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 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 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.

scholarly journals Interactions between the Outer Membrane Ferric Citrate Transporter FecA and TonB: Studies of the FecA TonB Box

2003 ◽  
Vol 185 (6) ◽  
pp. 1870-1885 ◽  
Author(s):  
Monica Ogierman ◽  
Volkmar Braun
Keyword(s):  
Outer Membrane ◽  
Disulfide Bond ◽  
Ferric Citrate ◽  
Cross Linking ◽  
Physical Interaction ◽  
Outer Membrane Receptor ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Citrate Transporter

ABSTRACT Both induction of transcription of the ferric citrate transport genes and transport of ferric citrate by the Escherichia coli outer membrane receptor FecA require energy derived from the proton motive force (PMF) of the inner membrane. The energy is transduced to FecA by the inner membrane complex, TonB, ExbB, and ExbD. Region 160 of TonB and the conserved TonB box of other TonB-dependent receptors are implicated as sites of interaction. In the present study, the postulated TonB box (D80A81L82T83V84) of FecA was deleted in frame, with a subsequent loss of both FecA functions. DALTV of FecA could be functionally replaced with the core TonB boxes of FhuA (DTITV) and FepA (DTIVV). Each residue of the TonB box of FecA was sequentially replaced with cysteine residues, and only the D80C replacement showed a loss (reduction) of both FecA functions. A physical interaction between TonB and FecA was demonstrated using both in vivo site-specific disulfide bond cross-linking and nonspecific formaldehyde (FA) cross-linking. Pairwise combinations of FecA (DALTV)/Cys substitutions were cross-linked via disulfide bond formation with TonBQ160C, TonBQ162C, and TonBY163C. Unexpectedly, this cross-linking was not enhanced by substrate (ferric citrate). In contrast, the TonB-FecA interaction was enhanced by ferric citrate in the FA-cross-linking assay. Energy derived from the PMF was not required for the TonB-FecA interaction in either the disulfide- or FA-cross-linking assay. TonB/CysExbB/ExbD(D25N) was still able to cross-link with the FecA (DALTV)/Cys derivatives in a tonB tolQ background, even though ExbD25N renders the TonB/ExbBD complex nonfunctional (V. Braun, S. Gaisser, C. Herrmann, K. Kampfenkel, H. Killmann, and I. Traub, J. Bacteriol. 178:2836-2845, 1996). TonB cross-linked to FecA via FA was not inhibited by either carbonylcyanide-m-chlorophenylhydrazone or 1 mM 2,4-dinitrophenol, which dissipate the electrochemical potential of the cytoplasmic membrane and disrupt both FecA functions. The studies shown here demonstrate the significance of the TonB box for FecA functions and are consistent with the view that it is the structure and not the sequence of the TonB box that is important for activity. Demonstrated here for the first time is the physical interaction of TonB and FecA, which is enhanced by ferric citrate.

scholarly journals Signaling via β2 Integrins Triggers Neutrophil-Dependent Alteration in Endothelial Barrier Function

2000 ◽  
Vol 191 (11) ◽  
pp. 1829-1840 ◽  
Author(s):  
Narinder Gautam ◽  
Heiko Herwald ◽  
Per Hedqvist ◽  
Lennart Lindbom
Keyword(s):  
Protein S ◽  
Endothelial Barrier ◽  
Cheek Pouch ◽  
Cross Linking ◽  
Endothelial Lining ◽  
Hamster Cheek Pouch ◽  
Cationic Protein ◽  
Edema Formation ◽  
Β2 Integrins

