Diffusion through channel derivatives of the Escherichia coli FhuA transport protein 1 Mikrobiologie/Membranphysiologie, Universita¨t Tu¨bingen, Germany;2Lehrstuhl fu¨r Biotechnologie, T
Trang 1Diffusion through channel derivatives of the Escherichia coli FhuA transport protein
1
Mikrobiologie/Membranphysiologie, Universita¨t Tu¨bingen, Germany;2Lehrstuhl fu¨r Biotechnologie, Theodor-Boveri-Institut (Biozentrum), Universita¨t Wu¨rzburg, Germany
FhuA is a multifunctional protein in the outer membrane of
the antibiotics albomycin and rifamycin CGP 4832, and
mediates sensitivity of cells to the unrelated phages T5, T1,
/80 and UC-1, and to colicin M and microcin J25 The
energy source of active transport is the proton motive force
of the cytoplasmic membrane that is required for all FhuA
functions except for infection by phage T5 The FhuA crystal
structure reveals 22 antiparallel transmembrane b-strands
that form a b-barrel which is closed by a globular N-terminal
domain FhuA still displays active transport and sensitivity
to all ligands except microcin J25 when the globular domain
(residues 5–160) is excised and supports weakly unspecific
diffusion of substrates across the outer membrane Here it is
shown that isolated FhuAD5–160 supported diffusion of
ions through artificial planar lipid bilayer membranes but
did not form stable channels The double mutant FhuAD5–
160 D322–336 lacking in addition to the globular domain
most of the large surface loop 4which partially constricts the
channel entrance, displayed an increased single-channel
conductance but formed no stable channels It transported
FhuA and did not increase sensitivity of cells to antibiotics
In contrast, a second FhuA double mutant derivative which
in addition to the globular domain contained a deletion of residues 335–355 comprising one-third of surface loop 4and half of the transmembrane b-strand 8 formed stable channels
in lipid bilayers with a large single-channel conductance of
D335–355 showed an increased sensitivity to antibiotics and supported diffusion of maltodextrins, SDS and ferrichrome across the outer membrane FhuAD5–160 D335–355 showed
no FhuA specific functions such as active transport of
It is concluded that FhuAD5–160 D335–355 assumes a conformation that is incompatible with any of the FhuA functions
Keywords: channel; Escherichia coli; FhuA transport protein
The outer membrane of Escherichia coli forms a
permeabi-lity barrier for hydrophilic substrates larger than 600 D [1]
Smaller substrates diffuse through water-filled pores formed
by the porins Most of the siderophores secreted by bacteria
low so that diffusion does not provide sufficient iron for
up by an energy-requiring transport process The proton
motive force of the cytoplasmic membrane drives their
transport across the outer membrane [3] and ATP energizes
uptake across the cytoplasmic membrane [4] FhuA
trans-ports ferrichrome, the structurally related antibiotic
albo-mycin, the unrelated antibiotic rifamycin CGP 4832, and
serves as receptor for the phages T5, T1, /80 and UC-1, for
the toxic colicin M protein and the toxic microcin J25
peptide [4]
The crystal structure of FhuA reveals 22 antiparallel transmembrane b-strands that form a b-barrel which is closed by a globular domain, also called cork [5] or plug [6]
It is thought that energy input from the cytoplasmic membrane opens the channel of the b-barrel in that the globular domain somehow moves and ferrichrome dissoci-ates from its binding site which is formed by 10 amino acid residues [5] The energy transfer from the cytoplasmic membrane to FhuA in the outer membrane is mediated by the TonB protein that is located in the periplasm and anchored by the N-proximal end in the cytoplasmic membrane [7] Two additional proteins, ExbB and ExbD, are associated with TonB and are required for TonB activity The subcellular localization of ExbD is similar to TonB [8], whereas ExbB is anchored with three transmem-brane segments in the cytoplasmic memtransmem-brane with most of the protein in the cytoplasm [9]
FhuA changes its conformation upon binding of ferri-chrome The crystal structure shows a small movement (1 A˚) of the globular domain towards the bound ferri-chrome but a large movement (17 A˚) of residues 19 and 22
of the globular domain that are exposed to the periplasm Binding of ferrichrome enhances interaction of FhuA with TonB [10], which may be facilitated by the structural transition of 17 A˚ Residues 1–18 are not seen in the crystal and therefore are thought to be flexible This segment contains the TonB box (residues 7–11) which has been
Correspondence to V Braun, Mikrobiologie/Membranphysiologie,
Universita¨t Tu¨bingen, Auf der Morgenstelle 28, D-72076 Tu¨bingen,
Germany Fax: + 49 7071 295843, Tel.: + 49 7071 2972096,
E-mail: volkmar.braun@mikrobio.uni-tuebingen.de
Abbreviations: LDAO, N,N-dimethyldodecylamine-N-oxide.
