From the Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
Received for publication, October 9, 2002, and in revised form, October 24, 2002
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ABSTRACT |
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We characterized the uptake of ferric
enterobactin (FeEnt), the native Escherichia coli ferric
siderophore, through its cognate outer membrane receptor protein, FepA,
using a site-directed fluorescence methodology. The experiments first
defined locations in FepA that were accessible to covalent modification
with fluorescein maleimide (FM) in vivo; among 10 sites
that we tested by substituting single Cys residues, FM labeled W101C,
S271C, F329C, and S397C, and all these exist within surface-exposed
loops of the outer membrane protein. FeEnt normally adsorbed to
the fluoresceinated S271C and S397C mutant FepA proteins in
vivo, which we observed as quenching of fluorescence intensity,
but the ferric siderophore did not bind to the FM-modified derivatives
of W101C or F329C. These in vivo fluorescence
determinations showed, for the first time, consistency with
radioisotopic measurements of the affinity of the FeEnt-FepA interaction; Kd was 0.2 nM by both
methods. Analysis of the FepA mutants with AlexaFluor680, a
fluorescein derivative with red-shifted absorption and emission spectra
that do not overlap the absorbance spectrum of FeEnt, refuted the
possibility that the fluorescence quenching resulted from resonance
energy transfer. These and other data instead indicated that the
quenching originated from changes in the environment of the fluor as a
result of loop conformational changes during ligand binding and
transport. We used the fluorescence system to monitor FeEnt uptake by
live bacteria and determined its dependence on ligand concentration,
temperature, pH, and carbon sources and its susceptibility to
inhibition by the metabolic poisons. Unlike cyanocobalamin
transport through the outer membrane, FeEnt uptake was sensitive to
inhibitors of electron transport and phosphorylation, in addition to
its sensitivity to proton motive force depletion.
Gram-negative bacteria recognize and transport a variety of ferric
siderophores (1, 2) through outer membrane
(OM)1 receptor proteins that
function as ligand-gated porins (LGP) (3). Some of the hallmarks of
these transport processes are the specificity with which LGP select
their iron-containing ligands (4-7), the high affinity of the
receptor-ligand interactions (8, 9), conformational changes in the
receptors during ligand binding (10-13) and transport
(14),2 the requirement for
the accessory proteins TonB (16), ExbB, and ExbD (17, 18), and the need
for cellular energy to accomplish active transport of the
metal-containing ligands through the OM (19-22). This latter energy
requirement is atypical in a bilayer that contains open channels
(23-26) and therefore cannot sustain an ion gradient. At present the
pathways through which both energy and TonB participate in metal
transport are unresolved (27-29). The crystal structures of FepA (30),
FhuA (31, 32), and FecA (13) revealed that their N-terminal 150 amino
acids fold into a 4-stranded Site-directed biophysical methodologies defined many of the biochemical
properties of FepA (10, 11, 14, 33) by derivatization of the
genetically engineered mutant FepAE280C with fluorescent or
paramagnetic labels. Residue E280C exists on the external surface of
the third surface loop (L3) (30, 33) of the receptor, and nitroxide
spin labels attached to it reflected structural changes in FepA when
FeEnt binds and different motion when it passes through the OM protein
(14). Similarly, the binding of either FeEnt or ColB quenched
fluorescein labels attached to purified FepAE280C, which allowed
determination of the thermodynamic and kinetic properties of the
binding reaction (10, 11). One of the conspicuous findings of those
experiments was that purification reduced the affinity of FepA for
FeEnt (9, 10) and the affinity of FhuA for its ligand, ferrichrome,
(35, 7) at least 100-fold. In this report we applied fluorescence
methodologies to FeEnt transport in vivo; the sensitivity
and specificity of the technique provided not only an explanation for
the discrepancies in affinity that were observed in vivo and
in vitro but also more detailed information on the ligand
internalization reaction through the OM, including its temperature, pH,
and energy dependence.
Bacterial Strains, Plasmids, and
Chemicals--
Escherichia coli K12 strains KDF541
(F Ferric Enterobactin--
Enterobactin was purified from E. coli strain AN102 (39), and FeEnt was prepared and purified as
previously described (40). Its concentration was determined from
absorbance at 495 nm ( Anti-iodoacetamide Fluorescein (IAF) Sera--
Rabbits were
immunized weekly for one month with 5-IAF (Molecular Probes) conjugated
to BSA and subsequently bled through the ear. Sera were titered by
enzyme-linked immunosorbent assay (> 10,000) and Western immunoblot
against ovalbumin-IAF conjugates.
