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Jan Tommassen - One of the best experts on this subject based on the ideXlab platform.
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Biogenesis of the gram-negative bacterial Outer Membrane
Annual Review of Microbiology, 2007Co-Authors: Martine P. Bos, Viviane Robert, Jan TommassenAbstract:The cell envelope of gram-negative bacteria consists of two Membranes, the inner and the Outer Membrane, that are separated by the periplasm. The Outer Membrane consists of phospholipids, lipopolysaccharides, integral Membrane proteins, and lipoproteins. These components are synthesized in the cytoplasm or at the inner leaflet of the inner Membrane and have to be transported across the inner Membrane and through the periplasm to assemble eventually in the correct Membrane. Recent studies in Neisseria meningitidis and Escherichia coli have led to the identification of several machineries implicated in these transport and assembly processes.
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Lipopolysaccharide Transport to the Bacterial Outer Membrane in Spheroplasts
Journal of Biological Chemistry, 2004Co-Authors: Boris Tefsen, Jan Tommassen, Jeroen Geurtsen, Frank Beckers, Hans De CockAbstract:The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner Membrane to the Outer Membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the Outer Membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner Membrane. The Outer Membrane localization of the LPS was confirmed by its modification by the Outer Membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the Outer Membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two Membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the Outer Membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the Outer Membrane.
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Biogenesis of the Gram-Negative Bacterial Outer Membrane
Current Opinion in Microbiology, 2004Co-Authors: Martine P. Bos, Jan TommassenAbstract:Gram-negative bacteria are bounded by two Membranes. The Outer Membrane consists of phospholipids, lipopolysaccharides, lipoproteins and integral Outer Membrane proteins, all of which are synthesized in the cytoplasm. Recently, much progress has been made in the elucidation of the mechanisms of transport of these molecules over the inner Membrane, through the periplasm and into the Outer Membrane, in part by exploiting the extraordinary capacity of Neisseria meningitidis to survive without lipopolysaccharide.
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role of a highly conserved bacterial protein in Outer Membrane protein assembly
Science, 2003Co-Authors: Rome Voulhoux, Jeroen Geurtsen, Martine P. Bos, Maarten Mols, Jan TommassenAbstract:After transport across the cytoplasmic Membrane, bacterial Outer Membrane proteins are assembled into the Outer Membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various Outer Membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of Outer Membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in Outer Membrane protein assembly.
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Folding of a bacterial Outer Membrane protein during passage through the periplasm
The EMBO Journal, 1997Co-Authors: Elaine F Eppens, Nico Nouwen, Jan TommassenAbstract:The transport of bacterial Outer Membrane proteins to their destination might be either a one-step process via the contact zones between the inner and Outer Membrane or a two-step process, implicating a periplasmic intermediate that inserts into the Membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the Outer Membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the Outer Membrane.
Meta J Kuehn - One of the best experts on this subject based on the ideXlab platform.
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Outer Membrane vesiculation facilitates surface exchange and in vivo adaptation of vibrio cholerae
Cell Host & Microbe, 2020Co-Authors: Franz G Zingl, Meta J Kuehn, Paul Kohl, Fatih Cakar, Deborah R Leitner, Fabian Mitterer, Katherine E Bonnington, Gerald Rechberger, Ziqiang Guan, Joachim ReidlAbstract:Summary Gram-negative bacteria release Outer Membrane vesicles into the external milieu to deliver effector molecules that alter the host and facilitate virulence. Vesicle formation is driven by phospholipid accumulation in the Outer Membrane and regulated by the phospholipid transporter VacJ/Yrb. We use the facultative human pathogen Vibrio cholerae to show that VacJ/Yrb is silenced early during mammalian infection, which stimulates vesiculation that expedites bacterial surface exchange and adaptation to the host environment. Hypervesiculating strains rapidly alter their bacterial Membrane composition and exhibit enhanced intestinal colonization fitness. This adaptation is exemplified by faster accumulation of glycine-modified lipopolysaccharide (LPS) and depletion of Outer Membrane porin OmpT, which confers resistance to host-derived antimicrobial peptides and bile, respectively. The competitive advantage of hypervesiculation is lost upon pre-adaptation to bile and antimicrobial peptides, indicating the importance of these adaptive processes. Thus, bacteria use Outer Membrane vesiculation to exchange cell surface components, thereby increasing survival during mammalian infection.