(Received 3 June 2002, revised 6 August 2002,
accepted 21 August 2002)
Trang 2named according to the finding that the amino acid
replacements I9P (isoleucine in position 9 replaced by
proline) and V11D abolished the TonB-dependent FhuA
activities [11] and reduced interaction of FhuA with TonB
[12] The Q160L and Q160K replacements in TonB partially
restored the activities of the FhuA TonB box mutants
These results supported the notion that FhuA interacts
through the TonB box with region 160 of TonB As in
FhuA the TonB box is contained near the N terminus of all
active outer membrane transport proteins and the group B
colicins which also require TonB to kill cells Further
support of the TonB box concept comes from the
sponta-neous disulfide formation between cysteine residues
intro-duced into the TonB box of the BtuB protein and cysteine
residues introduced into region 160 of TonB BtuB is similar
outer membrane [13] Site-directed spin labelling and
electron paramagnetic resonance studies revealed that
and the dynamics of the TonB box segment [14]
At the time when the crystal structure of FhuA was not
yet available we arrived at an early tentative transmembrane
model of FhuA that proposed a prominent loop at the cell
surface from residue 316 to residue 355 [15] The model was
experimentally derived from the proteolytic cleavage of
peptides of up to 16 amino acids which had been inserted
into FhuA, and by computer-based prediction programs In
support of this notion we showed that this region serves as
the binding site for the phages T1, T5 and /80, because
synthetic peptides covering this region inhibited infection by
the phages [16] Under the assumption that the surface loop
might also control a putative channel of FhuA we excised
residues 322–355 which indeed converted FhuA into an
open channel that exhibited stable single-channel
conduct-ance in artificial lipid bilayer membranes [17] Excision of
residues 322–336 and 335–355 resulted in no stable
single-channel conductance [18] The crystal structure later
revealed that the largest surface loop (L4), indeed extends
from residues 318–339 and that residues 340–355 are located
above the outer membrane lipid bilayer and form half of the
b-sheet number 8 The cork domain revealed by the crystal
structure could not be predicted by the methods used
To understand the role of the cork domain in channel
formation of FhuA we have constructed FhuAD5–160
based on the crystal structure under the assumption that
excision of the entire globular domain would convert FhuA
into an open channel similar to the channel formed by
FhuAD322–355 [17] and abolish all TonB-dependent FhuA
activities However, FhuAD5–160 still displayed all
TonB-related activities between 40 and 100% of wild-type FhuA
activity, depending on the function tested, and the
per-meability of the outer membrane for substrates and
antibiotics increased only slightly [19] These findings were
supported by a study using FhuAD5–160 derivatives of
Typhimurium which in addition showed that hybrid
proteins consisting of the b-barrel of one strain and the
globular domain of another strain were functional [20] An
investigation with an E coli deletion derivative of FepA, the
outer membrane transport protein for ferric enterobactin, in
which the cork domain was removed and comparison with
FhuAD5–160 supported the TonB dependent activities of
the b-barrels [21]
To gain further insights into the mode of action of FhuAD5–160, and in particular to the transport activity of the b-barrel, we determined in this report single-channel conductance of isolated FhuAD5–160 incorporated into artificial bilayer membranes We wanted to relate the
Since FhuAD5–160 formed no stable channels in vitro we deleted loop 4which constricts half the channel entrance of FhuA to about half the area of the total cross-section [6] As
no stable channels were formed by FhuAD5–160 D322–336,
we combined deletion D5–160 with deletion D335–355 which as a single deletion did not display stable single-channel conductance [18] FhuAD5–160 D335–355 formed large stable channels which were consistent with the in vivo
per-meability FhuAD5–160 D335–355 did not actively
D322–336 which displayed TonB-dependent transport activity
M A T E R I A L S A N D M E T H O D S
Bacterial strains, plasmids and growth conditions The E coli strains and plasmids used are listed in Table 1
when required
Plasmids pHK234and pHK237 were digested with MluI and SalI and ligated into MluI/SalI-cleaved plasmid pHK763 resulting in plasmids p7634and p7637, respect-ively Plasmids p7634and p7637 were digested with BspEI and EcoRI and ligated into BspEI/EcoRI-cleaved plasmid pBK7 resulting in plasmids pDM234and pDM237, respectively