Site-directed Mutagenesis--
We used QuikChange (Stratagene)
for site-directed mutagenesis (7, 12) on pITS449 or pT944 and
designated the mutant FepA proteins FepAXnY, where X represents the
one-letter abbreviation for the wild type residue at position n and Y
represents the one-letter abbreviation for the substituted amino acid.
We tested the functionality of each FepA mutant by evaluating its
expression (42), susceptibility to colicins B and D (40), 59FeEnt binding (9), and FeEnt transport in qualitative
siderophore nutrition assays (5) and quantitative determinations of
59FeEnt uptake (9).
Fluorescence Labeling of Live Cells--
For fluorescein
labeling of live bacteria, we grew the cells to stationary phase in LB
broth (43) and subcultured at 1% into MOPS medium (44)
containing appropriate nutritional supplements at 37 °C with
shaking to mid-log phase. For labeling in energy-deficient conditions, after growth in LB and subculture in MOPS the bacteria were
collected by centrifugation and resuspended in the same volume of MOPS
media but without glucose or casamino acids, and with half the usual
supplementation of required amino acids. The culture was then incubated
with shaking for 10 h at 37 °C.
Bacteria were collected by centrifugation (5000 × g,
20 min), washed with and resuspended in Tris-buffered saline (TBS), pH 7.4, to a final concentration of 5 × 108 cells/ml.
Fluorescein maleimide (FM) or AlexaFluor680 maleimide (AM)
in dimethyl formamide (less than 0.1% of final volume) was added to a
final concentration of 5 µM and incubated at room
temperature for 30 min in the dark with shaking. The labeled cells were
centrifuged, washed three times with 25 ml of ice-cold TBS plus 0.05%
Tween-20, washed once with and resuspended in ice-cold TBS, and stored
on ice for immediate use.
Labeling Specificity--
We evaluated labeling specificity with
anti-FepA and anti-IAF immunoblots of cell lysates and with
fluorescence emission scans of labeled experimental and control
bacterial cultures. In the former case, lysates from 108
cells were subjected to 10% SDS-PAGE and Western immunoblot with anti-BSA-IAF sera; the reactions were quantified on a Storm Scanner (Molecular Dynamics) after development with 125I-Protein A
(42). The immunoblots showed that expression of FepA did not
significantly vary under the culture conditions we employed. The
anti-BSA-IAF serum was equally effective against proteins and cells
modified by FM, demonstrating its recognition of the fluorescein moiety.
The characteristic orange color of fluorescein facilitated qualitative
evaluation of labeling specificity by visual inspection of treated cell
pellets. Strains expressing wild type FepA, which was not labeled by
either IAF or FM, were not colored after treatment with the reagents,
whereas bacteria expressing mutant FepA proteins containing single,
genetically engineered cysteines in accessible locations were
identified by the orange color of their cell pellets. To quantitatively
determine fluorescein labeling specificity, we performed emission scans
at 20 °C with an excitation wavelength of 490 nm and a 5 s
integration time. We compared scans for fluorescein-labeled strains
expressing wild type and mutant FepA proteins and used scans of
untreated bacteria to establish the background fluorescence.
Fluorescence Measurements--
Using an SLM-AMINCO 8000 fluorimeter (Rochester, NY) upgraded to 8100 functionality,
we recorded the fluorescence intensities of bacteria (5 × 107 cells) suspended in 2 ml of either TBS or MOPS media
and equilibrated at temperature. For fluorescence determinations
of FeEnt binding affinity, we added various concentrations of FeEnt to
the cell suspension and recorded fluorescence intensity with excitation and emission wavelengths of 490 nm and 518 nm, respectively, for fluorescein and 679 nm and 702 nm for AlexaFluor680. We
accounted for background fluorescence and volume changes and analyzed
the data with the bound (1-F/F0)
versus total function of GraFit 4 (Erithacus Software Ltd.,
Middlesex, UK).
Effects of Energy Inhibitors on FeEnt Transport--
To study
the effect of inhibitors on FeEnt uptake, 5 × 107
FM-labeled, energy-starved cells were first incubated for 40 min at
37 °C with shaking in TBS buffer plus glucose (0.4%) and the corresponding inhibitors. The cells were transferred to the sample cuvette, equilibrated at the measurement temperature, and the uptake
time course was recorded.
