scholarly journals Electrophysiological Characterization of Transport Across Outer‐Membrane Channels from Gram‐Negative Bacteria in Presence of Lipopolysaccharides

2020 ◽  
Vol 59 (22) ◽  
pp. 8517-8521 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter

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.


2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter

Multi-drug-resistance in Gram-negative bacteria is often associated with low permeability of outer membrane. To investigate the role of membrane protein channels in the passage of antibiotics, we extract, purify, reconstitute them into artificial bilayer. Here we demonstrate that using a fusion of native outer membrane vesicles (OMV) facilitates channel reconstitution into bilayer and allows to characterize them in their native environment. Proteins from <i>E. coli</i> (OmpF, OmpC) were overexpressed from the host, and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly only a few channel activities. The asymmetry of the OMV translates after fusion into bilayer with the LPS dominantly present at OMV addition side. Compared to conventional methods, channels fused from OMVs have similar conductance but broader distribution. The further addition of Enrofloxacin yielded higher association but lower dissociation rates attribute to the presence of LPS. We conclude using OMV is a robust approach for functional and structural studies of membrane channels in the native membrane.


2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter

Multi-drug-resistance in Gram-negative bacteria is often associated with low permeability of outer membrane. To investigate the role of membrane protein channels in the passage of antibiotics, we extract, purify, reconstitute them into artificial bilayer. Here we demonstrate that using a fusion of native outer membrane vesicles (OMV) facilitates channel reconstitution into bilayer and allows to characterize them in their native environment. Proteins from <i>E. coli</i> (OmpF, OmpC) were overexpressed from the host, and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly only a few channel activities. The asymmetry of the OMV translates after fusion into bilayer with the LPS dominantly present at OMV addition side. Compared to conventional methods, channels fused from OMVs have similar conductance but broader distribution. The further addition of Enrofloxacin yielded higher association but lower dissociation rates attribute to the presence of LPS. We conclude using OMV is a robust approach for functional and structural studies of membrane channels in the native membrane.


2005 ◽  
Vol 280 (43) ◽  
pp. 35961-35966 ◽  
Author(s):  
Boris Tefsen ◽  
Martine P. Bos ◽  
Frank Beckers ◽  
Jan Tommassen ◽  
Hans de Cock

The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.


2006 ◽  
Vol 189 (1) ◽  
pp. 244-253 ◽  
Author(s):  
Paola Sperandeo ◽  
Rachele Cescutti ◽  
Riccardo Villa ◽  
Cristiano Di Benedetto ◽  
Daniela Candia ◽  
...  

ABSTRACT The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.


2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  

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.


2019 ◽  
Vol 6 (3) ◽  
pp. 467-478 ◽  
Author(s):  
Miranda J. Wallace ◽  
Suresh Dharuman ◽  
Dinesh M. Fernando ◽  
Stephanie M. Reeve ◽  
Clifford T. Gee ◽  
...  

2010 ◽  
Vol 192 (24) ◽  
pp. 6329-6335 ◽  
Author(s):  
A. K. Fenton ◽  
M. Kanna ◽  
R. D. Woods ◽  
S.-I. Aizawa ◽  
R. E. Sockett

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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