scholarly journals Impact of FiuA Outer Membrane Receptor Polymorphism on the Resistance ofPseudomonas aeruginosatoward Peptidoglycan Lipid II-Targeting PaeM Pyocins

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Libera Latino ◽  
Delphine Patin ◽  
Dimitri Chérier ◽  
Thierry Touzé ◽  
Christine Pourcel ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Receptor ◽  
Site Directed Mutagenesis ◽  
Outer Membrane Receptor ◽  
Multiresistant Pathogens ◽  
Content Type ◽  
Lipid Ii ◽  
Molecular Determinants ◽  
Colicin M ◽  
Receptor Polymorphism

ABSTRACTCertainPseudomonas aeruginosastrains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptibleP. aeruginosastrains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against variousP. aeruginosastrains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization intoP. aeruginosacells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression offiuAgene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport inP. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination.IMPORTANCEBacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains ofP. aeruginosathat inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility ofP. aeruginosaclinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

scholarly journals Contributions of the heme coordinating ligands of the Pseudomonas aeruginosa outer membrane receptor HasR to extracellular heme sensing and transport

2020 ◽  
Vol 295 (30) ◽  
pp. 10456-10467 ◽  
Author(s):  
Alecia T. Dent ◽  
Angela Wilks
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Receptor ◽  
Site Directed Mutagenesis ◽  
Uptake System ◽  
Outer Membrane Receptor ◽  
Heme Uptake ◽  
Functional Link ◽  
Heme Transport ◽  
Σ Factor

Pseudomonas aeruginosa exhibits a high requirement for iron, which it can acquire via several mechanisms, including the acquisition and utilization of heme. The P. aeruginosa genome encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system, which encodes an extracytoplasmic function (ECF) σ factor system. Previous studies have shown that the transfer of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI and upregulation of has operon expression. Here, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting, and 13C-heme uptake experiments, we delineated the differential contributions of the extracellular FRAP/PNPNL loop residue His-624 in HasR and of His-221 in its N-terminal plug domain required for heme capture to heme transport and signaling, respectively. Specifically, we show that substitution of the N-terminal plug His-221 disrupts both signaling and transport, leading to dysregulation of both the Has and Phu uptake systems. Our results are consistent with a model wherein heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His-624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Our results provide critical insight into heme release, signaling, and transport in P. aeruginosa and suggest a functional link between the ECF σ factor and Phu heme uptake system.

scholarly journals Contributions of the Heme Coordinating Ligands of the Pseudomonas aeruginosa Outer Membrane Receptor HasR to Extracellular Heme Sensing and Transport

2020 ◽  
Author(s):  
Alecia T. Dent ◽  
Angela Wilks
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Receptor ◽  
Site Directed Mutagenesis ◽  
Outer Membrane Receptor ◽  
Heme Uptake ◽  
Functional Link ◽  
Heme Transport ◽  
Σ Factor ◽  
Factor System

ABSTRACTPseudomonas aeruginosa exhibits a high requirement for iron which it can acquire via several mechanisms including the acquisition and utilization of heme. P. aeruginosa encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonasheme utilization (Phu) system. Extracellular heme is sensed via the Has system that encodes an extra cytoplasmic function (ECF) σ factor system. Previous studies have shown release of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI. Herein, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting and 13C-heme uptake studies, we characterize the differential contributions of the outer membrane receptor HasR extracellular FRAP/PNPNL loop residue His-624 and the N-terminal plug residue His-221 to heme transport and signaling, respectively. Specifically, we show mutation of the N-terminal plug His-221 disrupts both signaling and transport. The data is consistent with a model where heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Furthermore, mutation of His-221 leads to dysregulation of both the Has and Phu uptake systems suggesting a possible functional link that is coordinated through the ECF σ factor system.

scholarly journals Vibrio cholerae VibF Is Required for Vibriobactin Synthesis and Is a Member of the Family of Nonribosomal Peptide Synthetases

2000 ◽  
Vol 182 (6) ◽  
pp. 1731-1738 ◽  
Author(s):  
Joan R. Butterton ◽  
Michael H. Choi ◽  
Paula I. Watnick ◽  
Patricia A. Carroll ◽  
Stephen B. Calderwood
Keyword(s):  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Consensus Sequence ◽  
Membrane Receptor ◽  
Transcriptional Level ◽  
Outer Membrane Receptor ◽  
Chromosomal Dna ◽  
E Coli ◽  
Binding Consensus Sequence

ABSTRACT A 7.5-kbp fragment of chromosomal DNA downstream of theVibrio cholerae vibriobactin outer membrane receptor,viuA, and the vibriobactin utilization gene,viuB, was recovered from a Sau3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the Escherichia coli enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli entmutants. Cross-feeding studies demonstrated that an E. coli entF mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of viuB which encoded a deduced protein of greater than 2,158 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, and E. coliEntF. A mutant with an in-frame deletion of this gene, namedvibF, was created with classical V. choleraestrain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned vibF fragment restored vibriobactin synthesis to normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the V. cholerae fur gene. Expression ofvibF was not autoregulatory and neither affected nor was affected by the expression of irgA or viuA. The promoter of vibF was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence. A footprint of purified V. cholerae Fur on the vibFpromoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.

Mapping the Interaction between the Hemophore HasA and Its Outer Membrane Receptor HasR Using CRINEPT−TROSY NMR Spectroscopy

2009 ◽  
Vol 131 (5) ◽  
pp. 1736-1744 ◽  
Author(s):  
Célia Caillet-Saguy ◽  
Mario Piccioli ◽  
Paola Turano ◽  
Nadia Izadi-Pruneyre ◽  
Muriel Delepierre ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Outer Membrane ◽  
Membrane Receptor ◽  
Outer Membrane Receptor

TonB or not TonB: is that the question?This paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 87-97 ◽  
Author(s):  
Karla D. Krewulak ◽  
Hans J. Vogel
Keyword(s):  
Outer Membrane ◽  
Metal Ion ◽  
Peer Review Process ◽  
Three Dimensional ◽  
Membrane Receptor ◽  
Membrane Receptors ◽  
Dimensional Structure ◽  
Outer Membrane Receptor ◽  
Different Strains ◽  
Low Molecular Weight Iron

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.

A new mechanism for membrane iron transport in Pseudomonas aeruginosa

2002 ◽  
Vol 30 (4) ◽  
pp. 702-705 ◽  
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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