Analysis of Escherichia coli TonB membrane topology by use of PhoA fusions

Roof, S K; Allard, John; Bertrand, Kevin P.; Postle, Kathleen

doi:10.1128/jb.173.17.5554-5557.1991

Cited by 88 publications

(76 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since TonB has been demonstrated to interact physically with ExbB (20,32) and since TonB and ExbD have similar topologies (12,15,29), we wanted to determine whether TonB-ExbD interactions could be further characterized. Unfortunately, neither in vivo cross-linking nor competition studies with ExbD-PhoA revealed any interactions.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability

et al. 1995

Self Cite

View full text Add to dashboard Cite

TonB protein appears to couple the electrochemical potential of the cytoplasmic membrane to active transport across the essentially unenergized outer membrane of gram-negative bacteria. ExbB protein has been identified as an auxiliary protein in this process. In this paper we show that ExbD protein, encoded by an adjacent gene in the exb cluster at 65, was also required for TonB-dependent energy transduction and, like ExbB, was required for the stability of TonB. The phenotypes of exbB exbD ؉ strains were essentially indistinguishable from the phenotypes of exbB ؉ exbD strains. Mutations in either gene resulted in the degradation of TonB protein and in decreased, but not entirely absent, sensitivities to colicins B and Ia and to bacteriophage 80. Evidence that the absence of ExbB or ExbD differentially affected the half-lives of newly synthesized and steady-state TonB was obtained. In the absence of ExbB or ExbD, newly synthesized TonB was degraded with a half-life of 5 to 10 min, while the half-life of TonB under steady-state conditions was significantly longer, approximately 30 min. These results were consistent with the idea that ExbB and ExbD play roles in the assembly of TonB into an energy-transducing complex. While interaction between TonB and ExbD was suggested by the effect of ExbD on TonB stability, interaction of ExbD with TonB was detected by neither in vivo cross-linking assays nor genetic tests for competition. Assays of a chromosomally encoded exbD::phoA fusion showed that exbB and exbD were transcribed as an operon, such that ExbD-PhoA levels in an exbB::Tn10 strain were reduced to 4% of the levels observed in an exbB ؉ strain under iron-limiting conditions. Residual ExbD-PhoA expression in an exbB::Tn10 strain was not iron regulated and may have originated from within the Tn10 element in exbB.The acquisition of Fe(III) siderophores and vitamin B 12 by gram-negative bacteria appears to be energized by the transduction of the cytoplasmic membrane proton motive force to a set of outer membrane proteins (termed receptors) that actively transport those nutrients into the periplasmic space. In this process, the cytoplasmic membrane protein, TonB, serves as the energy transducer (1). An auxiliary cytoplasmic membrane protein, ExbB, is required for the stability of TonB (9, 33) and plays a direct role in energy transduction (20,32).ExbB is encoded by the first of a closely positioned pair of genes, with the second gene encoding ExbD (8). ExbB is a 26-kDa protein that traverses the cytoplasmic membrane three times, with the majority of the protein occupying the cytoplasm (9,16,17). ExbD, a 17.8-kDa cytoplasmic membrane protein, has a single transmembrane region at its amino terminus, with the majority of the protein extending into the periplasmic space (15). On the basis of this topological partitioning, it has been proposed that the two proteins may function in concert as a signal transduction molecule (26).The process of transcriptional regulation of the two proteins is not clear. Since the open readin...

show abstract

Section: Discussionmentioning

confidence: 99%

Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability

et al. 1995

Self Cite

View full text Add to dashboard Cite

show abstract

“…Studies carried out primarily in Escherichia coli indicate that the receptors form gated pores in the outer membrane, and iron transport via these receptors requires TonB, ExbB and ExbD (Klebba et al, 1993). TonB is a periplasmic protein that is anchored in the cytoplasmic membrane by its hydrophobic N-terminal amino acids (Postle and Skare, 1988;Skare et al, 1989;Roof et al, 1991). TonB is associated with two accessory proteins, ExbB and ExbD.…”

Section: Introductionmentioning

confidence: 99%

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Occhino

Wyckoff

Henderson

et al. 1998

Molecular Microbiology

161

203

View full text Add to dashboard Cite

SummaryVibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set (tonB1, exbB1, exbD1) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system (tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes (hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1. A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae, the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae.

show abstract

“…TonB protein is anchored in the cytoplasmic membrane by its uncleaved N-terminus, which serves as a signal anchor (Postle and Skare, 1988;Roof et al, 1991;Skare et al, 1989), a probable connection to the pmf (Jaskula et al, 1994), and a region of contact with one of the three transmembrane domains of ExbB (Karlsson et al, 1993a;Larsen et al, 1994;Traub et al, 1993). Although TonB has been localized to the cytoplasmic membrane by several groups (Hannavy et al, 1990;Plastow and Holland, 1979;Roof et al, 1991), its association with the outer membrane has never been investigated even though there have been numerous suggestions that such an interaction takes place (Bell et al, 1990;Braun et al, 1991;Gü nter and Braun, 1990;Reynolds et al, 1980;Schö ffler and Braun, 1989;Tuckman and Osburne, 1992), including the detection, by in vivo cross-linking, of direct physical interaction with the outer membrane receptor FepA (Skare et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

“…Although TonB has been localized to the cytoplasmic membrane by several groups (Hannavy et al, 1990;Plastow and Holland, 1979;Roof et al, 1991), its association with the outer membrane has never been investigated even though there have been numerous suggestions that such an interaction takes place (Bell et al, 1990;Braun et al, 1991;Gü nter and Braun, 1990;Reynolds et al, 1980;Schö ffler and Braun, 1989;Tuckman and Osburne, 1992), including the detection, by in vivo cross-linking, of direct physical interaction with the outer membrane receptor FepA (Skare et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli

Letain

Postle

1997

Molecular Microbiology

142

196

View full text Add to dashboard Cite

SummaryThe energy source for active transport of iron-siderophore complexes and vitamin B12 across the outer membrane in Gram-negative bacteria is the cytoplasmic membrane proton-motive force (pmf). TonB protein is required in this process to transduce cytoplasmic membrane energy to the outer membrane. In this study, Escherichia coli TonB was found to be distributed in sucrose density gradients approximately equally between the cytoplasmic membrane and the outer membrane fractions, while two proteins with which it is known to interact, ExbB and ExbD, as well as the NADH oxidase activity characteristic of the cytoplasmic membrane, were localized in the cytoplasmic membrane fraction. Neither the N-terminus of TonB nor the cytoplasmic membrane pmf, both of which are essential for TonB activity, were required for TonB to associate with the outer membrane. When the TonB C-terminus was absent, TonB was found associated with the cytoplasmic membrane, suggesting that the C-terminus was required for outer membrane association. When ExbB and ExbD, as well as their cross-talk-competent homologues TolQ and TolR, were absent, TonB was found associated with the outer membrane. TetA-TonB protein, which cannot interact with ExbB/D, was likewise found associated with the outer membrane. These results indicated that the role of ExbB/D in energy transduction is to bring TonB that has reached the outer membrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One possibility is that TonB transduces energy by shuttling between membranes, and, at some stages in the energy-transduction cycle, is associated with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alternative interpretation that TonB remains localized in the cytoplasmic membrane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observations into two new models, consistent with the novel aspects of this system, that describe a mechanism for TonB-dependent energy transduction.

show abstract

Analysis of Escherichia coli TonB membrane topology by use of PhoA fusions

Cited by 88 publications

References 21 publications

Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability

Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli

Contact Info

Product

Resources

About