From the School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4233
Received for publication, August 16, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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The cytoplasmic membrane protein TonB couples the
proton electrochemical potential of the cytoplasmic membrane to
transport events at the outer membrane of Gram-negative bacteria. The
amino-terminal signal anchor of TonB and its interaction with the
cytoplasmic membrane protein ExbB are essential to this process. The
TonB signal anchor is predicted to form an The outer membrane of Gram-negative bacteria serves as a diffusion
barrier, that, by virtue of the lipopolysaccharide in the outer
leaflet, presents a polar, negatively charged surface to the external
surroundings, hindering the passage of detergents, hydrophobic
antibiotics, and other toxic agents soluble in standard phospholipid
bilayers. This barrier contains aqueous channels that are formed by
general and substrate-specific porin proteins that enable diffusion of
small, hydrophilic nutrients (<600 Da in Escherichia coli),
across the outer membrane.
Simple diffusion is not sufficient for the acquisition of either the
nutrient cobalamin or Fe(III)-siderophore complexes. Instead, these
nutrients are efficiently harvested at the outer membrane surface by
high affinity receptors, which catalyze the active transport of bound
ligand into the periplasmic space (1-3). The release of cobalamin and
Fe(III)-siderophores into the periplasmic space is
energy-dependent, requiring an intact proton gradient at
the cytoplasmic membrane (4-6). The separation of transport events
from their energy source suggests the need for an energy transducer;
this need is met by the protein TonB (5, 7-9). TonB is anchored to the
cytoplasmic membrane presumably by a single transmembrane domain (amino
acids 12-32), derived from an uncleaved signal sequence (10), with the
bulk of the protein occupying the periplasmic space (11, 12). TonB
spans the periplasm and directly contacts outer membrane active
transport proteins (13, 14). Roughly one-third to one-half of the total
cellular TonB partitions exclusively with the outer membrane upon
fractionation, suggesting that TonB functions as an intermembrane
energy shuttle (15). The outer membrane components and specific
features of TonB that support shuttling to the outer membrane are only
defined in part, whereas the requisites for TonB interaction with the cytoplasmic membrane are clear: an intact, wild type signal anchor and
the presence of two proteins, ExbB and ExbD (15, 16). Both of these
integral cytoplasmic membrane proteins physically interact with TonB,
as evident by in vivo cross-linking (17), which, in the case
of ExbB, appears to involve the TonB signal anchor (18).
The TonB residues Ser16 and His20 occur on a
conserved face of the predicted Bacterial Strains and Plasmids--
All bacteria used in this
study were derived from the E. coli K-12 strain W3110 (20).
The majority of experiments used the ampicillin-resistant
tonB deletion strain KP1344 and its
exbB::Tn10 derivative, KP1347 (16).
Transport assays used an aroB derivative of KP1344, KP1351
(16). When necessitated by the use of ampicillin-selected plasmids,
experiments were performed with the ampicillin-sensitive tonB deletion strains KP1229 (21) and KP1304 (constructed
here by P1vir-mediated transduction (22) of an
exbB::Tn10 originating in H1388 (23)
into KP1229).
A pACYC184 derivative (pKP325) carrying araC and the
tonB gene under control of the arabinose promoter and an
isogenic construct encoding TonB
Site-directed mutagenesis was performed in pKP325 and pKP390 by a
method adapted from that described by Michael (27), wherein a third,
5'-phosphorylated oligonucleotide primer bearing the desired base
changes is introduced into a modified polymerase chain reaction
reaction that includes a thermostabile ligase, such that the mutagenic
primer is incorporated in the extending strand. In this study, the
primers used for amplification flanked two BamHI sites, thus
the resultant amplimers could be restricted with BamHI and
then reintroduced to the parent vectors cut at these same sites.
Because the mutagenic primers were designed to also introduce silent
mutations that altered characteristic restriction patterns, products
bearing desired mutations could be detected by restriction mapping.
Candidates thus identified were further mapped to determine insert
orientation, with the inserts ultimately verified by sequencing.
Plasmids generated in this fashion are described in Table
I.
