scholarly journals Toxin import through the antibiotic efflux channel TolC

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicholas G. Housden ◽  
Melissa N. Webby ◽  
Edward D. Lowe ◽  
Tarick J. El-Baba ◽  
Renata Kaminska ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Proton Motive Force ◽  
Target Cells ◽  
Gram Negative Bacteria ◽  
Drug Efflux ◽  
High Affinity ◽  
Helical Hairpin ◽  
Protein Toxins ◽  
Antibiotic Efflux

AbstractBacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.

scholarly journals High affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Outer Membrane ◽  
Single Molecule ◽  
Protein C ◽  
Outer Membrane Proteins ◽  
Correlation Spectroscopy ◽  
Major Protein ◽  
Gram Negative Bacteria ◽  
Fluorescence Correlation ◽  
High Affinity ◽  
Quality Control Mechanism

AbstractOuter membrane proteins (OMPs) are essential to Gram-negative bacteria, and they need molecular chaperones to prevent from aggregation in periplasm during the OMPs biogenesis. Seventeen kilodalton protein (Skp) is the major protein for this purpose. Here we used singlemolecule detection (SMD) to study the stoichiometry modulation of Skp in binding with outer membrane protein C (OmpC) from Escherichia coli. To accomplish our task, we developed the tool of portion selectively chosen fluorescence correlation spectroscopy (pscFCS). We found that Skp binds OmpC with high affinity. The half concentration for Skp to form homotrimer Skp3 (C1/2) was measured to be 250 nM. Under the Skp concentrations far below C1/2 OmpC can recruit Skp monomers to form OmpC·Skp3. The affinity of the process is in picomolar range, indicating that the trimerization of Skp in OmpC·Skp3 complex is induced by OmpC-Skp interaction even though free Skp3 is rarely present. In the concentration range that Skp3 is the predominant form, OmpC may directly interact with Skp3. Under micro-molar concentrations of Skp, the formation of OmpC·(Skp3)2 was observed. Our results suggest that the fine-tuned modulation of Skp composition stoichiometry plays an important role in the safe-guarding and quality control mechanism of OMPs in the periplasm.

scholarly journals The quantitative basis for the redistribution of immobile bacterial lipoproteins to division septa

2021 ◽  
Vol 17 (12) ◽  
pp. e1009756
Author(s):  
Lara Connolley ◽  
Joanna Szczepaniak ◽  
Colin Kleanthous ◽  
Seán M. Murray
Keyword(s):  
Outer Membrane ◽  
Physical Mechanism ◽  
Underlying Mechanism ◽  
Proton Motive Force ◽  
Motive Force ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Division Site ◽  
Quantitative Basis ◽  
Mutant Phenotypes

The spatial localisation of proteins is critical for most cellular function. In bacteria, this is typically achieved through capture by established landmark proteins. However, this requires that the protein is diffusive on the appropriate timescale. It is therefore unknown how the localisation of effectively immobile proteins is achieved. Here, we investigate the localisation to the division site of the slowly diffusing lipoprotein Pal, which anchors the outer membrane to the cell wall of Gram-negative bacteria. While the proton motive force-linked TolQRAB system is known to be required for this repositioning, the underlying mechanism is unresolved, especially given the very low mobility of Pal. We present a quantitative, mathematical model for Pal relocalisation in which dissociation of TolB-Pal complexes, powered by the proton motive force across the inner membrane, leads to the net transport of Pal along the outer membrane and its deposition at the division septum. We fit the model to experimental measurements of protein mobility and successfully test its predictions experimentally against mutant phenotypes. Our model not only explains a key aspect of cell division in Gram-negative bacteria, but also presents a physical mechanism for the transport of low-mobility proteins that may be applicable to multi-membrane organelles, such as mitochondria and chloroplasts.

scholarly journals Redefining the essential trafficking pathway for outer membrane lipoproteins

