginine residues were likewise slowly incorporated into the OM in a strain that lacks the periplasmic protease DegP. In contrast to the EspP variants, however, the OmpLA mutants were degraded before they were inserted into the OM in a wild-type strain. The OmpLA arginine variants also folded slowly into large unilamellar vesicles in vitro. Taken together, the results suggest that lipid-facing arginine residues are strongly disfavored because proteins that assemble slowly are captured by the periplasmic quality control system through a kinetic partitioning mechanism or form prolonged interactions with the Bam complex that are potentially deleterious. Our results not only explain a key constraint on the sequence of OMPs, but also reveal important differences in the early stages of assembly of distinct classes of these proteins.

Modulating bilayer mechanical properties to promote the coupled folding and insertion of an integral membrane protein.

Bilayer mechanical properties are not only of crucial importance to the mechanism of action of mechanosensation in lipid membranes but also affect preparative laboratory taskssuch as membrane-protein refolding. We report this for coupled refolding and bilayer insertion of outer membrane phospholipaseA (OmpLA), an integral membrane enzyme that catalyses the hydrolytic cleavage of glycerophospholipids. OmpLA can be refolded into a variety of detergent micelles and unilamellar vesicles composed of short-chain phospholipids but, in the absence of chemical or molecular chaperones, not into thicker membranes. Controlled modulation of bilayer mechanical properties by judicious use of subsolubilising concentrations of detergents induces monolayer curvature strain, acyl chain fluidisation, membrane thinning, and transient aqueous bilayer defects. This enables quantitative and functional refolding of OmpLA even into bilayer membranes composed of long-chain phospholipids to yield enzymatically active proteoliposomes without requiring membrane solubilisation.

Evolutionary Avoidance of Transmembrane Embedded Arginines is due to Slowed Folding Kinetics

Conserved transmembrane (TM) arginines are essential for the transport function of many membrane proteins, such as voltage-gated ion channels. However, among the lipid-facing residues in TM regions, the statistical propensity of arginines is extremely low. The minimal occurrence of TM arginines is often attributed to the thermodynamic cost of inserting an arginine into a hydrophobic lipid environment. But the energetic cost of TM arginine insertion measured by the Moon-Fleming Hydrophobicity scale is only 3 kcal mol-1, implying that Arg should be allowed in the bilayer. To investigate the discrepancy between this moderate thermodynamic cost and the evolutionary avoidance of TM arginines, we utilized Outer Membrane Phospholipase A (OmpLA) as a host for lipid-facing arginine mutations, at three different depths in the bilayer. We probed the effects of the introduction of TM arginines on the secondary structure and enzymatic activity of OmpLA and found that Arg has a minimal impact on the structure and function of OmpLA. We also determined that a TM Arg does not affect the thermodynamic binding of OmpLA to the periplasmic chaperones, SurA and Skp. Molecular dynamics simulations revealed that all TM Args studied form hydrogen bonds with water and phospholipid head groups. Kinetics of folding into large unilamellar vesicles of varying head group compositions reveal that the R-variants exhibit slow folding, even in the presence of the OMP assembly machinery protein, BamA. Slow kinetics are additionally observed for in vivo folding of these R-variants. In addition to a thermodynamic cost for TM arginine insertion our results indicate that a pronounced kinetic barrier may play a role in the evolutionary pressure against TM arginines.

Aromatic Residues Display an Energetic Depth-Dependence in Lipid Bilayers that can be Modulated by Nearest-Neighbor Interactions

The interfacial region of the bilayer is a complex environment composed of lipid head groups, phosphates, diglycerides and a gradient of water molecule density. Due to the statistical preference of the aromatic residues for this area of the bilayer, it is generally believed that these residues are important for both folding and positioning of proteins within the membrane. To interrogate if this preference has a thermodynamic basis, we measured the water-to-lipid free energy changes for the aromatic side chains as a function of depth in the lipid bilayer. We used outer membrane phospholipase A (OmpLA) as a host membrane protein to introduce lipid-facing amino acid substitutions. At twelve sites, we mutated host side chains to alanine, tryptophan, tyrosine and phenylalanine. Chemical denaturation and thermodynamic linkage were employed to measure side chain water-to-lipid free energy changes. These quantities were calculated by taking the difference between stabilities (ΔΔGow,l) of OmpLA aromatic variants from that of alanine, which was used as a reference state. The experimental data can be interpreted as a sum of water-to-lipid transfer free energies and specific nearest neighbor interactions between certain side chains. We used molecular dynamics simulations and double variant cycles to model and quantify these nearest neighbor interactions. Altogether, our findings indicate that the tryptophan and tyrosine aromatic side chains exhibit a depth-dependence in ΔΔGow,l values whereas phenylalanine shows no such trend. These data demonstrate that the enrichment of tryptophan and tyrosine in interfacial regions of bilayers is due to their favorable contributions to the thermodynamic stabilities of membrane proteins.

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.

