scholarly journals Determination of the molecular basis for coprogen import by Gram-negative bacteria

IUCrJ ◽  
2019 ◽  
Vol 6 (3) ◽  
pp. 401-411 ◽  
Author(s):  
Rhys Grinter ◽  
Trevor Lithgow
Keyword(s):  
Outer Membrane ◽  
Molecular Basis ◽  
Structural Data ◽  
Structural Basis ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Highly Active ◽  
Hydroxamate Siderophores ◽  
Membrane Barrier

In order to survive in mixed microbial communities, some species of fungi secrete coprogens, siderophores that facilitate capture of the scarce nutrient iron. The TonB-dependent transporter FhuE is integrated in the outer membrane of Gram-negative bacteria and has been reported to scavenge these fungally produced coprogens. In this work, anEscherichia colistrain was engineered that is dependent solely on FhuE for its access to siderophore-sequestered iron. Using this tool, it is shown that while FhuE is highly active in the import of coprogens, it has some level of promiscuity, acting as a low-affinity transporter for related siderophores. The crystal structure of FhuE in complex with coprogen was determined, providing a structural basis to explain this selective promiscuity. The structural data, in combination with functional analysis, presented in this work show that FhuE has evolved to specifically engage with planar siderophores. A potential evolutionary driver, and a critical consequence of this selectivity, is that it allows FhuE to exclude antibiotics that mimic nonplanar hydroxamate siderophores: these toxic molecules could otherwise cross the outer membrane barrier through a Trojan horse mechanism.

scholarly journals Trans-envelope multidrug efflux pumps of Gram-negative bacteria and their synergism with the outer membrane barrier

2018 ◽  
Vol 169 (7-8) ◽  
pp. 351-356 ◽  
Author(s):  
Helen I. Zgurskaya ◽  
Valentin V. Rybenkov ◽  
Ganesh Krishnamoorthy ◽  
Inga V. Leus
Keyword(s):  
Outer Membrane ◽  
Efflux Pumps ◽  
Gram Negative Bacteria ◽  
Multidrug Efflux ◽  
Gram Negative ◽  
Multidrug Efflux Pumps ◽  
Membrane Barrier

The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3982-3991 ◽  
Author(s):  
Ana Sauri ◽  
Zora Soprova ◽  
David Wickström ◽  
Jan-Willem de Gier ◽  
Roel C. Van der Schors ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Critical Role ◽  
Structural Data ◽  
Intermediate Stage ◽  
Gram Negative Bacteria ◽  
Passenger Domain ◽  
Gram Negative ◽  
Periplasmic Chaperone ◽  
Folded Structures

Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms a β-barrel pore in the outer membrane implicated in translocation of the upstream ‘passenger’ domain across the outer membrane. However, recent structural data suggest that the diameter of the β-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage the β-domain was not inserted and folded as an integral β-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Bam (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane.

scholarly journals Genetic and Chemical-Genetic Interactions Map Biogenesis and Permeability Determinants of the Outer Membrane of Escherichia coli

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Kristina Klobucar ◽  
Shawn French ◽  
Jean-Philippe Côté ◽  
James R. Howes ◽  
Eric D. Brown
Keyword(s):  
Outer Membrane ◽  
Genetic Interactions ◽  
Gram Negative Bacteria ◽  
Gene Deletions ◽  
Gram Negative ◽  
Content Type ◽  
Double Deletion ◽  
Nonessential Gene ◽  
Membrane Barrier ◽  
Outer Membrane Permeability

ABSTRACT Gram-negative bacteria are intrinsically resistant to many antibiotics due to their outer membrane barrier. Although the outer membrane has been studied for decades, there is much to uncover about the biology and permeability of this complex structure. Investigating synthetic genetic interactions can reveal a great deal of information about genetic function and pathway interconnectivity. Here, we performed synthetic genetic arrays (SGAs) in Escherichia coli by crossing a subset of gene deletion strains implicated in outer membrane permeability with nonessential gene and small RNA (sRNA) deletion collections. Some 155,400 double-deletion strains were grown on rich microbiological medium with and without subinhibitory concentrations of two antibiotics excluded by the outer membrane, vancomycin and rifampin, to probe both genetic interactions and permeability. The genetic interactions of interest were synthetic sick or lethal (SSL) gene deletions that were detrimental to the cell in combination but had a negligible impact on viability individually. On average, there were ∼30, ∼36, and ∼40 SSL interactions per gene under no-drug, rifampin, and vancomycin conditions, respectively; however, many of these involved frequent interactors. Our data sets have been compiled into an interactive database called the Outer Membrane Interaction (OMI) Explorer, where genetic interactions can be searched, visualized across the genome, compared between conditions, and enriched for gene ontology (GO) terms. A set of SSL interactions revealed connectivity and permeability links between enterobacterial common antigen (ECA) and lipopolysaccharide (LPS) of the outer membrane. This data set provides a novel platform to generate hypotheses about outer membrane biology and permeability. IMPORTANCE Gram-negative bacteria are a major concern for public health, particularly due to the rise of antibiotic resistance. It is important to understand the biology and permeability of the outer membrane of these bacteria in order to increase the efficacy of antibiotics that have difficulty penetrating this structure. Here, we studied the genetic interactions of a subset of outer membrane-related gene deletions in the model Gram-negative bacterium E. coli. We systematically combined these mutants with 3,985 nonessential gene and small RNA deletion mutations in the genome. We examined the viability of these double-deletion strains and probed their permeability characteristics using two antibiotics that have difficulty crossing the outer membrane barrier. An understanding of the genetic basis for outer membrane integrity can assist in the development of new antibiotics with favorable permeability properties and the discovery of compounds capable of increasing outer membrane permeability to enhance the activity of existing antibiotics.