Site-directed Covalent Modification with Fluorescein Maleimide in
Vivo: Effect of Lipopolysaccharide--
OM proteins contain few
unpaired cysteines, and we tested the possibility that genetically
engineered single Cys residues at sites of interest may be
covalently modified in live bacteria with -SH-specific fluorescent
probes. Although we previously modified residue E280C in FepA L3 with
nitroxide compounds (14, 33, 34), we could not label it with
fluorescent probes (45), probably because of the inaccessibility of
sites close to the hydrophilic OM surface to hydrophobic molecules like
fluorescein (Fig. 1A). Therefore, from the FepA crystal structure we designed and introduced 10 more individual, unpaired Cys residues at positions either on the
cell surface or within the periplasm: W101C, S150C, S211C, Y260C,
S271C, F329C, S397C, S423C, S575C, and S595C. Among these 10 target Cys
residues, FM specifically labeled W101C, S271C, F329C, and S397C, which
all reside in cell surface-exposed loops of FepA (Fig. 1A)
above the level of the LPS core sugars; W101 lies in the second loop of
the N-domain, whereas S271, F329, and S397 exist in loops 3, 4, and 5, respectively, of the C-domain. FM also specifically modified, at lower
levels, sites S63C and S150C, which reside in NL1 and on the
periplasmic rim of the FepA
Further study showed that two circumstances affected the specificity
and efficiency of the fluorescence labeling procedure: the nature of
the LPS O-antigen and the bacterial culture conditions. In our initial
experiments the bacteria adsorbed fluorescence probes, but we saw
little difference in the fluorescence intensity of FM-treated
KDF541/pFepAS271C and KDF541/pITS449
(fepA+). The use of two different
reagents, IAEDANS and coumarin maleimide, did not remedy this lack of
specificity (data not shown). KDF541, the E. coli strain
that was the usual host for plasmids, produces rough LPS without any
O-antigen, and we considered the possibility that LPS was a determining
factor in the specificity of the fluorescence labeling reactions. The
use of deep rough mutant strains did not improve the labeling
specificity of surface-exposed Cys residues (data not shown). Although
Western blots revealed specific covalent modification of some of the
Cys mutants, FM nonspecifically adsorbed to deep rough strains, which
was apparent in the yellow color of the cell pellets that was not
diminished by repeated washing. These results suggested that in deep
rough strains, and to a lesser extent in rough strains, the fluorescent
reagents penetrated into the OM bilayer and resisted washing
procedures. The converse experiment confirmed this inference; the use
of bacterial cells synthesizing full-length LPS minimized nonspecific
adsorption of fluorescent dyes. We achieved this result by introducing
pMF19, which carries a rhamnosyl transferase that allows production of
the LPS O-chain (wbbL+) (37). Strains harboring pMF19 and
one of the four accessible Cys substitution mutants of FepA were
specifically labeled by FM, as seen by fluorescence intensity
measurements of live bacteria and in Western immunoblots (Fig.
1B).
FeEnt Binding to Fluorescein-labeled Live Bacteria--
We
measured the ability of the four surface-localized, Cys substitution
mutants of FepA to bind 59FeEnt before and after
fluoresceination. Adsorption of the ferric siderophore was weak and
barely detectable in W101C- and F329C-FM; attachment of fluorescein at
these sites sterically hindered the adsorption of the siderophore.
These data concurred with the prior conclusion that residue
Phe329 exists in close proximity to, or is a
component of, an initial FeEnt binding site (B1) (11); the crystal
structure shows W101C in the middle of the FepA vestibule (Fig.
1A). On the other hand, FM modification of S271C or S397C
did not interfere with FeEnt recognition and binding; bacteria
expressing either of these mutant proteins manifested normal
(subnanomolar) binding affinities (Fig. 2) even after fluoresceination. These
experiments showed, for the first time, correspondence between the
binding affinities measured in vivo by radioisotopic and
fluorescence methodologies (Kd = 0.3 nM). In subsequent experiments we exclusively studied the
S271C site, located at the extremity of L3.
FeEnt Binding to Alexa Fluor680-labeled
Bacteria--
Ligand binding diminished the fluorescence of probes
attached to FepA at E280C, and our modifications and analyses of S271C and S397C showed the same quenching phenomenon in vivo.