Media and Culture Conditions--
Tryptone plates and top agar,
and LB broth and agar were made as described (22). Liquid cultures were
grown with aeration at 37 °C in M9 minimal salts; supplemented with
0.4% w/v glycerol, 0.2% vitamin-free casamino acids, 40 µg·ml Chemicals and Reagents--
Spot Titer Assay--
Cells were grown in supplemented M9 to an
A550 of 0.4 (as determined with a Spectronic 20 spectrophotometer; path length, 1.5 cm), harvested in 100-µl
aliquots, suspended in 2 ml of molten top agar (60 °C) containing
1.30 mM L-arabinose and antibiotics as
appropriate, and overlaid onto room temperature T plates. Serial 10-fold dilutions of bacteriophage Proteinase K Accessibility Assays--
Cells were grown in
supplemented M9 (containing chloramphenicol and arabinose as indicated)
to an A550 of 0.4 and spheroplasts prepared as
previously described (18). The effects of the protonophore CCCP was
examined as previously described (16). Briefly, CCCP (or an equal
volume of ethanol carrier) was added to spheroplast suspensions to a
final concentration of 50 µM, with samples then incubated
with or without proteinase K (25 µg·ml In Vivo Chemical Cross-linking--
Cells were grown in
supplemented M9 (containing antibiotics and arabinose as indicated) to
an A550 of 0.4, harvested in 1.0-ml aliquots,
centrifuged, and suspended in 938 µl of room temperature 100 mM phosphate buffer (pH 6.8), followed by the addition of 62 µl of fresh 16% formaldehyde monomer. Suspensions were incubated for 15 min at room temperature, centrifuged, suspended in 50 µl of
Laemmli sample buffer, incubated for 5 min at 60 °C, and then stored
at Electrophoretic Analysis of TonB Protein--
Samples prepared
as above were subjected to electrophoresis on SDS 11%
polyacrylamide gels, and the resolved proteins were electrotransferred
to polyvinylidiene fluoride membranes. Immunoblot analyses were
subsequently performed using TonB-specific mAbs and ECL, as previously
described (30).
Transport of [55Fe]Ferrichrome--
Ferrichrome
transport assays were adapted from a study by Köster and Braun
(31). The strain KP1351 ( Single Residue Deletions at Positions 16-20 Result in the Loss of
TonB Activity--
Certain bacteriophage and colicins have evolved to
exploit the TonB-dependent transport system to gain entry
into E. coli (32, 33), and their toxicity provides indirect
assays for TonB function. In this study, the activity of TonB proteins
carrying single residue deletions in the transmembrane domain was
assayed by spot titer against the TonB-dependent
bacteriophage Single Residue Deletions at Positions 16-20 Result in the Loss of
TonB pmf Responsiveness--
We had previously demonstrated that the
conformation of TonB varies with the presence or absence of pmf (16).
Comparison of spheroplasts in the presence and absence of a
protonophore (CCCP or dinitrophenol) revealed that upon collapse
of the cytoplasmic membrane proton gradient, wild type TonB acquires a
distinct conformation wherein the amino-terminal 152-156 residues
exhibit an enhanced resistance to digestion by proteinase K. This
ability to respond to pmf changes is dependent upon the presence of
ExbB/D and is lost upon mutation of either TonB residues
Ser16 or His20 or the deletion of residue
Val17 (16). Here, identical assays of pmf responsiveness
were performed on the present deletion series (Fig.