Activation of polymorphonuclear leukocytes (PMNs) and adhesion to the endothelial lining is a major cause of edema formation. Although known to be dependent on the function of β2 integrins (CD11/CD18), the precise mechanisms by which adherent PMNs may impair endothelial barrier capacity remain unclear. Here, the role of transmembrane signaling by β2 integrins in PMN-induced alterations in tight junctional permeability of cultured endothelial cell (EC) monolayers was investigated. PMN activation, in the absence of proinflammatory stimuli, was accomplished through antibody cross-linking of CD11b/CD18, mimicking adhesion-dependent receptor engagement. CD18 cross-linking in PMNs added to the EC monolayer provoked a prompt increase in EC permeability that coincided with a rise in EC cytosolic free Ca2+ and rearrangement of actin filaments, events similar to those evoked by chemoattractant PMN activation. Cell-free supernatant obtained after CD18 cross-linking in suspended PMNs triggered an EC response indistinguishable from that induced by direct PMN activation, and caused clear-cut venular plasma leakage when added to the hamster cheek pouch in vivo preparation. The PMN-evoked EC response was specific to β2 integrin engagement inasmuch as antibody cross-linking of l-selectin or CD44 was without effect on EC function. Our data demonstrate a causal link between outside-in signaling by β2 integrins and the capacity of PMNs to induce alterations in vascular permeability, and suggest a paracrine mechanism that involves PMN-derived cationic protein(s) in the cellular crosstalk between PMNs and ECs.

scholarly journals Pyoverdine-Mediated Iron Uptake in Pseudomonas aeruginosa: the Tat System Is Required for PvdN but Not for FpvA Transport

2006 ◽  
Vol 188 (9) ◽  
pp. 3317-3323 ◽  
Author(s):  
Romé Voulhoux ◽  
Alain Filloux ◽  
Isabelle J. Schalk
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Iron Uptake ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Iron Release ◽  
Outer Membrane Receptor ◽  
Uptake Pathway ◽  
Uptake Process ◽  
Tat System

ABSTRACT Under iron-limiting conditions, Pseudomonas aeruginosa PAO1 secretes a fluorescent siderophore called pyoverdine (Pvd). After chelating iron, this ferric siderophore is transported back into the cells via the outer membrane receptor FpvA. The Pvd-dependent iron uptake pathway requires several essential genes involved in both the synthesis of Pvd and the uptake of ferric Pvd inside the cell. A previous study describing the global phenotype of a tat-deficient P. aeruginosa strain showed that the defect in Pvd-mediated iron uptake was due to the Tat-dependent export of proteins involved in Pvd biogenesis and ferric Pvd uptake (U. Ochsner, A. Snyder, A. I. Vasil, and M. L. Vasil, Proc. Natl. Acad. Sci. USA 99:8312-8317, 2002). Using biochemical and biophysical tools, we showed that despite its predicted Tat signal sequence, FpvA is correctly located in the outer membrane of a tat mutant and is fully functional for all steps of the iron uptake process (ferric Pvd uptake and recycling of Pvd on FpvA after iron release). However, in the tat mutant, no Pvd was produced. This suggested that a key element in the Pvd biogenesis pathway must be exported to the periplasm by the Tat pathway. We located PvdN, a still unknown but essential component in Pvd biogenesis, at the periplasmic side of the cytoplasmic membrane and showed that its export is Tat dependent. Our results further support the idea that a critical step of the Pvd biogenesis pathway involving PvdN occurs at the periplasmic side of the cytoplasmic membrane.

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 Mechanism of Bactericidal Activity of Microcin L inEscherichia coliandSalmonella enterica

2010 ◽  
Vol 55 (3) ◽  
pp. 997-1007 ◽  
Author(s):  
Natacha Morin ◽  
Isabelle Lanneluc ◽  
Nathalie Connil ◽  
Marie Cottenceau ◽  
Anne Marie Pons ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bactericidal Activity ◽  
Salmonella Enterica ◽  
Cytoplasmic Membrane ◽  
Genetic System ◽  
Proton Motive Force ◽  
Motive Force ◽  
Membrane Properties ◽  
Outer Membrane Receptor ◽  
E Coli

ABSTRACTFor the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced byEscherichia coliLR05 that exhibits a strong antibacterial activity against relatedEnterobacteriaceae, includingSalmonella entericaserovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set ofE. coliandSalmonella entericamutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL onE. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential ofE. colicells. However, no permeabilization of the membrane was detected.

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