In vivo evidence for FhuA outer membrane receptor interaction with the TonB inner membrane protein ofEscherichia coli

ABSTRACT Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonBpp) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonBpp was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonBpp interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.

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Detection of soluble expression and in vivo interactions of the inner membrane protein OppC using green fluorescent protein

Genetics and Molecular Research ◽

10.4238/2015.december.22.8 ◽

2015 ◽

Vol 14 (4) ◽

pp. 17834-17846 ◽

Cited By ~ 2

Author(s):

Q.J. Xiang ◽

J.F. Zhai ◽

M. Zhang ◽

B. Zhang

Keyword(s):

Green Fluorescent Protein ◽

Membrane Protein ◽

Fluorescent Protein ◽

Soluble Expression ◽

Inner Membrane ◽

Inner Membrane Protein ◽

Green Fluorescent

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

Journal of Bacteriology ◽

10.1128/jb.182.6.1731-1738.2000 ◽

2000 ◽

Vol 182 (6) ◽

pp. 1731-1738 ◽

Cited By ~ 38

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.

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Mutagenesis and Molecular Modeling Reveal Three Key Extracellular Loops of the Membrane Receptor HasR That Are Involved in Hemophore HasA Binding

Journal of Bacteriology ◽

10.1128/jb.00251-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5379-5382 ◽

Cited By ~ 10

Author(s):

Clément Barjon ◽

Karine Wecker ◽

Nadia Izadi-Pruneyre ◽

Philippe Delepelaire

Keyword(s):

Molecular Modeling ◽

Outer Membrane ◽

Three Dimensional ◽

Membrane Receptor ◽

Dimensional Model ◽

Outer Membrane Receptor ◽

Three Dimensional Model ◽

Extracellular Loops

ABSTRACT On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro.

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Deletion of the proline-rich region of TonB disrupts formation of a 2:1 complex with FhuA, an outer membrane receptor ofEscherichia coli

Protein Science ◽

10.1110/ps.051342505 ◽

2005 ◽

Vol 14 (5) ◽

pp. 1266-1273 ◽

Cited By ~ 12

Author(s):

Cezar M. Khursigara ◽

Gregory De Crescenzo ◽

Peter D. Pawelek ◽

James W. Coulton

Keyword(s):

Outer Membrane ◽

Membrane Receptor ◽

Ofescherichia Coli ◽

Outer Membrane Receptor ◽

Rich Region ◽

Proline Rich Region

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Identification and characterization of an iron-regulated gene, chtA, required for the utilization of the xenosiderophores aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.28552-0 ◽

2006 ◽

Vol 152 (4) ◽

pp. 945-954 ◽

Cited By ~ 19

Author(s):

Páraic Ó Cuív ◽

Paul Clarke ◽

Michael O'Connell

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Membrane Receptor ◽

Outer Membrane Receptor ◽

Significant Similarity ◽

Hydroxamate Siderophores ◽

Insertional Inactivation ◽

Titration Assay

Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.

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Crystallization and preliminary X-ray diffraction analysis of YejM from Salmonella typhimurium: an essential inner membrane protein involved in outer membrane directed cardiolipin transport

F1000Research ◽

10.12688/f1000research.8647.2 ◽

2017 ◽

Vol 5 ◽

pp. 1086

Author(s):

Uma Gabale ◽

Gene Qian ◽

Elaina Roach ◽

Susanne Ressl

Keyword(s):

Membrane Protein ◽

Diffraction Analysis ◽

Outer Membrane ◽

Drug Targets ◽

Essential Gene ◽

The United States ◽

X Ray Diffraction ◽

Inner Membrane ◽

X Ray ◽

Inner Membrane Protein

Salmonella typhimurium is responsible for over 35% of all foodborne illness related hospitalizations in the United States. This Gram-negative bacterium possesses an inner and an outer membrane (OM), the latter allowing its survival and replication within host tissues. During infection, OM is remodeled by transport of glycerophospholipids across the periplasm and into the OM. Increased levels of cardiolipin in the OM were observed upon PhoPQ activation and led to the discovery of YejM; an inner membrane protein essential for cell growth involved in cardiolipin binding and transport to the OM. Here we report how YejM was engineered to facilitate crystal growth and X-ray diffraction analysis. Successful structure determination of YejM will help us understand how they interact and how YejM facilitates cardiolipin transport to the OM. Ultimately, yejm, being an essential gene, may lead to new drug targets inhibiting the pathogenic properties of S. typhimurium.

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The essential inner membrane protein YejM is a metalloenzyme

Scientific Reports ◽

10.1038/s41598-020-73660-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Uma Gabale ◽

Perla Arianna Peña Palomino ◽

HyunAh Kim ◽

Wenya Chen ◽

Susanne Ressl

Keyword(s):

Antibiotic Resistance ◽

Membrane Protein ◽

Outer Membrane ◽

Critical Role ◽

Membrane Properties ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Periplasmic Domain ◽

Inner Membrane Protein

Abstract Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

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Mutations in a tRNA Import Signal Define Distinct Receptors at the Two Membranes of LeishmaniaMitochondria

Molecular and Cellular Biology ◽

10.1128/mcb.20.19.7410-7417.2000 ◽

2000 ◽

Vol 20 (19) ◽

pp. 7410-7417 ◽

Cited By ~ 13

Author(s):

Subhendra Nath Bhattacharyya ◽

Shankar Mukherjee ◽

Samit Adhya

Keyword(s):

Outer Membrane ◽

Mitochondrial Matrix ◽

Outer Membrane Receptor ◽

Inner Membrane ◽

Stem Loop ◽

In Organello ◽

The Matrix ◽

Biochemical Fractionation ◽

Membrane Transfer

ABSTRACT Nucleus-encoded tRNAs are selectively imported into the mitochondrion of Leishmania, a kinetoplastid protozoan. An oligoribonucleotide constituting the D stem-loop import signal of tRNATyr(GUA) was efficiently transported into the mitochondrial matrix in organello as well as in vivo. Transfer through the inner membrane could be uncoupled from that through the outer membrane and was resistant to antibody against the outer membrane receptor TAB. A number of mutations in the import signal had differential effects on outer and inner membrane transfer. Some mutants which efficiently traversed the outer membrane were unable to enter the matrix. Conversely, restoration of the loop-closing GC pair in reverse resulted in reversion of transfer through the inner, but not the outer, membrane, and binding of the RNA to the inner membrane was restored. These experiments indicate the presence at the two membranes of receptors with distinct specificities which mediate stepwise transfer into the mitochondrial matrix. The combination of oligonucleotide mutagenesis and biochemical fractionation may provide a general tool for the identification of tRNA transport factors.

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