2). The results obtained paralleled those
of the Some Single Residue Deletions Alter the in Vivo Chemical
Cross-linking Profile of TonB--
Wild type TonB can be cross-linked
in vivo with formaldehyde to form a set of characteristic
higher molecular mass complexes that includes a TonB·FepA complex
that migrates with an apparent mass of 195 kDa, a cluster of
uncharacterized complexes that migrate at 77 kDa, an additional
unidentified complex that migrates at 43.5 kDa (13), and a TonB·ExbB
heterodimer with an apparent molecular mass of 59 kDa (13, 17). Initial
studies with TonB Alanine Replacement of Residues 17-19 Does Not Alter the TonB
Phenotype--
The deletion of either residue 16 or 20 or of the
individual amino acids that reside between these positions resulted in
essentially the same phenotype; inability to support TonB functions as an energy transducer, apparently harvesting the
pmf of the cytoplasmic membrane to drive active transport at the outer
membrane of Gram-negative bacteria (2, 34-36). TonB-dependent processes can be blocked by protonophores
(4-6) and, in unc strains (which lack membrane-bound ATP
synthase), by cyanide (6). Together these results suggest pmf is
necessary, but whether it is the direct, or even the sole energy
source, is unresolved. Regardless of the energy source, recent
observations suggest a dynamic model of energy transduction, wherein
TonB transits through a set of conformations that alternately store and
release potential energy, with transition to a higher energy isomer
coupled to the energy source and transition to a lower energy isomer
coincident to energy release to a ligand-occupied outer membrane
receptor (16). The means by which TonB is coupled to the pmf of the
cytoplasmic membrane is unknown, but it is clear that the transmembrane
signal anchor of TonB (residues 12-32) and its ability to interact
with ExbB are required. Beyond targeting and tethering TonB to the energy source (10), the TonB signal anchor is essential for both
activity (37, 38) and formation of the formaldehyde cross-linked TonB·ExbB heterodimer (38). More recently, we noted that TonB could
assume a pmf-sensitive conformation. This required both a competent
signal anchor and ExbB/D, with mutants such as TonBS16L and
TonBH20Y unable to achieve the conformation unless an ExbB suppressor allele was present (16).
Further evidence for the importance of the TonB signal anchor stems
from the ability of TolQ and TolR to substitute in part for ExbB and
ExbD (39-41). These respective protein sets are similar in both
primary amino acid sequence (25, 42) and topology (43-46), with the
greatest identity evident in their transmembrane domains. Although TonB
and TolA also have a common topology (12, 24), the similarity does not
extend to the primary amino acid sequences, which are highly dissimilar
(28, 47), except for their signal anchors, which share a conserved set
of residues comprising one face of the putative transmembrane The conserved face common to the signal anchors of TonB and TolA
consists of the E. coli TonB residues Ser16,
His20, Leu27, and Ser31 (depicted
in red, Fig. 6). Of these
residues, mutation of Ser31 did not appear to overtly
affect TonB activity (49). In our hands, mutations involving either
Leu27 or Ser31 have not been recovered in
extensive mutant hunts using both high and low stringency selections
for TonB activity (16, 18, 50). Similar mutations of the corresponding
residues in TolA do not disrupt function (51). Thus, residues
Leu27 and Ser31 do not appear to be essential
for energy transduction. Conversely, mutations involving either
Ser16 or His20 have a profound impact on TonB
activity. When expressed at wild type levels, TonBS16L,
TonBH20Y (16), and TonBH20A2 lack
demonstrable activity, whereas for TonBH20R (assayed under conditions of overexpression) activity was greatly diminished (49).
Similarly, substitutions at the corresponding S and H residues of TolA
render it inactive (51).
-helix, with a conserved face comprised of residues Ser16, His20,
Leu27, and Ser31. Deletion of either
Ser16 or His20 or of individual intervening but
not flanking residues rendered TonB inactive and unable to assume a
proton motive force-dependent conformation. In
vivo formaldehyde cross-linking experiments revealed that the
ability of this subset of mutants to form a characteristic heterodimer
with ExbB was greatly diminished. Replacement of residues 17-19 by
three consecutive alanines produced a wild type TonB allele, indicating
that the intervening residues (Val, Cys, and Ile) contributed only to
spacing. These data indicated that the spatial relationship of
Ser16 to His20 was essential to function and
suggested that the motif HXXXS defines the minimal
requirement for the coupling of TonB to the cytoplasmic membrane
electrochemical gradient. Deletion of Trp11 resulted in a
TonB that remained active yet was unable to cross-link with ExbB.