Previous determinations of binding kinetics found a biphasic
association reaction between purified FepAE280C-FM and FeEnt and ColB
(10, 11). We interpreted these data as initial ligand adsorption to an
external site (B1), followed by movement to a second site deeper in the
vestibule (B2). The ability of E280C-FM to reflect these two binding
phases presumably arose from changes in the local environment of the
fluor that occurred as the receptor protein underwent conformational
dynamics during ligand adsorption. However, because its absorption
spectrum overlaps the emission spectrum of fluorescein, the possibility
existed that during its binding FeEnt quenched fluorescence by energy
transfer between the excited fluor and the ferric siderophore. Studies
with a red-shifted fluorescein derivative that does not overlap the
absorption spectrum of FeEnt (AM: 679 nm and 702 nm, respectively),
refuted this explanation (Fig. 2); during FeEnt binding, FepA proteins
modified with AM exhibited equivalent reductions in intensity to those
modified with FM. The comparable quenching of AM, without the
possibility of energy transfer to FeEnt, indicated that the reductions
in intensity did not derive from close proximity of the ferric
siderophore to the fluor. Rather, the data favored the notion that
binding triggers conformational dynamics that alter the environment of the reporter molecules.
Temperature-dependent Changes in Polarization--
At
physiological temperatures we expected a decrease in the polarization
of fluorescent labels attached to the surface loops of FepA because of
increased Brownian motion (46, 47). The polarization of FepAS397C-FM
(in L5) decreased as the temperature of the system was raised, but that
of FepAS271C-FM (in L3) increased (Fig.
3). These data confirmed that the
temperature affected the motion of the attached fluor; L3 became less
mobile and L5 became more mobile in response to increased temperature.
These states were reversible as the temperature of the system decreased
again.
Fluorescence Measurements of FeEnt Uptake--
When exposed to
FeEnt at 20 °C, KDF541/pMF19/pS271C-FM bound and transported it, and
we spectroscopically monitored these reactions. Subsequent to its
binding, we observed FeEnt transport as a recovery of fluorescence
intensity, but only when the extracellular free ligand was depleted by
its uptake into the cells. At that time, when the population of
receptor proteins was vacated, its original fluorescence intensity
returned. The consumption of FeEnt was necessary to observe
fluorescence changes because, when present, it bound and quenched
again. Thus in the presence of sufficient excess of the ferric
siderophore, we saw no renewal of fluorescence.2 When it
occurred, the resurgence of fluorescence paralleled the kinetics of
FeEnt uptake, independently measured in 59Fe uptake
experiments (Fig. 4). In those studies,
the increase in fluorescence intensity occurred between 1200 and
1400 s. The corresponding radioisotope uptake experiment at
20 °C showed that accumulation of 59FeEnt stopped at the
same time as a result of depletion of the substrate from the media. So
the recovery of fluorescence intensity precisely mirrored the depletion
of the ligand from the culture.
Consistent with prior observations of the energy-dependent
uptake reaction (9, 48-51), the spectroscopic measurement of FeEnt
transport was affected by media composition, FeEnt concentration, and
temperature. It occurred more slowly in bacteria deprived of
nutritional requirements, either in minimal media lacking auxotrophic amino acid supplements or suspended in TBS (Fig.
5A). As discussed above, at
20 °C the transport of FeEnt was visible as a return of fluorescence
intensity, and the concentration-dependence of the phenomenon was
striking. For bacteria exposed to 2, 5, 10, and 20 nM
FeEnt, the lag time preceding transport was 250, 550, 1000, and
2050 s, respectively (Fig. 5B). Furthermore, AM-labeled cells showed the same spectroscopic response as FM-labeled bacteria. These data indicated that the intensity changes that occurred during
uptake also derived from conformational motion in the surface loops, in
this case associated with ligand internalization. At 37 °C, in the
presence of equimolar or subsaturating FeEnt, the reaction occurred
most rapidly (Fig. 5B, inset), too rapidly to permit a detailed analysis. However, the fluorescence intensity changes
were fully TonB-dependent, whether the experiments were conducted at 20 °C (Fig. 5B) or 37 °C. A complete
description of the fluorescence changes associated with the 37 °C
transport reaction, including its energy- and TonB-dependence, is given elsewhere.2
The uptake reaction was pH-dependent. We attempted to study
the process at pH 10 but found that FeEnt does not adsorb to FepA at
high pH. This result concurred with the fact that basic residues (Arg286, Arg316, Ref. 42; Lys483,
Ref. 12) play a role in the binding of the acidic ferric siderophore. At pH 4, FeEnt bound and quenched the fluorescence intensity of KDF541/pMF19/pS271C-FM; however, the magnitude of the quenching was
slightly less, and the recovery of fluorescence that normally occurred
as the temperature approached 37 °C did not take place at pH 4 (Fig.
