An exposed outer membrane haemin-binding protein facilitates haemin transport by a TonB-dependent receptor in Riemerella anatipestifer

Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes haemoglobin-derived haemin as an iron source; however, the mechanism by which this bacterium acquires haemin from haemoglobin is largely unknown. Here, RhuA disruption was shown to impair iron utilization from duck haemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane haemin-binding protein, but it could not extract haemin from duck haemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A and RhuAH149A lost haemin-binding ability, suggesting that amino acid sites tyrosine 144 (Y144), Y177 and histidine 149 (H149) are crucial for haemin binding. Furthermore, RhuR, the gene adjacent to RhuA, encodes a TonB2-dependent haemin transporter. The function of RhuA in duck haemoglobin utilization was abolished in the RhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1ΔRhuA rather than R. anatipestifer CH-1ΔRhuRΔRhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA haemin utilization locus exhibits no similarity with those of characterized haemin transport systems. Thus, this locus is a novel haemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, haemin from haemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain haemin from haemoglobin, but the mechanisms of haemin acquisition from haemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of haemin acquisition from mammalian haemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in haemin acquisition from duck haemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane haemin-binding protein, and RhuR was identified as a TonB2-dependent haemin transporter. Moreover, the function of RhuA in haemoglobin utilization is RhuR dependent, not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel haemin transportation system of gram-negative bacteria. This study not only was important for understanding haemin uptake in R. anatipestifer but also enriched the knowledge about the haemin transportation pathway in gram-negative bacteria.

Download Full-text

Development of transport systems of quinolone class antibiotics through outer membrane vesicles (OMVs) in Gram-negative bacteria

Revista dos Trabalhos de Iniciação Científica da UNICAMP ◽

10.20396/revpibic262018276 ◽

2018 ◽

Author(s):

Carlos Fernando Macedo da Silva ◽

Marcelo Lancellotti

Keyword(s):

Outer Membrane ◽

Bacterial Growth ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Extracellular Medium ◽

Gram Negative ◽

Resistance To Antibiotics ◽

Cell Reproduction

Multi-resistance to antibiotics in Gram-negative bacteria has been reported in several studies, which make more effective methods of controlling and eliminating these bacteria necessary. To overcome multiresistant profiles, we used OMVs (Outer Membrane Vesicles) as carriers of levofloxacin to encapsulate and transport the drug from the extracellular medium into the cell, overcoming resistance barriers and inhibiting cell reproduction machinery. Prepackaged formulations in this manner were quite effective and, in some cases, totally inhibited bacterial growth by making the drug efficient again.

Download Full-text

Structure of LetB reveals a tunnel for lipid transport across the bacterial envelope

10.1101/748145 ◽

2019 ◽

Author(s):

Georgia L. Isom ◽

Nicolas Coudray ◽

Mark R. MacRae ◽

Collin T. McManus ◽

Damian C. Ekiert ◽

...

Keyword(s):

Outer Membrane ◽

Lipid Transport ◽

Transport Systems ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Outer Membranes ◽

Hydrophobic Tunnel ◽

Outer Membrane Permeability

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide (LPS), which acts as a barrier to the environment and contributes to antibiotic resistance. While mechanisms of LPS transport have been well characterised, systems that translocate phospholipids across the periplasm, such as MCE (Mammalian Cell Entry) transport systems, are less well understood. Here we show that E. coli MCE protein LetB (formerly YebT), forms a ∼0.6 megadalton complex in the periplasm. Our cryo-EM structure reveals that LetB consists of a stack of seven modular rings, creating a long hydrophobic tunnel through the centre of the complex. LetB is sufficiently large to span the gap between the inner and outer membranes, and mutations that shorten the tunnel abolish function. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Together, our results support a model in which LetB establishes a physical link between the bacterial inner and outer membranes, and creates a hydrophobic pathway for the translocation of lipids across the periplasm, to maintain the integrity of the outer membrane permeability barrier.

Download Full-text

Bacterial Adaptor Membrane Fusion Proteins and the Structurally Dissimilar Outer Membrane Auxiliary Proteins Have Exchanged Central Domains inα-Proteobacteria

International Journal of Microbiology ◽

10.1155/2010/589391 ◽

2010 ◽

Vol 2010 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Anthony Y. Xiao ◽

Jing Wang ◽

Milton H. Saier

Keyword(s):

Membrane Fusion ◽

Outer Membrane ◽

Fusion Proteins ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Transporter Protein ◽

Functional Implications ◽

Membrane Fusion Proteins ◽

Helical Region

Transport systems frequently include auxiliary proteins that perform subfunctions within the transporter protein complex. Two such proteins found in Gram-negative bacteria are the Membrane Fusion Proteins (MFPs) and the Outer Membrane Auxiliary (OMA) proteins. We here demonstrate that OMAs present inα-proteobacteria (but not in other bacterial types) contain a longα-helical region that is homologous to corresponding regions in the MFPs. The results suggest that during their evolution, OMAs, specifically fromα-proteobacteria, exchanged their ownα-helical domain for one derived from an MFP. The structural and functional implications of these findings are discussed.

Download Full-text

Correct Sorting of Lipoproteins into the Inner and Outer Membranes ofPseudomonas aeruginosaby theEscherichia coliLolCDE Transport System

mBio ◽

10.1128/mbio.00194-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 4

Author(s):

Christian Lorenz ◽

Thomas J. Dougherty ◽

Stephen Lory

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Outer Membranes

ABSTRACTBiogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. InEnterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that inPseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those fromEscherichia coli. TheP. aeruginosalipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either theE. coliorP. aeruginosaLolCDE. We further demonstrate that an inhibitor ofE. coliLolCDE is active againstP. aeruginosaonly when expressing theE. coliorthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCEGram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways ofEscherichia coliandPseudomonas aeruginosaare interchangeable, with theE. coliorthologues correctly sorting theP. aeruginosalipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified inE. colias aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

Download Full-text

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

10.26434/chemrxiv.8282204.v1 ◽

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 Escherichia coli, 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 (kon) and lower dissociation (koff) 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.

Download Full-text

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

International Journal of Molecular Sciences ◽

10.3390/ijms22105328 ◽

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00914-10 ◽

2010 ◽

Vol 192 (24) ◽

pp. 6329-6335 ◽

Cited By ~ 40

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.

Download Full-text