Because Trp11 was demonstrably not involved in the actual
cross-linking, these results suggest that the TonB/ExbB interaction
detected by cross-linking occurred at a step in the energy transduction
cycle distinct from the coupling of TonB to the electrochemical gradient.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical TonB transmembrane domain
(19). We previously noted that the deletion of a residue
(Val17) occurring between the two conserved residues
rendered TonB inactive (18), suggesting either that residue
Val17 made a side chain-specific contribution to energy
transduction or conversely that the spatial relationship between
Ser16 and His20 was essential to function. To
resolve this dichotomy, we constructed a series of single residue
deletions that spanned the amino-terminal two-thirds of the TonB
transmembrane domain. Characterization of the resultant deletion
mutants and of several substitution mutants suggested by our initial
findings indicated that the positional relationship of
Ser16 to His20 was an essential feature for the
apparent coupling of TonB to the proton motive force
(pmf).1
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
V17 (pKP362)
have been described (24). In this study, a compatible pBR322 derivative
carrying a similarly regulated exb operon was constructed by
ligation of a 5'-phosphorylated polymerase chain reaction amplimer
(generated from a pKP298 (13) template and corresponding to residues
585-1748 of the exb operon (25)) into a pBAD18 vector (26)
that had been restricted at the unique SmaI site and
dephosphorylated. Constructs were mapped for orientation, and inserts
were sequenced in entirety to verify identity. The resultant construct
(pKP390) used the vector-provided start site to encode an ExbB with an amino terminus containing a valyl and a prolyl residue between the
methionine initiation and what would normally be the second residue of
ExbB. The second gene in the operon, encoding ExbD, was unaltered by
the cloning strategy, retaining its native initiation and termination codons.
Plasmids generated by site-directed mutagenesis
1 tryptophan, 0.4 µg·ml
1
thiamin, 1.0 mM MgSO4, 0.5 mM
CaCl2, 1.85 µM iron (provided as FeCl3·6H2O), and L-arabinose as
indicated below. For transport assays, vitamin-free casamino acids were
replaced by a defined amino acid mix (40 µg·ml
1 each,
except aspartic acid and glutamic acid (30 µg·ml
1
each), and tyrosine (10 µg·ml
1)) Antibiotics were
used where required at a concentration of 100 µg·ml
1
for ampicillin and 34 µg·ml
1 for chloramphenicol. In
initial cross-linking and proteinase K accessibility experiments, all
strains were grown in supplemented M9 containing 67 µM
L-arabinose to induce tonB expression, which resulted in steady state TonB levels near that of chromosomal controls
(i.e. 6-8-fold greater than the noninduced) for all
constructs except those encoding deletions at positions 14/15-20,
where levels were greater than noninduced but less than the chromosomal
control (data not shown). In subsequent experiments
16-20 were
induced with 130 µM L-arabinose, and
14/15
with 260 µM L-arabinose to attain steady
state levels equivalent to or slightly greater than that of
chromosomally encoded TonB.
-TonB mAb were prepared as
previously described (29). The
-FhuA mAbs 6.9 and 6.14 were the
generous gift of James Coulton. Media components were purchased from
Difco Laboratories (Detroit, MI). L-Arabinose was purchased
from Pfanstiehl Laboratories, Inc. (Waukegan, IL). Monomeric
formaldehyde was purchased from Electron Microscopy Sciences (Fort
Washington, PA). ECL immunoblot kits and [55Fe], provided
as iron chloride, were purchased from PerkinElmer Life Sciences.
Horseradish peroxidase-conjugated goat-
-mouse IgG was purchased from
Caltag Laboratories (Burlingame, CA). Polyvinylidiene fluoride
membranes (Immobilon-P) were purchased from Millipore Corp. (Bedford,
MA). Oligonucleotides were synthesized by Life Technologies, Inc.
Thermolabile alkaline phosphatase was purchased from Epicentre
Technologies (Madison, WI). All other DNA-modifying enzymes were
purchased from either New England Biolabs (Beverly, MA) or Life
Technologies, Inc. Proteinase K, lysozyme, and phenymethylsulfonyl fluoride were purchased from Roche Molecular Biochemicals. All other
reagents were purchased from Sigma.
80vir (initial
concentration, 5.5 × 1010 plaque-forming
units·ml
1) were applied to the plate surface in 5-µl
aliquots and then incubated for 16 h at 37 °C. Results were
recorded as the reciprocal of the highest dilution at which clearing of
the lawn was evident.
1) for 15 min
at 4 °C, treated for 2 min with 1.0 mM
phenymethylsulfonyl fluoride to inactivate the proteinase, and then
precipitated with trichloroacetic acid and processed as described
(16).