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Outer Membrane vesicles from gram negative bacteria biogenesis and functions
Nature Reviews Microbiology, 2015Co-Authors: Carmen Schwechheimer, Meta J KuehnAbstract:In this Review, Schwechheimer and Kuehn describe recent developments in elucidating the mechanisms of biogenesis and cargo selection of the Outer-Membrane vesicles (OMVs) produced by Gram-negative bacteria. They also discuss the functions of OMVs in bacterial physiology and during pathogenesis. Outer-Membrane vesicles (OMVs) are spherical buds of the Outer Membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles.
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genome wide assessment of Outer Membrane vesicle production in escherichia coli
PLOS ONE, 2015Co-Authors: Adam Kulp, Andrew J. Manning, Bo Sun, Nichole Orenchrivera, Amy K Schmid, Meta J KuehnAbstract:The production of Outer Membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate Outer Membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for Outer Membrane vesiculation in E. coli.
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contribution of bacterial Outer Membrane vesicles to innate bacterial defense
BMC Microbiology, 2011Co-Authors: Andrew J. Manning, Meta J KuehnAbstract:Background Outer Membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate Outer Membrane vesiculation as a bacterial mechanism for immediate short-term protection against Outer Membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection.
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contribution of bacterial Outer Membrane vesicles to innate bacterial defense
BMC Microbiology, 2011Co-Authors: Andrew J. Manning, Meta J KuehnAbstract:Outer Membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate Outer Membrane vesiculation as a bacterial mechanism for immediate short-term protection against Outer Membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified Outer Membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the Outer Membrane of Gram-negative bacteria.
Shelley Hasenoehrl - One of the best experts on this subject based on the ideXlab platform.
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a β barrel Outer Membrane protein facilitates cellular uptake of polychlorophenols in cupriavidus necator
Biodegradation, 2010Co-Authors: Sara M Belchik, Scott Schaeffer, Shelley HasenoehrlAbstract:The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a Membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a β-barrel Outer Membrane protein, similar to the Escherichia coli Outer Membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an Outer Membrane protein that facilitates the passing of polychlorophenols across the Outer Membrane of C. necator. Similarly, other COG4313 proteins are possibly Outer Membrane transporters of hydrophobic aromatic compounds.
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a β barrel Outer Membrane protein facilitates cellular uptake of polychlorophenols in cupriavidus necator
Biodegradation, 2010Co-Authors: Sara M Belchik, Scott Schaeffer, Shelley HasenoehrlAbstract:The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a Membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a β-barrel Outer Membrane protein, similar to the Escherichia coli Outer Membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an Outer Membrane protein that facilitates the passing of polychlorophenols across the Outer Membrane of C. necator. Similarly, other COG4313 proteins are possibly Outer Membrane transporters of hydrophobic aromatic compounds.
Susan K. Buchanan - One of the best experts on this subject based on the ideXlab platform.
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structural insight into mitochondrial beta barrel Outer Membrane protein biogenesis
bioRxiv, 2020Co-Authors: Kathryn A Diederichs, Istvan Botos, Sarah E Rollauer, Xiaofeng Tan, Martin S King, Edmund R S Kunji, Jiansen Jiang, Susan K. BuchananAbstract:In mitochondria, {beta}-barrel Outer Membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold {beta}-barrel proteins and insert them into the mitochondrial Outer Membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transMembrane {beta}-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the interMembrane space. Sam35 and Sam37 are located on the cytosolic side of the Outer Membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 {beta}-barrel opens a lateral gate to accommodate its substrates. The SAM complex structure suggests how it interacts with other mitochondrial Outer Membrane proteins to create supercomplexes.
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structural insight into mitochondrial β barrel Outer Membrane protein biogenesis
bioRxiv, 2020Co-Authors: Kathryn A Diederichs, Istvan Botos, Sarah E Rollauer, Xiaofeng Tan, Martin S King, Edmund R S Kunji, Jiansen Jiang, Susan K. BuchananAbstract:Abstract In mitochondria, β-barrel Outer Membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold β-barrel proteins and insert them into the mitochondrial Outer Membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transMembrane β-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the interMembrane space. Sam35 and Sam37 are located on the cytosolic side of the Outer Membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 β-barrel opens a lateral gate to accommodate its substrates. The SAM complex structure suggests how it interacts with other mitochondrial Outer Membrane proteins to create supercomplexes.