5B), suggesting that uptake was impaired under these conditions.
We investigated the range of permissive temperatures for FeEnt uptake
(Fig. 5C). As temperature decreased, so also did the slope
of the fluorescence recovery. Conversely, the bacteria experienced a
lag prior to the rebound of fluorescence increase, the magnitude of
which was inversely proportional to temperature. These data supported
the conclusion that the recovery of fluorescence derived from the
transport reaction. Somewhat unexpectedly, the bacteria transported
FeEnt, although very slowly, even at 5 °C (Fig. 5C).
Energy-dependence and Susceptibility to Poisons--
The proton
ionophore CCCP, and the electron transport inhibitors cyanide and azide
completely and indefinitely abrogated uptake of the ferric siderophore
(Fig. 6A); glucose starvation
produced comparable effects (Fig. 6B). On the other hand,
the proton ionophore DNP and the phosphate analog arsenate inhibited
uptake but not to the same extent. Transport occurred in their
presence, but the slope of the fluorescence increase was significantly
reduced in both cases, such that the bacteria utilized the quantity
of extrinsic FeEnt (2 nM) in 1 or 2 h,
respectively. In the absence of inhibitors the cells exhausted the
exogenous ligand within 10 min. The effects of arsenate specifically
related to its inhibition of phosphate metabolism because the presence
of 30 mM phosphate effectively suppressed arsenate
inhibition (Fig. 6C). Finally, in the absence of glucose,
ascorbic acid and succinate effectively restored transport activity,
although in the latter they supported transport only at a much slower
rate.
Variations in the fluorescence intensity of bacteria labeled with
fluorescein or AlexaFluor680 at FepA residue S271C
reflected the active transport of FeEnt through the OM layer of the
cell envelope. The spectroscopic phenomena recapitulated the
characteristics of the energy-dependent active transport
reaction: high affinity, occurring at concentrations less than 2 nM; a direct relationship between lag time and initial
ligand concentration; temperature-dependence of the rate of intensity
change; consistency with uptake measurements by radioisotopic
determinations; susceptibility to energy poisons. These data
unambiguously demonstrate that the recovery of fluorescence after FeEnt
binding originates from its uptake reaction through the OM. The
spectroscopic system offers an alternative to existing methods for
observation of OM transport that is sensitive, precise, continuous, and
amenable to quantitative analysis.
The evidence that the fluorescence measurements describe conformational
motion during the uptake reaction stems from several independent
observations. (i) In prior work utilizing FepAE280C-FM, we concluded
that conformational change occurs in L3 in vitro because the
modified side chain of E280C resides on the outside of the surface
vestibule where it cannot contact FeEnt and similar quenching was
engendered by binding of colicin B, which contains no metal complex.
These data made collisional quenching unlikely for either ligand and
excluded energy transfer as a mechanism to explain the effects of ColB,
which has no chromophores. (ii) In the current experiments, the FepA
crystal structure suggests that fluorescein attached to S271C at the
extremity of L3 likely projects toward the interior of the vestibule.
The observation that FeEnt binding quenches S271C-AM in the same way
that it quenches S271C-FM eliminates energy transfer as an explanation
of the inhibition. (iii) These data do not rule out collisional
quenching of fluors attached to S271C, but this possibility seems
unlikely. Modification of S271C with fluorescein had no effects on the
affinity, capacity, or transport of 59FeEnt, indicating
that this residue does not likely contact FeEnt during binding or
uptake. Fluoresceination of Phe329, on the other hand,
abrogated FeEnt adsorption and transport (45), reinforcing this
inference. (iv) In addition, given the extensive spectral overlap of
FeEnt and fluorescein, it seems unlikely that significant collisional
quenching can occur without significant energy transfer, which was not
found. (v) From a logistics perspective, FeEnt binds to the outside of
a closed channel, so some sort of conformational change is necessary to
promote its internalization. (vi) Finally, the loops of FepA (L7) (12)
and FecA (L7 and L8) (13) are conformationally active during FeEnt binding, and loop motion was previously seen in vivo (14).
In summary, a body of evidence argues against either collisional or
energy transfer mechanisms of quenching, leaving conformational dynamics that relocalize the fluor to a quenching environment as a
single viable explanation for the quenching phenomena. This conclusion
concurs with data acquired by a variety of methodologies (7, 10,
12-14, 32-34).