20 °C until analysis.
tonB,
aroB), carrying either a control plasmid (pACYC184), or
plasmids encoding either wild type TonB (pKP325) or
TonBVCI17-19AAA (pKP441) was grown to an
A550 of 0.5 in supplemented M9 salts with
chloramphenicol, defined amino acids, and 67 µM
L-arabinose. Cells were centrifuged and then suspended to
2 × 108 colony-forming units
·ml
1 in M9 salts containing 0.1 mM
nitrilotriacetate, followed by incubation with shaking for 5 min. at
30 °C. Samples of 0.5 ml were removed and precipitated with
trichloroacetic acid preparative to electrophoresis. Transport was
initiated by the addition of 150 pmol of
[55Fe]ferrichrome (generated by preincubation of
deferriferrichrome with [55Fe]Cl3 at a 6.7:1
molar ratio in 10 mM HCl at 37 °C for 15 min) to 2 × 108 colony-forming units, with incubation
continued by shaking at 30 °C. Samples of 0.5 ml were harvested at
the indicated time points by filtration onto glass filters, which were
then washed three times with 5 ml of 0.1 M LiCl and dried.
Incorporated [55Fe] was determined by liquid
scintillation counting.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80. Every attempt was made to express the
plasmid-encoded proteins at chromosomal levels. TonB-encoding plasmids
and controls were carried in the tonB deletion strain
KP1344, with TonB expression induced by the presence of 1.3 mM L-arabinose in the top agar. All assays were performed in triplicate. Because this is a solid phase assay, direct
determination of expression levels is problematic; however, this
concentration of L-arabinose resulted in a level of
activity for wild type plasmid-encoded TonB (pKP325) that was
indistinguishable from that obtained with the chromosomally encoded
TonB of W3110 (in each case clearing/plaque formation was evident at
the 10
8 dilution; data not shown). In the absence of
induction, clearing/plaque formation with plasmid-encoded wild type
TonB was reduced 10-fold (data not shown). In a prior study (16),
induction with higher levels of L-arabinose resulted in a
similar reduction of activity for wild type TonB, presumably a dominant
negative gene dosage effect. Thus, although we do not presume that the
levels of TonB achieved in this assay precisely match that encoded by
the chromosome, any activity differences caused by variation in
expression level were less than the resolving power of this assay, with
one possible exception as described below. This noted, the effects of
single residue deletion, when present, were unambiguous (Fig.
1). Deletion of single residues from both
positions 10-14/15 and positions 21-23/24 of TonB had no detectable
impact on sensitivity to
80. Conversely, single residue deletions in
the region of positions 16-20 completely eliminated TonB activity,
with the exception of
V17, where slight clearing with undiluted
phage (~2.75 × 108 plaque-forming units) was
evident in two of three cases (Fig. 1). We have previously found
chromosomally encoded TonB
V17 to be
completely inactive in this assay, with traces of activity evident upon
overexpression (16). Thus it is likely that in the present assay
TonB
V17 was somewhat overexpressed relative
to chromosomally encoded TonB.
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Fig. 1.
Activity of TonB and TonB-
alleles. Activities are expressed on a log scale as the
reciprocal of the highest dilution of bacteriophage
80vir at which a complete clearing of the cell lawn was
evident. The absence of detectable activity is indicated by the
open bars. All strains were assayed in triplicate, with
identical values obtained within each set, except for
TonB
V17, where two plates indicated
sensitivity to undiluted phage and one plate was scored as fully
resistant.
80 spot titer assays (Fig. 1), with proteins bearing single
residue deletions involving positions 16-20 rendered unresponsive to
pmf, whereas proteins with deletions on either flank of this region
(residues 10-14/15 and 21-23/24) retained pmf responsiveness (Fig.
2). In addition, each TonB derivative examined was susceptible to
proteinase K in spheroplasts, indicating that negative phenotypes did
not merely reflect improper secretion.
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Fig. 2.
Identification of a pmf-responsive TonB
conformation in spheroplasts. Immunoblots are shown of
spheroplasts (Sph) and CCCP-treated spheroplasts
(Sph/CCCP), generated from cells in which tonB
genes were induced with L-arabinose to levels approximating
that of chromosomally encoded TonB. Samples were resolved on SDS-11%
polyacrylamide gels, transferred to polyvinylidiene fluoride membranes,
and probed with 1:5,000 mAb 4H4 (specific for TonB residues 79-84;
Ref. 29). The positions and apparent molecular masses of intact TonB
and the proteinase K-resistant product are indicated on the
left. The presence of full-length TonB in the
proteinase-treated lanes is most likely due to incomplete spheroplast
formation. Panels representing S16 through
H20 were overexposed
relative to the other panels to more stringently establish absence of
the pmf-dependent band. The relative amount of proteinase
K-resistant product varies among the positively scored strains. Whether
or not this variation correlates to subtle differences in TonB activity
remains to be established.
