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 Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Frédéric Lauber ◽  
Guy Richard Cornelis ◽  
Francesco Renzi
Keyword(s):  
Outer Membrane ◽  
Mutational Analysis ◽  
Signal Sequence ◽  
Underlying Mechanism ◽  
Gram Negative Bacteria ◽  
Reporter System ◽  
Gram Negative ◽  
Export Pathway ◽  
Export Signal ◽  
Bacterial Lipoprotein

ABSTRACTBacteria of the phylumBacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread inBacteroidetes. Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogenCapnocytophaga canimorsuscorresponding to K-(D/E)2or Q-A-(D/E)2. We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposedC. canimorsuslipoprotein. Furthermore, we identify a LES inBacteroides fragilisandFlavobacterium johnsoniaesurface lipoproteins that allowC. canimorsussurface protein exposure, thus suggesting thatBacteroidetesshare a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane.IMPORTANCEBacteria of the phylumBacteroidetesare important human commensals and pathogens. Understanding their biology is therefore a key question for human health. A main feature of these bacteria is the presence of abundant lipoproteins at their surface that play a role in nutrient acquisition. To date, the underlying mechanism of lipoprotein transport is unknown. We show for the first time thatBacteroidetessurface lipoproteins share an N-terminal signal that drives surface localization. The localization and overall negative charge of the lipoprotein export signal (LES) are crucial for its role. Overall, our findings provide the first evidence thatBacteroidetesare endowed with a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane.

scholarly journals Hexameric and pentameric complexes of the ExbBD energizer in the Ton system

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Saori Maki-Yonekura ◽  
Rei Matsuoka ◽  
Yoshiki Yamashita ◽  
Hirofumi Shimizu ◽  
Maiko Tanaka ◽  
...  
Keyword(s):  
Proton Motive Force ◽  
Motive Force ◽  
Membrane Receptors ◽  
Proton Channel ◽  
Gram Negative Bacteria ◽  
Transmembrane Helices ◽  
Gram Negative ◽  
X Ray ◽  
X Ray Crystallography ◽  
Channel Current

Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. Recently, crystal structures suggest that the ExbB pentamer is the scaffold. Here, we present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation/inactivation associated with hexamer and pentamer formation and utilization of proton motive force.

scholarly journals Proton motive force underpins respiration-mediated potentiation of aminoglycoside lethality in pathogenic Escherichia coli

2022 ◽  
Vol 204 (1) ◽  
Author(s):  
Calum M. Webster ◽  
Ayrianna M. Woody ◽  
Safura Fusseini ◽  
Louis G. Holmes ◽  
Gary K. Robinson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Respiratory Chain ◽  
Proton Motive Force ◽  
Motive Force ◽  
Gram Negative Bacteria ◽  
Ros Production ◽  
Gram Negative ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Aminoglycoside Toxicity

AbstractIt is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli. DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.

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.

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.

scholarly journals Mechanism of Bactericidal Activity of Microcin L inEscherichia coliandSalmonella enterica

2010 ◽  
Vol 55 (3) ◽  
pp. 997-1007 ◽  
Author(s):  
Natacha Morin ◽  
Isabelle Lanneluc ◽  
Nathalie Connil ◽  
Marie Cottenceau ◽  
Anne Marie Pons ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bactericidal Activity ◽  
Salmonella Enterica ◽  
Cytoplasmic Membrane ◽  
Genetic System ◽  
Proton Motive Force ◽  
Motive Force ◽  
Membrane Properties ◽  
Outer Membrane Receptor ◽  
E Coli

ABSTRACTFor the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced byEscherichia coliLR05 that exhibits a strong antibacterial activity against relatedEnterobacteriaceae, includingSalmonella entericaserovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set ofE. coliandSalmonella entericamutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL onE. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential ofE. colicells. However, no permeabilization of the membrane was detected.

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