Because L3, L5, and L7 in FepA change in conformation during the
interaction with ligand, it appears likely that such motion is a
general property of ligand-gated porin surface loops. We envision a
model of FepA conformation in which the receptor exists in an extended,
open form in the absence of ligand but closes around ligands when they
bind. At either 4 °C or 20 °C, FeEnt adsorption creates the
closed state, in which the fluor undergoes maximal quenching from
increased collisions with water or other polar or charged molecules in
the environment created by the aggregation of the loops around the
ligand. This mechanism agrees with the observation of biphasic binding
kinetics for ligand adsorption to FepA and with contractions from open
to closed states of FecA loops 7 and 8 and FepA L7. As the temperature
warms and FepA transports FeEnt, fluorescence rebounds because the now
empty receptor proteins revert from the closed state to the open state.
At 37 °C, further motion occurs that results in additional quenching
(18).
One perplexing finding about FepA and FhuA was that purification from
the OM quite drastically decreased their ability to bind ligands. This
decrease questioned the relevance of the crystallographic data to the
structures of the siderophore receptors in vivo. However, two different techniques, 59FeEnt binding in
vivo (9) and fluorescence spectroscopy in vitro (10),
defined the discrepancy, raising the possibility of methodological
bias. The results we report eliminate this contingency by determining
with a fluorescence methodology that the affinity of FepA for FeEnt in
live bacteria is in fact subnanomolar: Kd = 0.1-0.2
nM, as now established by two independent methods. Thus these data confirm that extraction from the OM reduces the affinity of
FepA for FeEnt about 100-fold (Kd = 10 nM; Ref. 10). Furthermore, the affinity of
native FepA for FeEnt in vivo is 20,000-fold greater than
that of the isolated FepA N-domain in vitro
(Kd = 5 µM; Ref. 52). The result of
these considerations is that at the Kd measured
in vivo, purified FepA is less than 1% saturated with
FeEnt, but why? The crystal structures of both ligand-free and
ligand-bound FepA and FhuA revealed a closed form of the proteins with
loops condensed together above the membrane channel, whereas later
crystallographic and cross-linking studies in vivo showed
that the loops may extend into an open conformation. Thus, the likely
explanation for the discrepancy in binding affinities between the
native and purified receptors is that in the living cell, in the
absence of ligands, ligand-gated porins adopt an open conformation but
when complexed with a ferric siderophore, or removed from the OM, their
loops close.
Besides its prior application to the binding reactions between FepA and
its ligands, site-directed fluorescence spectroscopy defined
conformational changes in FhuA L5 during ferrichrome binding (53).
Furthermore, the intrinsic fluorescence of pyoverdin facilitated characterization of its adsorption to the pseudomonad OM protein, FpvA
(54-56). Our experiments are the first to use site-directed fluorescence to directly observe protein motion associated with uptake
of a ligand through a membrane. It is important to note that the
methodology does not necessarily reflect exclusively OM transport.
Although the intensity changes in fluors attached to FepA derive from
fluctuations of its loops between open and closed states, we only
observed the recovery of fluorescence when the exogenous ligand was
completely exhausted. In our experiments the bacteria had an
59FeEnt binding capacity of 80 pmol/109 cells
(data not shown); when we provided 10 nM FeEnt in the
medium, the 2.5 × 107 cells/ml underwent five rounds
of transport to fully deplete the external ferric siderophore. This
quantity of FeEnt exceeds the concentration of FepB in the periplasm
(15), so its complete utilization by the bacteria requires transport
through the inner membrane to the cytoplasm.
On the other hand, 2 nM FeEnt, the amount that we employed
to study the susceptibility of the fluorescence phenomena to energy inhibitors, is a concentration that precisely saturates the bacteria. Therefore, its depletion from the media involves only a single round of
transport by the FepA proteins of the OM, without requirement for
uptake through the inner membrane. The fact that we saw an immediate
recovery of fluorescence, without a lag period, when we provided 2 nM FeEnt supports this inference. At this stoichiometric concentration, the assay exclusively measured the activity of the OM
component. The ability to monitor OM transport without the need for
null mutants in the subsequent inner membrane permease components (i.e., fepC, fepD, or
fepG) is a significant advantage of the spectroscopic
approach. Our characterization of the energetics of the OM transport
stage diverged from those obtained for cyanocobalamin uptake (21, 22)
in that inhibitors of electron transport (cyanide and azide),
phosphorylation (arsenate), and proton motive force (CCCP and DNP) all
impaired FeEnt uptake through FepA, in some cases abrogating transport.
