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

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.

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Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906158116 ◽

2019 ◽

Vol 116 (34) ◽

pp. 17013-17022 ◽

Cited By ~ 8

Author(s):

Florence Caro ◽

Nicole M. Place ◽

John J. Mekalanos

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Drug Target ◽

Membrane Vesicle ◽

Essential Genes ◽

Gram Negative Bacteria ◽

Human Pathogen ◽

Membrane Reorganization ◽

A Cell ◽

Classical Genetics

Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen Vibrio cholerae. We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or “MOMV” encased by OM which dynamically emerges specifically at plasmolysis sites. Our work identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.

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A new mechanism for membrane iron transport in Pseudomonas aeruginosa

Biochemical Society Transactions ◽

10.1042/bst0300702 ◽

2002 ◽

Vol 30 (4) ◽

pp. 702-705 ◽

Cited By ~ 14

Author(s):

I.J. Schalk ◽

M. A. Abdallah ◽

F. Pattus

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Iron Uptake ◽

Iron Transport ◽

Membrane Receptor ◽

Gram Negative Bacteria ◽

Outer Membrane Receptor ◽

Uptake Mechanism ◽

Atp Binding Cassette Transporter ◽

Biophysical Studies

Various biochemical and biophysical studies have demonstrated the existence of a novel iron-uptake mechanism in Pseudomonas aeruginosa, different from that generally described for ferrichrome and ferric-enterobactin in Escherichia coli. This new iron-uptake mechanism involves all the proteins generally reported to be involved in the uptake of ferric-siderophore complexes in Gram-negative bacteria (i.e. the outer membrane receptor, periplasmic binding protein and ATP-binding-cassette transporter), but differs in the behaviour of the siderophore. One of the key features of this process is the binding of iron-free pyoverdin to the outer membrane receptor FpvA in conditions of iron deficiency.

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Pathogenic Neisseriae Can Use Hemoglobin, Transferrin, and Lactoferrin Independently of the tonBLocus

Journal of Bacteriology ◽

10.1128/jb.182.19.5586-5591.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5586-5591 ◽

Cited By ~ 15

Author(s):

Pragnya Jasvantrai Desai ◽

Eric Garges ◽

Caroline Attardo Genco

Keyword(s):

Neisseria Gonorrhoeae ◽

Neisseria Meningitidis ◽

Outer Membrane ◽

Deletion Mutant ◽

Iron Transport ◽

Gram Negative Bacteria ◽

Wild Type ◽

Chromosomal Dna ◽

Gram Negative ◽

Type Strains

ABSTRACT Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis andNeisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb+ variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb+ variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the NeisseriaHb+ variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously describedtonB locus.

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Toxin import through the antibiotic efflux channel TolC

Nature Communications ◽

10.1038/s41467-021-24930-y ◽

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.

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Novel Role of the Lipopolysaccharide O1 Side Chain in Ferric Siderophore Transport and Virulence of Vibrio anguillarum

Infection and Immunity ◽

10.1128/iai.73.9.5864-5872.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5864-5872 ◽

Cited By ~ 23

Author(s):

Timothy J. Welch ◽

Jorge H. Crosa

Keyword(s):

Iron Transport ◽

Vibrio Anguillarum ◽

Fish Host ◽

Side Chain ◽

Reading Frame ◽

Transposon Insertion ◽

Protein Receptor ◽

Defective Mutant ◽

Concomitant Reduction ◽

Siderophore Transport

ABSTRACTFrom a library of approximately 20,000 transposon mutants, we have identified mutants affected in chromosomal genes involved in synthesis of the siderophore anguibactin, as well as in ferric anguibactin utilization. Genetic and sequence analyses of one such transport-defective mutant revealed that the transposon insertion occurred in an open reading frame (ORF) with homology tormlC, a dTDP-rhamnose biosynthetic gene. This ORF resides within a cluster of four ORFs, all of which are predicted to function in the biosynthesis of this O side chain precursor. The same phenotype was seen in a mutant obtained by allelic exchange inrmlD, another ORF in this dTDP-rhamnose biosynthetic cluster. This mutation could be complemented with the wild-typermlDgene, restoring both production of the O1 antigen side chain and ferric anguibactin transport. Presence of the O1 side chain was crucial for the resistance ofVibrio anguillarumto the bactericidal action of nonimmune serum from the fish host. Surprisingly, further analysis demonstrated that these mutations were pleiotropic, leading to a dramatic decrease in the levels of FatA, the outer membrane protein receptor for ferric anguibactin transport, and a concomitant reduction in iron transport. Thus, our results in this work demonstrate that the lipopolysaccharide O1 side chain is required for the operation of two critical virulence factors inV. anguillarum: serum resistance and anguibactin-mediated iron transport. These factors allowV. anguillarumto survive in serum and multiply in the iron-limiting milieu of the host vertebrate.