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Insertion of proteins and lipopolysaccharide into the bacterial Outer Membrane
Philosophical Transactions of the Royal Society B: Biological Sciences, 2017Co-Authors: Istvan Botos, Nicholas Noinaj, Susan K. BuchananAbstract:The bacterial Outer Membrane contains phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the Outer leaflet. Both proteins and LPS must be frequently inserted into the Outer Membrane to preserve its integrity. The protein complex that inserts LPS into the Outer Membrane is called LptDE, and consists of an integral Membrane protein, LptD, with a separate globular lipoprotein, LptE, inserted in the barrel lumen. The protein complex that inserts newly synthesized Outer-Membrane proteins (OMPs) into the Outer Membrane is called the BAM complex, and consists of an integral Membrane protein, BamA, plus four lipoproteins, BamB, C, D and E. Recent structural and functional analyses illustrate how these two complexes insert their substrates into the Outer Membrane by distorting the Membrane component (BamA or LptD) to directly access the lipid bilayer. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.
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heat modifiability of Outer Membrane proteins from gram negative bacteria
Methods of Molecular Biology, 2015Co-Authors: Nicholas Noinaj, Adam Kuszak, Susan K. BuchananAbstract:β-barrel Membrane proteins are somewhat unique in that their folding states can be monitored using semi-native SDS-PAGE methods to determine if they are folded properly or not. This property, which is commonly referred to as heat modifiability, has been used for many years on both purified protein and on whole cells to monitor folded states of proteins of interest. Additionally, heat modifiability assays have proven indispensable in studying the BAM complex and its role in folding and inserting β-barrel Membrane proteins into the Outer Membrane. Here, we describe the protocol our lab uses for performing the heat modifiability assay in our studies on Outer Membrane proteins.
Martine P. Bos - One of the best experts on this subject based on the ideXlab platform.
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Biogenesis of the gram-negative bacterial Outer Membrane
Annual Review of Microbiology, 2007Co-Authors: Martine P. Bos, Viviane Robert, Jan TommassenAbstract:The cell envelope of gram-negative bacteria consists of two Membranes, the inner and the Outer Membrane, that are separated by the periplasm. The Outer Membrane consists of phospholipids, lipopolysaccharides, integral Membrane proteins, and lipoproteins. These components are synthesized in the cytoplasm or at the inner leaflet of the inner Membrane and have to be transported across the inner Membrane and through the periplasm to assemble eventually in the correct Membrane. Recent studies in Neisseria meningitidis and Escherichia coli have led to the identification of several machineries implicated in these transport and assembly processes.
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Biogenesis of the Gram-Negative Bacterial Outer Membrane
Current Opinion in Microbiology, 2004Co-Authors: Martine P. Bos, Jan TommassenAbstract:Gram-negative bacteria are bounded by two Membranes. The Outer Membrane consists of phospholipids, lipopolysaccharides, lipoproteins and integral Outer Membrane proteins, all of which are synthesized in the cytoplasm. Recently, much progress has been made in the elucidation of the mechanisms of transport of these molecules over the inner Membrane, through the periplasm and into the Outer Membrane, in part by exploiting the extraordinary capacity of Neisseria meningitidis to survive without lipopolysaccharide.
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role of a highly conserved bacterial protein in Outer Membrane protein assembly
Science, 2003Co-Authors: Rome Voulhoux, Jeroen Geurtsen, Martine P. Bos, Maarten Mols, Jan TommassenAbstract:After transport across the cytoplasmic Membrane, bacterial Outer Membrane proteins are assembled into the Outer Membrane. Meningococcal Omp85 is a highly conserved protein in Gram-negative bacteria, and its homolog Toc75 is a component of the chloroplast protein-import machinery. Omp85 appeared to be essential for viability, and unassembled forms of various Outer Membrane proteins accumulated upon Omp85 depletion. Immunofluorescence microscopy revealed decreased surface exposure of Outer Membrane proteins, which was particularly apparent at the cell-division planes. Thus, Omp85 is likely to play a role in Outer Membrane protein assembly.