2017 ◽  
Vol 114 (18) ◽  
pp. 4769-4774 ◽  
Author(s):  
Marcin Grabowicz ◽  
Thomas J. Silhavy
Keyword(s):  
Stress Response ◽  
Outer Membrane ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Secretion Systems ◽  
Trafficking Pathway ◽  
Envelope Stress ◽  
Periplasmic Chaperone ◽  
Membrane Lipoproteins ◽  
Antibiotic Efflux

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins’ key function is to prevent lethal accumulation of mislocalized lipoproteins.

scholarly journals The multifarious roles of Tol-Pal in Gram-negative bacteria

2020 ◽  
Vol 44 (4) ◽  
pp. 490-506
Author(s):  
Joanna Szczepaniak ◽  
Cara Press ◽  
Colin Kleanthous
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Physiological Role ◽  
Proton Motive Force ◽  
The Other ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
The 1960S ◽  
Shed Light

ABSTRACT In the 1960s several groups reported the isolation and preliminary genetic mapping of Escherichia coli strains tolerant towards the action of colicins. These pioneering studies kick-started two new fields in bacteriology; one centred on how bacteriocins like colicins exploit the Tol (or more commonly Tol-Pal) system to kill bacteria, the other on the physiological role of this cell envelope-spanning assembly. The following half century has seen significant advances in the first of these fields whereas the second has remained elusive, until recently. Here, we review work that begins to shed light on Tol-Pal function in Gram-negative bacteria. What emerges from these studies is that Tol-Pal is an energised system with fundamental, interlinked roles in cell division – coordinating the re-structuring of peptidoglycan at division sites and stabilising the connection between the outer membrane and underlying cell wall. This latter role is achieved by Tol-Pal exploiting the proton motive force to catalyse the accumulation of the outer membrane peptidoglycan associated lipoprotein Pal at division sites while simultaneously mobilising Pal molecules from around the cell. These studies begin to explain the diverse phenotypic outcomes of tol-pal mutations, point to other cell envelope roles Tol-Pal may have and raise many new questions.

scholarly journals A Novel Protein, TtpC, Is a Required Component of the TonB2 Complex for Specific Iron Transport in the Pathogens Vibrio anguillarum and Vibrio cholerae

2006 ◽  
Vol 189 (5) ◽  
pp. 1803-1815 ◽  
Author(s):  
Michiel Stork ◽  
Ben R. Otto ◽  
Jorge H. Crosa
Keyword(s):  
Outer Membrane ◽  
General Feature ◽  
Iron Transport ◽  
Vibrio Anguillarum ◽  
Proton Motive Force ◽  
Gram Negative Bacteria ◽  
Human Pathogen ◽  
Transposon Insertion ◽  
Similar Gene ◽  
Novel Protein

ABSTRACT Active transport across the outer membrane in gram-negative bacteria requires the energy that is generated by the proton motive force in the inner membrane. This energy is transduced to the outer membrane by the TonB protein in complex with the proteins ExbB and ExbD. In the pathogen Vibrio anguillarum we have identified two TonB systems, TonB1 and TonB2, the latter is used for ferric-anguibactin transport and is transcribed as part of an operon that consists of orf2, exbB2, exbD2, and tonB2. This cluster was identified by a polar transposon insertion in orf2 that resulted in a strain deficient for ferric-anguibactin transport. Only the entire cluster (orf2, exbB2, exbD2 and tonB2) could complement for ferric-anguibactin transport, while just the exbB2, exbD2, and tonB2 genes were unable to restore transport. This suggests an essential role for this Orf2, designated TtpC, in TonB2-mediated transport in V. anguillarum. A similar gene cluster exists in V. cholerae, i.e., with the homologues of ttpC-exbB2-exbD2-tonB2, and we demonstrate that TtpC from V. cholerae also plays a role in the TonB2-mediated transport of enterobactin in this human pathogen. Furthermore, we also show that in V. anguillarum the TtpC protein is found as part of a complex that might also contain the TonB2, ExbB2, and ExbD2 proteins. This novel component of the TonB2 system found in V. anguillarum and V. cholerae is perhaps a general feature in bacteria harboring the Vibrio-like TonB2 system.

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.

Sign in / Sign up

Export Citation Format

Share Document