V17 indicated that the
mutant could not form a detectable cross-linked complex with ExbB upon
formaldehyde treatment yet retained the ability to form the other
characteristic complexes (18). These experiments were extended here to
include the entire set of single residue deletions (Fig.
3). Several minor deviations in the
cross-linking profile were evident, including variations in the signal
intensity of the several bands comprising the 77-kDa cluster of the
14/15-
20 mutants, increased signal intensity of the 43.5-kDa
complex for the
13-
20 mutants, and the appearance of a novel
66-kDa complex (indicated by the arrow in the right
panel) for the
17-
19 mutants. Because the non-TonB
components of these complexes are not as yet determined, the
implications of these differences were unclear. More readily
interpretable were the effects of the mutations on the ability of TonB
to form the 59-kDa heterodimer with ExbB. These results paralleled the
results of the activity and pmf responsiveness assays, with the ability
of the
16-
20 mutants to form the characteristic cross-linked
complex with ExbB greatly diminished or absent. This phenotype was also
evident with the
14/15 mutant and, surprisingly, with the
11
mutant. Preliminary studies2
suggest the inability to detect cross-linked complexes of the
14/15
mutant with ExbB may be related to its apparent instability (see
"Experimental Procedures"). Because tryptophan 11 is theoretically formaldehyde-reactive, the inability to detect cross-linked complexes of the
11 mutant with ExbB suggested the possibility that the actual
cross-linking occurred through this residue. To test this possibility,
two additional mutations were generated. First, the Trp11
residue of TonB was conservatively replaced by phenylalanine, a residue
nonreactive with formaldehyde. Second, Trp38 of ExbB was
replaced with a phenylalanine, because topological comparisons
suggested that this was the nearest formaldehyde-reactive residue with
which TonB Trp11 could interact. The TonB activity of
strains carrying one or both of these alleles, as determined by spot
titer assays, was indistinguishable from wild type (data not shown).
Alleles were paired and evaluated by in vivo formaldehyde
cross-linking (Fig. 4). Unexpectedly,
replacement of TonB Trp11 with a nonreactive residue did
not result in the loss of formaldehyde-mediated cross-linking of TonB
to ExbB. Similarly, ExbBW38F did not lose the ability to
cross-link to TonB; however, the relative amount of complex formed
appeared to be diminished with both wild type TonB and
TonBW11F.
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Fig. 3.
In vivo chemical formaldehyde
cross-linking of tonB strains expressing
plasmid-encoded wild type or deletion mutant TonB derivatives.
Wild type TonB was expressed in both the ExbB+ strain
KP1344 (labeled ExbB+) and the ExbB
strain
KP1347 (labeled ExbB-) to verify the TonB/ExbB heterodimer.
Mutant TonB derivatives were all expressed in the ExbB+
strain KP1344. To normalize small differences in expression level,
samples were first run in 10-µl aliquots, and the relative amount of
TonB monomer was estimated visually (gel not shown). Samples were then
loaded on a second gel in volumes of 5, 10, or 15 µl (except for
TonB
L14/15, for which 25 µl was loaded) to
provide equivalent TonB signals for each sample. Samples were resolved
and visualized as with Fig. 2, except that probing was performed with
1:2,500 mAb 4F1 (specific for TonB residues 120-128; Ref. 29). The
position of TonB monomer and of the standard TonB-containing complexes
are indicated on the left. The position of the novel 66-kDa
complex present in samples
V17-
I19 is indicated by the
arrow on the right.
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Fig. 4.
In vivo chemical formaldehyde
cross-linking of the tonB,
exbB::Tn10 strain KP1304
expressing plasmid-encoded wild type (wt) TonB or
TonBW11F paired with wild type ExbB,
ExbBW38F, or the parent plasmid of the ExbB
derivatives. Samples were resolved and visualized as with Fig. 3.