A Molecular Dynamics Simulation Study of Outer Membrane Phospholipase a (OMPLA) Structure and Dynamics in an Asymmetric Lipopolysaccharide Membrane

The outer membrane of Gram-negative bacteria is a unique and highly asymmetric lipid bilayer composed of phospholipids in the inner leaflet and mostly lipopolysaccharide (LPS) in the outer leaflet. Outer membrane phospholipase A (OmpLA) is an integral membrane enzyme in Escherichia coli. The structure of monomeric OmpLA consists of a 12-stranded antiparallel β-strands with a convex and a flat side, six loops at the extracellular side and five turns at the periplasmic side of the membrane. Utilizing the latest C36 CHARMM lipid and carbohydrate force field, we have constructed a model of OmpLA embedded in an asymmetric lipid bilayer with rough LPS molecules (without O-antigen) in one leaflet and phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in the other leaflet to model the realistic outer membrane environment. The simulation results will be discussed in terms of the key structural properties of the bacterial outer membrane including hydrophobic thickness, area per lipid, and acyl chain order parameter. We will also show the difference of OmpLA structure and dynamics compared to that in a DLPC bilayer. At the same time a comparison between simulations with different numbers of LPS molecules on the outer leaflet will elucidate the potential technical difficulties in building asymmetric bilayer.

Interleukin-8 is the single most up-regulated gene in whole genome profiling of H. pylori exposed gastric epithelial cells.

BackgroundThe association between Helicobacter pylori infection and upper gastrointestinal disease is well established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the inflammatory response to OMPLA+ and OMPLA- H. pylori variants.ResultsInterleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the epithelial cell response to H. pylori infection and in the pathological processes leading to gastric disease. MAPK and NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response. There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation. We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory epithelial cell gene response in this experiment.ConclusionIn whole genome analysis of the epithelial response to H. pylori exposure, IL-8 demonstrated the most marked up-regulation, and was involved in many of the most important cellular response processes to the infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24 h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA+/-did not affect the acute inflammatory response to H. pylori.

In silico evolutionary analysis of Helicobacter pylori outer membrane phospholipase A (OMPLA)

BackgroundIn the past decade, researchers have proposed that the pldA gene for outer membrane phospholipase A (OMPLA) is important for bacterial colonization of the human gastric ventricle. Several conserved Helicobacter pylori genes have distinct genotypes in different parts of the world, biogeographic patterns that can be analyzed through phylogenetic trees. The current study will shed light on the importance of the pldA gene in H. pylori. In silico sequence analysis will be used to investigate whether the bacteria are in the process of preserving, optimizing, or rejecting the pldA gene. The pldA gene will be phylogenetically compared to other housekeeping (HK) genes, and a possible origin via horizontal gene transfer (HGT) will be evaluated through both intra- and inter-species evolutionary analyses.ResultsIn this study, pldA gene sequences were phylogenetically analyzed and compared with a large reference set of concatenated HK gene sequences. A total of 246 pldA nucleotide sequences were used; 207 were from Norwegian isolates, 20 were from Korean isolates, and 19 were from the NCBI database. Best-fit evolutionary models were determined with MEGA5 ModelTest for the pldA (K80 + I + G) and HK (GTR + I + G) sequences, and maximum likelihood trees were constructed. Both HK and pldA genes showed biogeographic clustering. Horizontal gene transfer was inferred based on significantly different GC contents, the codon adaptation index, and a phylogenetic conflict between a tree of OMPLA protein sequences representing 171 species and a tree of the AtpA HK protein for 169 species. Although a vast majority of the residues in OMPLA were predicted to be under purifying selection, sites undergoing positive selection were also found.ConclusionsOur findings indicate that the pldA gene could have been more recently acquired than seven of the HK genes found in H. pylori. However, the common biogeographic patterns of both the HK and pldA sequences indicated that the transfer occurred long ago. Our results indicate that the bacterium is preserving the function of OMPLA, although some sites are still being evolutionarily optimized.

Delineation of the translocation of colicin E7 across the inner membrane of Escherichia coli

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.

Measuring Side-Chain Hydrophobicity using a Whole Transmembrane Protein in Lipid Bilayers

Membrane protein behavior should be predictable from first principles of physical chemistry. One such first principle is the hydrophobic effect, which may be the foremost reason membrane proteins are stable in lipid bilayers. Several methods exist for estimating the hydrophobicity of proteins in membranes by modeling the energies felt by individual amino acid side-chains in hydrophobic environments. However, none of these existing methods have measured side-chain energetics directly from a whole multi-pass membrane protein in a lipid bilayer. Consequently, the different hydrophobicity scales that have been proposed to be relevant for membrane proteins disagree on the magnitude of the first principles, especially the principles controlling the interaction of ionizable side-chains (such as Arginine) with membranes. Here, we establish a new whole protein hydrophobicity scale, which we measured using rigorous equilibrium thermodynamic methods with a multi-pass transmembrane protein that was folded and inserted in a phospholipid bilayer. Our protein was the Outer Membrane Phospholipase A (OMPLA) from E. coli . This beta-barrel protein yields results that are relevant for all membrane proteins, even alpha-helical ones, because the contribution of the backbone conformation is subtracted from our final measurements. We were also able to use OMPLA to measure how the energetics of side-chain insertion vary with depth in the bilayer. Both Arginine and Leucine have their most extreme insertion energies when they are closest to the middle of the hydrophobic region of OMPLA. Further, we carried out a double mutant cycle with Arginines and discovered that the insertion of a second Arginine is aided by the insertion of the first. This result is particularly relevant for understanding the function of the voltage sensing domains of some ion channels, which may involve multiple Arginines penetrating the lipid bilayer.