scholarly journals Synergy between Active Efflux and Outer Membrane Diffusion Defines Rules of Antibiotic Permeation into Gram-Negative Bacteria

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Ganesh Krishnamoorthy ◽  
Inga V. Leus ◽  
Jon W. Weeks ◽  
David Wolloscheck ◽  
Valentin V. Rybenkov ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Efflux Pumps ◽  
Antibacterial Activities ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux ◽  
Membrane Barrier ◽  
Biological Determinants ◽  
Resistant Strains

ABSTRACT Gram-negative bacteria are notoriously resistant to antibiotics, but the extent of the resistance varies broadly between species. We report that in significant human pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia spp., the differences in antibiotic resistance are largely defined by their penetration into the cell. For all tested antibiotics, the intracellular penetration was determined by a synergistic relationship between active efflux and the permeability barrier. We found that the outer membrane (OM) and efflux pumps select compounds on the basis of distinct properties and together universally protect bacteria from structurally diverse antibiotics. On the basis of their interactions with the permeability barriers, antibiotics can be divided into four clusters that occupy defined physicochemical spaces. Our results suggest that rules of intracellular penetration are intrinsic to these clusters. The identified specificities in the permeability barriers should help in the designing of successful therapeutic strategies against antibiotic-resistant pathogens. IMPORTANCE Multidrug-resistant strains of Gram-negative pathogens rapidly spread in clinics. Significant efforts worldwide are currently directed to finding the rules of permeation of antibiotics across two membrane envelopes of these bacteria. This study created the tools for analysis of and identified the major differences in antibacterial activities that distinguish the permeability barriers of P. aeruginosa, A. baumannii, Burkholderia thailandensis, and B. cepacia. We conclude that synergy between active efflux and the outer membrane barrier universally protects Gram-negative bacteria from antibiotics. We also found that the diversity of antibiotics affected by active efflux and outer membrane barriers is broader than previously thought and that antibiotics cluster according to specific biological determinants such as the requirement of specific porins in the OM, targeting of the OM, or specific recognition by efflux pumps. No universal rules of antibiotic permeation into Gram-negative bacteria apparently exist. Our results suggest that antibiotic clusters are defined by specific rules of permeation and that further studies could lead to their discovery. IMPORTANCE Multidrug-resistant strains of Gram-negative pathogens rapidly spread in clinics. Significant efforts worldwide are currently directed to finding the rules of permeation of antibiotics across two membrane envelopes of these bacteria. This study created the tools for analysis of and identified the major differences in antibacterial activities that distinguish the permeability barriers of P. aeruginosa, A. baumannii, Burkholderia thailandensis, and B. cepacia. We conclude that synergy between active efflux and the outer membrane barrier universally protects Gram-negative bacteria from antibiotics. We also found that the diversity of antibiotics affected by active efflux and outer membrane barriers is broader than previously thought and that antibiotics cluster according to specific biological determinants such as the requirement of specific porins in the OM, targeting of the OM, or specific recognition by efflux pumps. No universal rules of antibiotic permeation into Gram-negative bacteria apparently exist. Our results suggest that antibiotic clusters are defined by specific rules of permeation and that further studies could lead to their discovery.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

scholarly journals Shadowing the Actions of a Predator: Backlit Fluorescent Microscopy Reveals Synchronous Nonbinary Septation of Predatory Bdellovibrio inside Prey and Exit through Discrete Bdelloplast Pores

2010 ◽  
Vol 192 (24) ◽  
pp. 6329-6335 ◽  
Author(s):  
A. K. Fenton ◽  
M. Kanna ◽  
R. D. Woods ◽  
S.-I. Aizawa ◽  
R. E. Sockett
Keyword(s):  
Cell Division ◽  
Outer Membrane ◽  
Fluorescent Microscopy ◽  
Gram Negative Bacteria ◽  
Bacterial Cell Division ◽  
Prey Cell ◽  
Gram Negative ◽  
A Genome ◽  
Predatory Bacteria ◽  
First Time

ABSTRACT The Bdellovibrio are miniature “living antibiotic” predatory bacteria which invade, reseal, and digest other larger Gram-negative bacteria, including pathogens. Nutrients for the replication of Bdellovibrio bacteria come entirely from the digestion of the single invaded bacterium, now called a bdelloplast, which is bound by the original prey outer membrane. Bdellovibrio bacteria are efficient digesters of prey cells, yielding on average 4 to 6 progeny from digestion of a single prey cell of a genome size similar to that of the Bdellovibrio cell itself. The developmental intrabacterial cycle of Bdellovibrio is largely unknown and has never been visualized “live.” Using the latest motorized xy stage with a very defined z-axis control and engineered periplasmically fluorescent prey allows, for the first time, accurate return and visualization without prey bleaching of developing Bdellovibrio cells using solely the inner resources of a prey cell over several hours. We show that Bdellovibrio bacteria do not follow the familiar pattern of bacterial cell division by binary fission. Instead, they septate synchronously to produce both odd and even numbers of progeny, even when two separate Bdellovibrio cells have invaded and develop within a single prey bacterium, producing two different amounts of progeny. Evolution of this novel septation pattern, allowing odd progeny yields, allows optimal use of the finite prey cell resources to produce maximal replicated, predatory bacteria. When replication is complete, Bdellovibrio cells exit the exhausted prey and are seen leaving via discrete pores rather than by breakdown of the entire outer membrane of the prey.

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