The positions of TonB monomer and the TonB/ExbB heterodimer are
indicated on the left.
80 infection
(Fig. 1), inability to assume a pmf-dependent conformation
(Fig. 2), and greatly decreased ability to form formaldehyde-mediated
cross-links with ExbB in vivo (Fig. 3). Although these
results appeared to exclude the possibility that residues flanking
residues 16-20 made direct contributions to energy transduction, they
did not distinguish whether any of the residues between positions 16 and 20 were of specific consequence or merely functioned as spacers to
provide the proper degree of helix twist between Ser16 and
His20. To address this issue, we replaced these intervening
residues (Val17, Cys18, and Ile19)
with three alanines. Initial characterization of the resultant TonBVCI17-19AAA found it to be indistinguishable from wild type TonB on the basis of
80 spot titer-determined activity, ability
to assume a pmf-dependent conformation, and in
vivo formaldehyde cross-linking profile (data not shown). To more
rigorously address function, transport assays using
55Fe-loaded ferrichrome were performed. The ability of
TonBVCI17-19AAA to transport radiolabeled ferrichrome did
not significantly differ from that of wild type TonB (Fig.
5). Transport was not evident for cells
carrying a control plasmid (pACYC184) alone or when pmf was collapsed
in cells encoding wild type TonB by the addition of CCCP, with counts
remaining consistently below 1,000 cpm/1 × 108 cells
throughout the experiment (data not shown). Immunoblot analysis of TonB
and the ferrichrome receptor FhuA levels in the assayed cultures
indicated that the levels of both were essentially identical in cells
expressing either wild type TonB or TonBVCI17-19AAA (data
not shown).
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Fig. 5.
Transport of
[55Fe]ferrichrome. KP1351 carrying plasmids encoding
either wild type TonB or TonBVCI17-19AAA were grown and
assayed for uptake of [55Fe]ferrichrome, as described
under "Experimental Procedures." Data are expressed as cpm/cells.
In four separate experiments, counting efficiency ranged from 23.5 to
34.6% (data not shown). Combined with a radiochemical purity of
99.0%, this predicts that 10,000 cpm corresponds to 0.63-0.93 pmol of
[55Fe]ferrichrome transported. Values for wild type TonB
are indicated by the filled triangles, whereas values for
TonBVCI17-19AAA are indicated by the open
squares.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix
(19).
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Fig. 6.
Thermodynamically minimized
-helical (3.6 residues per turn, 5.4 angstrom
pitch) prediction for the TonB amino-terminal transmembrane domain
region (residues 9-32), modeled in and adapted from Ref. 18.
Residues 12-32 are predicted by average hydrophobicity (48) and
topology (12) to represent the transmembrane domain. The orientation of
the structure relative to the cytoplasm and the periplasmic space is
indicated. Residues comprising the conserved face are indicated in
red and identified by label. The data presented in Figs.
2-4 are summarized at the right of the figure at individual
positions corresponding to the position of the involved residue
depicted in the modeled structure. Activity is scored as the highest
log dilution of
80vir at which clearing or plaque
formation was evident,
indicates no clearing with undiluted
80vir. For pmf sensitivity, the ability to form
the pmf-dependent proteinase K-resistant product is
indicated by +, whereas the apparent absence of such a product is
indicated by
. For in vivo cross-linking to ExbB, the
ability to form an identifiable TonB/ExbB heterodimer is indicated by
+, whereas the apparent inability to efficiently form such a product is
indicated by (
).
We previously isolated a tonB mutation where loss of
function resulted from the deletion of a single residue
(Val17) in the region separating Ser16 and
His20 (18). The phenotype of this mutant was similar to
that of the later isolated Ser16 and His20
mutations, to the extent that ExbB suppressors isolated for
TonV17 and TonBS16L were
essentially interchangeable (16, 18). When considered together, these
and the above-cited results led us to consider whether or not
transmembrane domain residues exclusive of positions 16-20 were of
direct consequence to the coupling of TonB to the cytoplasmic membrane
proton gradient. That hypothesis was examined using a set of single
residue deletions, the phenotypes of which are summarized in Fig.
6.
Not surprisingly, deletion of either Ser16 or
His20 resulted in a negative phenotype by all measures,
similar to the results obtained with point mutations involving these
residues (16). Deletion of any single intervening residue also produced
a negative phenotype, consistent with the previous V17 results (18).