In the vitamin B12 transport system, DNP-mediated depletion of PMF
inhibited OM transport, but the process was insensitive to cyanide.
Bradbeer (22, 38) conducted those experiments by radioisotopic methods
in an atp genetic background, whereas our strains were
atp+. Most other considerations were similar
between his experiments and ours, except for the preincubation times of
the bacteria with the poisons, which were 10 min (22) and 40 min (this
report). Our data almost exactly reprise, nevertheless, a prior
characterization of the energy dependence of FeEnt uptake by E. coli (50) that was performed by radioisotopic methods. The
explanation for these differences and similarities awaits further experimentation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-sheet domain that lodges within
otherwise typical transmembrane
-barrels, creating a third
mechanistic paradox: how do ligands pass through such closed pores?
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, thi, entA, pro, trp, rpsL, recA,
fepA, fhuA, cir) (3) and KDF571 (KDF541, but tonB) (3)
were hosts for fepA+ pUC19 derivatives
pITS449 (36) and pT944 (11). Both plasmids encode wild type FepA, but
pT944 contains a series of genetically engineered restriction sites
that facilitate cloning procedures and restriction fragment exchange.
pMF19 (provided by M. A. Valvano) (37) is a pEXT21 derivative that
carries wbbL, the structural gene for a rhamnosyltransferase
involved in O16 LPS biosynthesis. Fluorescent reagents were purchased
from Molecular Probes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-barrel, respectively.
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Fig. 1.
Accessibility of cysteine substitution
mutants to fluorescein labeling in vivo.
A, we engineered 10 cysteine substitutions in pITS449
(fepA+ on pUC18), expressed them in
KDF541/pMF19 in MOPS minimal media, and labeled them with FM. The
structural model (30) shows the Cys substitutions relative to the OM
bilayer and to the position of LPS (32) associated with the exterior of
the FepA -barrel. The N-domain backbone is red, the
-barrel backbone is green. The sites of mutation
and LPS, in space-filling format, are shown in CPK
(Corey-Pauling-Kolun) colors. The positions of residues
Ser271 and Phe329, which were not defined in
the crystal structure, are only estimated as delimited by the last
solved residues of L3 (orange, Gly323,
Gln335) and L4 (red, Ala383,
Asp400), respectively. B, after fluorescent
labeling, 108-labeled cells were lysed and subjected to
Western immunoblot with anti-BSA-IAF, developed with
125I-Protein A.
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Fig. 2.
Ligand binding by fluorescein-labeled
FepA. KDF541 expressing S271C ( ) or S397C (
) and labeled
with FM was diluted to 2.5 × 106/ml in TBS buffer,
and fluorescence intensity was measured in the presence of varying
amounts of FeEnt. KDF541 expressing wild type FepA was subjected to the
same procedure and used as a control for nonspecific adsorption of FM.
After background and dilution correction,
1-F/F0 was fit against the FeEnt
concentration, using GraFit 4 (Erithacus Software Ltd.) "Bound
versus Total" equation. The Kd values
for FepAS271C and FepAS397C were 0.263 nM (S.E., 0.032 nM) and 0.292 nM (0.021 nM),
respectively. The inset shows the ability of FeEnt (added at
200 s) to quench the fluorescence of cells labeled with AM in a
comparable manner to its quenching of cells labeled with FM.
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Fig. 3.
Effects of temperature on the polarization of
FepAS271C-FM and FepAS397C-FM. Bacteria were grown and
fluorescently labeled and washed; 5 × 107 bacteria
were deposited into a 2-ml cuvette for polarization measurements.
Polarization was determined at 4 °C, and at 500 s the
temperature of the sample cuvette jacket was raised to 37 °C.
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Fig. 4.
Transport of FeEnt by KDF541/pS271C-FM.
The time courses of spectroscopic intensity change (A) and
59FeEnt uptake (B) were compared in
fluorescently labeled bacteria. The cells were suspended to 2.5 × 107 cells/ml in MOPS minimal media with glucose and
casamino acids at 20 °C. For the fluorescence measurements, at
t = 0 FeEnt was added to 10 nM, and
intensity changes were measured in the fluorescence spectrometer. In
the radioactivity determinations, at t = 0 59FeEnt was added to 10 nM, and aliquots were
removed at the indicated times, filtered, and the filters were counted
(o-o). The data monitor the accumulation of 59FeEnt by the
bacteria.
