scholarly journals Energy transduction between membranes. TonB, a cytoplasmic membrane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA

1993 ◽  
Vol 268 (22) ◽  
pp. 16302-16308
Author(s):  
J.T. Skare ◽  
B.M. Ahmer ◽  
C.L. Seachord ◽  
R.P. Darveau ◽  
K. Postle
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Energy Transduction ◽  
Membrane Receptor ◽  
Outer Membrane Receptor

scholarly journals Interactions in the TonB-Dependent Energy Transduction Complex: ExbB and ExbD Form Homomultimers

1998 ◽  
Vol 180 (22) ◽  
pp. 6031-6038 ◽  
Author(s):  
Penelope I. Higgs ◽  
Paul S. Myers ◽  
Kathleen Postle
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protein S ◽  
Energy Transduction ◽  
The Other ◽  
Receptor Protein ◽  
Cross Linking ◽  
Vitamin B ◽  
Outer Membrane Receptor

ABSTRACT The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane ofEscherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.

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.

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

2007 ◽  
Vol 189 (14) ◽  
pp. 5379-5382 ◽  
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.

scholarly journals 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 ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 945-954 ◽  
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.

scholarly journals TonB Interacts with Nonreceptor Proteins in the Outer Membrane of Escherichia coli

2002 ◽  
Vol 184 (6) ◽  
pp. 1640-1648 ◽  
Author(s):  
Penelope I. Higgs ◽  
Tracy E. Letain ◽  
Kelley K. Merriam ◽  
Neal S. Burke ◽  
HaJeung Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Receptors ◽  
Strong Interactions ◽  
Cross Linking ◽  
Outer Membrane Receptor ◽  
Outer Membranes

ABSTRACT The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12 across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.

scholarly journals Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
Michael G. Gresock ◽  
Kathleen Postle
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Current Model ◽  
Membrane Transporters ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Interaction Sites ◽  
Outer Membrane Transporters

ABSTRACT In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ∼158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligand-sensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivo. IMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as FepA. Only one contact between TonB and outer membrane transporters has been identified to date: the TonB box at the transporter amino terminus. The TonB box has low information content, raising the question of how TonB can discriminate among multiple different TonB-dependent transporters present in the bacterium if it is the only means of contact. Here we identified several additional sites through which FepA contacts TonB in vivo, including two neighboring residues that may explain how FepA signals to TonB that ligand has bound.

In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study—the outer membrane receptor FpvA

2004 ◽  
Vol 38 (1) ◽  
pp. 79-83 ◽  
Author(s):  
Nicolas Folschweiller ◽  
Karine Pacaud ◽  
Hervé Celia ◽  
Noëlle Potier ◽  
David Cobessi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Receptor ◽  
Outer Membrane Receptor ◽  
Expression Host

scholarly journals The Intrinsically Disordered Region of ExbD Is Required for Signal Transduction

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Dale R. Kopp ◽  
Kathleen Postle
Keyword(s):  
Signal Transduction ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Intrinsic Disorder ◽  
Energy Transduction ◽  
Cross Linking ◽  
Gram Negative ◽  
Conserved Motif ◽  
Tonb System

ABSTRACT The TonB system actively transports vital nutrients across the unenergized outer membranes of the majority of Gram-negative bacteria. In this system, integral membrane proteins ExbB, ExbD, and TonB work together to transduce the proton motive force (PMF) of the inner membrane to customized active transporters in the outer membrane by direct and cyclic binding of TonB to the transporters. A PMF-dependent TonB-ExbD interaction is prevented by 10-residue deletions within a periplasmic disordered domain of ExbD adjacent to the cytoplasmic membrane. Here, we explored the function of the ExbD disordered domain in more detail. In vivo photo-cross-linking through sequential pBpa substitutions in the ExbD disordered domain captured five different ExbD complexes, some of which had been previously detected using in vivo formaldehyde cross-linking, a technique that lacks the residue-specific information that can be achieved through photo-cross-linking: two ExbB-ExbD heterodimers (one of which had not been detected previously), previously detected ExbD homodimers, previously detected PMF-dependent ExbD-TonB heterodimers, and for the first time, a predicted, ExbD-TonB PMF-independent interaction. The fact that multiple complexes were captured by the same pBpa substitution indicated the dynamic nature of ExbD interactions as the energy transduction cycle proceeded in vivo. In this study, we also discovered that a conserved motif—V45, V47, L49, and P50—within the disordered domain was required for signal transduction to TonB and to the C-terminal domain of ExbD and was the source of motif essentiality. IMPORTANCE The TonB system is a virulence factor for Gram-negative pathogens. The mechanism by which cytoplasmic membrane proteins of the TonB system transduce an electrochemical gradient into mechanical energy is a long-standing mystery. TonB, ExbB, and ExbD primary amino acid sequences are characterized by regions of predicted intrinsic disorder, consistent with a proposed multiplicity of protein-protein contacts as TonB proceeds through an energy transduction cycle, a complex process that has yet to be recapitulated in vitro. This study validates a region of intrinsic disorder near the ExbD transmembrane domain and identifies an essential conserved motif embedded within it that transduces signals to distal regions of ExbD suggested to configure TonB for productive interaction with outer membrane transporters.

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