Deletion of single residues to the carboxyl-terminal flank of
His20 generated a wild type TonB, indicating that amino
acids 21-24 have no role in energy transduction and suggesting that
the position of Ser16 and His20 relative to the
other conserved residues (Leu27 and Ser31) was
also not essential to function. Deletion of single residues to the
amino-terminal flank of Ser16 also generated essentially
wild type TonB (except for formaldehyde-mediated cross-link formation
with ExbB by TonB
L14/15 (which may be
unstable) and TonB
W11, discussed below),
indicating that amino acids 10-15 have no role in energy transduction
and suggesting that the position of Ser16 and
His20 relative to the TonB cytoplasmic domain and the first
several residues of the signal anchor was also not essential to function.
These results suggested two alternatives, either the position of
Ser16 and His20 relative to each other was
sufficient for effective coupling of TonB to the energy gradient or the
side chains of one or more intervening residues contributed to the
energization mechanism. This was resolved by the substitution of
residues 17-19 by three consecutive alanines. The resultant
TonBVCI17-19AAA was wild type by all measures, indicating
that the intervening residues did not make side chain-specific
contributions essential for the energization of TonB. Beyond supporting
the hypothesis that the positional relationship of Ser16 to
His20 is necessary to function, these results suggest that
the motif SXXXH (where residues S and H occupy a common face
of the -helix) may define the minimal structural requirement for the
coupling of TonB to the cytoplasmic membrane proton electrochemical
gradient. It should be noted that the initial suspicion that
Ser16 and His20 were of consequence came from
their conservation in TolA and the suggestion that these residues would
occupy the same face of an
-helix (19). In the absence of a solved
crystal structure, the present data represent the strongest advocacy to
date for such a structure.
The inability of TonBW11 to form
formaldehyde-mediated cross-links with ExbB, while otherwise remaining ostensibly wild type, is intriguing. Our initial suspicion that the
explanation for this observation would prove to be trivial (i.e. the deleted tryptophan was the site of cross-link
formation) was not borne out. Thus, we are left with the more exciting
interpretation that this lack of detectable cross-linking could be
indicative of an altered TonB conformation wherein the actual
cross-linking site on TonB is no longer proximal to a
formaldehyde-reactive site on ExbB. This suggests that the TonB/ExbB
interaction detected by cross-linking is not essential to energy
transduction. One possibility is that cross-linking detects an
interaction that defines the recycling process by which spent TonB is
shuttled back to a form competent to store energy. Thus, the reduction or absence of cross-linking to ExbB by TonB mutants unable to support
energy transduction might reflect the fact that they are blocked in a
portion of the cycle that occurs prior to the stage marked by
cross-linking to ExbB.
Studies concerning the contribution of the TonB signal anchor to the
recycling phase of the shuttling cycle are ongoing. The present study
has addressed the role of the TonB signal anchor in the energization
step of the cycle and has defined what appears to be the minimal
structural requirement of TonB for efficient coupling to the
cytoplasmic membrane proton gradient. It is evident that this process
is facilitated by ExbB and ExbD; however, the means by which this
occurs remain obscure. ExbB and ExbD can each form, at minimum,
homotrimers (17). Because ExbB and ExbD can interact in
vitro (52), they may occur as a heterohexamer, structurally consistent with the idea that they could be a proton translocator. They
are also required for TonB to respond to the presence or absence of pmf
(16). Understanding ExbB and ExbD will be essential for elucidating the
mechanism by which TonB becomes energized. A likely first step would
involve determining whether or not ExbB (or ExbD) directly interacts
with the TonB SXXXH motif and, if so, which features of ExbB
participate in such an interaction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank James Coulton for the -FhuA mAb
and Camari Ferguson for construction of KP1304. We also thank
Penelope Higgs, Tracy Letain, and Hema Vakharia for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant GM42146 from the National Institutes of General Medical Sciences (to K. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
509-335-5614; E-mail: postle@mail.wsu.edu.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M007479200
2 R. A. Larsen and K. Postle, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pmf, proton motive force; CCCP, carbonylcyanide m-chlorophenylhydrazone; mAb, monoclonal antibody.
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