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Fig. 5.
Effects of environmental conditions on the
fluorescence time course. KDF541
(tonB+)/pMF19/S271C was cultured in complete
MOPS media (containing 0.4% glucose, 0.2% casamino acids, and
supplementary auxotrophic amino acids), labeled with FM, and diluted to
2.5 × 107 cells/ml in physiological buffers and
different conditions. A, media: The fluorescence response of
the cells to 2 nM FeEnt (added at 300 s) was observed
at 20 °C in complete MOPS media (green) or MOPS media
without amino acids (teal) or TBS (blue); all
media contained 0.4% glucose. The response of bacteria to which
fluorescein was nonspecifically adsorbed is also shown
(black). KDF541/pMF19/pITS449 was grown in LB for 14 h
and treated with FM as usual; the fluorescence intensity of 5 × 107 cells in 2 ml of MOPS media was monitored at 20 °C.
FeEnt was added at 425 s. B, concentration,
TonB-deficiency, and pH: Bacteria were diluted into complete MOPS media
at 20 °C and pH 7.4, FeEnt was added (at 300 s) to 2 nM (green), 5 nM (teal),
10 nM (blue), or 20 nM
(black), and fluorescence intensity changes were recorded.
KDF541/pMF19/S271C-AM was also tested in the same media with 2 nM FeEnt (magenta). The red curve
derives from KDF571 (tonB)/pMF19/S271C-FM, subjected to the
same regimen in the same media; the gray curve derives from
KDF541/pMF19/S271C-FM tested in complete MOPS media that was adjusted
to pH 4. C, temperature: Bacteria were diluted into complete
MOPS media and equilibrated at 20 °C (green), 15 °C
(teal), 10 °C (blue), or 5 °C
(black). FeEnt was added to 10 nM, and the
changes in fluorescence were recorded. The experiment was also
performed with 2 nM FeEnt (inset) at 37 °C
(purple) or 20 °C (green).
View larger version (22K):
[in a new window]
Fig. 6.
Effects of metabolic poisons. In all
panels, KDF541/pMF19/S271C was grown in complete MOPS media, labeled
with FM, washed, and resuspended at 2.5 × 107/ml.
A, energy poisons: The labeled cells were suspended in MOPS
media containing 0.4% glucose and shaken for 40 min at 37 °C in the
absence of any poison (green) or the presence of CCCP (0.1 mM, red), cyanide (20 mM,
magenta), azide (10 mM, black), DNP
(2 mM, blue), or arsenate (10 mM,
teal). The cells were equilibrated at 20 °C, FeEnt was
added to 2 nM (at 800 s), and fluorescence changes
were monitored. B, carbon sources: The cells were
resuspended in MOPS media without glucose, shaken for 10 h at
37 °C, and equilibrated at 20 °C in the presence of 0.4% glucose
(green), succinate (teal), ascorbate
(blue), or the absence of an extraneous carbon source
(black). FeEnt was added to 2 nM (at 1000 s), and fluorescence changes were monitored. C, arsenate and
phosphate: Labeled cells were resuspended in MOPS media plus glucose
and shaken for 1 h at 37 °C in the absence of test compounds
(green) or the presence of 30 mM sodium
phosphate (magenta), 30 mM sodium phosphate and
10 mM sodium arsenate (blue), or 10 mM sodium arsenate (teal). The cells, in the
same buffers, were re-equilibrated at 20 °C, FeEnt was added to 2 nM (at 800 s), and fluorescence intensity changes were
recorded.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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.:
405-325-4969; Fax: 405-325-6111; E-mail: peklebba@ou.edu.
§ Present address: Faculte de Medecine, Necker Enfants Malades, INSERM U570 156, rue de Vaugirard, Paris, 75730 France.
¶ Present address: Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M210360200
2 Z. Cao, S. M. Newton, and P. E. Klebba, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: OM, outer membrane; FeEnt, ferric enterobactin; FM, fluorescein maleimide; LPS, lipopolysaccharide; IAF, iodoacetamide fluorescein; LB, Luria Bertani; AM, AlexaFluor680 maleimide; L3, third surface loop; MOPS, 4-morpholinepropanesulfonic acid; CCCP, carbonyl cyanide p-chlorophenylhydrazone; DNP, 2,4-dinitrophenol; PMF, proton motive force; BSA, bovine serum albumin.
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