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

PLoS Computational Biology ◽

10.1371/journal.pcbi.1009756 ◽

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.

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

FEMS Microbiology Reviews ◽

10.1093/femsre/fuaa018 ◽

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.

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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.

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Ultraviolet Treatment of Water for Destruction of Five Gram-Negative Bacteria Pathogenic to Fishes

Journal of the Fisheries Research Board of Canada ◽

10.1139/f77-183 ◽

1977 ◽

Vol 34 (8) ◽

pp. 1244-1249 ◽

Cited By ~ 14

Author(s):

G. L. Bullock ◽

H. M. Stuckey

Keyword(s):

Particulate Matter ◽

Ultraviolet Irradiation ◽

Vibrio Anguillarum ◽

Spring Water ◽

Aeromonas Salmonicida ◽

Water Disinfection ◽

Gram Negative Bacteria ◽

Fish Pathogens ◽

Gram Negative ◽

Ultraviolet Treatment

Filtration (25 nm) and ultraviolet irradiation dosages of 13,100–29,400 microwatt seconds per square centimetre (μW∙s∙cm−2) effected a 99.98–100% reduction of five gram-negative fish pathogens — Aeromonas salmonicida, A. hydrophila, Vibrio anguillarum, Pseudomonas fluorescens, and the enteric redmouth organism in 12.5 °C clear spring water or spring water containing particulate matter. Filtration and a dosage of 4500 μW∙s∙cm−2 killed 99.83–100% of test strains in spring water and 4000–4750 μW∙s∙cm−2 killed 99.33–99.99% in water with particulate matter. Irradiation of unfiltered water containing particulate matter was less effective, especially at dosages of 5000 μW∙s∙cm−2 or less, which killed 97–99.94% of strains. Filtration and 13,100 μW∙s∙cm−2 irradiation of water containing A. salmonicida prevented transmission of furunculosis. Key words: ultraviolet irradiation, bacterial fish pathogens, water disinfection

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Packaging Covered with Antiviral and Antibacterial Coatings Based on ZnO Nanoparticles Supplemented with Geraniol and Carvacrol

International Journal of Molecular Sciences ◽

10.3390/ijms22041717 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1717

Author(s):

Małgorzata Mizielińska ◽

Paweł Nawrotek ◽

Xymena Stachurska ◽

Magdalena Ordon ◽

Artur Bartkowiak

Keyword(s):

Synergistic Effect ◽

Moderate Activity ◽

Gram Negative Bacteria ◽

Human Pathogen ◽

Safety Measures ◽

Gram Negative ◽

Airborne Viruses ◽

Respiratory Droplets ◽

Active Substances ◽

External Coating

The purpose of the study was to obtain an external coating based on nanoparticles of ZnO, carvacrol, and geraniol that could be active against viruses such as SARS-Co-V2. Additionally, the synergistic effect of the chosen substances in coatings was analyzed. The goal of the study was to measure the possible antibacterial activity of the coatings obtained. Testing antiviral activity with human pathogen viruses, such as SARS-Co-V2, requires immense safety measures. Bacteriophages such as phi 6 phage represent good surrogates for the study of airborne viruses. The results of the study indicated that the ZC1 and ZG1 coatings containing an increased amount of geraniol or carvacrol and a very small amount of nanoZnO were found to be active against Gram-positive and Gram-negative bacteria. It is also important that a synergistic effect between these active substances was noted. This explains why polyethylene (PE) films covered with the ZC1 or ZG1 coatings (as internal coatings) were found to be the best packaging materials to extend the quality and freshness of food products. The same coatings may be used as the external coatings with antiviral properties. The ZC1 and ZG1 coatings showed moderate activity against the phi 6 phage that has been selected as a surrogate for viruses such as coronaviruses. It can be assumed that coatings ZG1 and ZC1 will also be active against SARS-CoV-2 that is transmitted via respiratory droplets.

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