Plasmid pSKF405-04 was digested with MluI and BstEII and ligated into MluI/BstEII-cleaved plasmid pHK763
of the primers His1 5¢-GATCATCACCATCACCATCAC-3¢ and His2 5¢-GATCGTGATGGTGATGGTGAT-5¢-GATCATCACCATCACCATCAC-3¢
30 min followed by an incubation for 5 min at room temperature The annealed His1 and His2 primers were ligated into BglII-cleaved plasmid p76405 resulting in plasmid pHK763H
Plasmid pHK763H was digested with SalI and EcoRI and ligated into SalI/EcoRI-cleaved plasmids pBK7, p7634, pDM234, p7637, and pDM237 resulting in plasmids pBK7H, p7634H, pDM234H, p7637H, and pDM237H, respectively
Recombinant DNA techniques Isolation of plasmids, use of restriction enzymes, liga-tion, agarose gel electrophoresis, and transformation followed standard techniques [22] All genetic construc-tions were examined by DNA sequencing using the dideoxy chain-termination method with
Trang 3fluorescence-labelled or unfluorescence-labelled nucleotides (Auto Read Sequencing
Kit, Pharmacia Biotech) and the A.L.F sequencer
(Pharmacia)
Protein analytical methods
To show the FhuA proteins in cells that were grown under
the same conditions as the phenotype assays were
per-formed, E coli HK97 fhuA was transformed with fhuA
wild-type and fhuA mutant genes and grown overnight in
NB medium The overnight cultures were used to inoculate
isolated by lysing cells with lysozyme–EDTA, followed by
solubilization of the cytoplasmic membrane in 0.2% Triton
X-100 and differential centrifugation The proteins of the
undissolved outer membrane fraction were dissolved by
heating in sample buffer, separated by SDS/PAGE and
stained with Serva blue [23]
Phenotype assays
All phenotype assays were carried out with freshly
trans-formed E coli K-12 strains HK97 aroB fhuA fhuE and
HK99 aroB fhuA tonB These strains carry the same four
amino acid replacements and an amino acid deletion in fhuA
and contain the mutated FhuA protein in the outer
membrane [23] The plasmid-encoded fhuA genes in the transformants were transcribed from the fhuA promoter Sensitivity of cells to the FhuA ligands was tested by spotting 10-fold (phages T1, T5, /80, and colicin M) or 3-fold (microcin J25, rifamycin CGP 4832, and albomycin) diluted solutions (3 lL) on TY agar plates overlaid with
tested The colicin M solution was a crude extract of a strain carrying plasmid pTO4 cma cmi [24] The microcin J25 solution was a supernatant of E coli MC4100 carrying the plasmid pTUC203 mcjABCD [25] after growth of the
Growth inhibition by SDS and various antibiotics was determined by placing filter paper discs supplemented with
10 lL of the agents in concentrations as indicated on TY
cells of the strain to be tested The tests were performed in parallel with TY agar plates and TY soft agar both
Growth promotion by siderophores was tested by placing filter paper discs containing 10 lL of a siderophore solution
of different concentrations on NBD agar plates overlaid
be tested After overnight incubation, the diameter and the growth density around the filter paper discs were determined
Table 1 E coli strains and plasmids used in this study.
Strains
ptsF25 rbsR aroB thi fhuE::kplac Mu53fhuA
[23]
BL21 (DE3) omp8 F – hsdS B B(r B–m B–) gal ompT dcm (DE3) DlamB
ompF::Tn5 DompA T7 polymerase under lacUV5 control
[35]
Plasmids
pDM234H pT7-6 fhuA D5–160 D322–336 E3D (P 321 PDL K 337 ) (P 405 PDH 6 DLA V 406 ) This study
Trang 4Growth promotion by maltodextrins was tested with the
transformed with plasmids encoding various FhuA
deriv-atives Overnight cultures were washed twice with M9
medium The test was performed by placing filter paper
discs supplemented with 10 lL of a 40% solution of
maltodextrins (maltose to maltohexaose) on M9 minimal
agar plates that contained no other carbon source overlaid
with M9 minimal top agar containing 100 lL of the strain
diameter and the growth density around the filter discs were
determined
Ferrichrome uptake assays
fhuA tonB, freshly transformed with the plasmids to be
tested were grown overnight on TY plates Cells were
washed and suspended in transport medium (M9 salts [26],
nitrilotriacetate, pH 7.0 to 1 mL cells After incubation for
diffusion of ferrichrome into cells was tested In the latter
case, E coli HK99 aroB fhuA tonB was used as the test
strain and a 150-fold surplus of nonradioactive ferrichrome
was added as a chase after 17 min to remove adsorbed
50 lL or 100 lL were withdrawn and added to 10 mL
(pore size 0.4 5 lm; Sartorius AG) and washed twice with
radio-activity was determined by liquid scintillation counting
Ferrichrome binding assay
with the plasmids to be tested were grown overnight on TY
was overlaid with 80 lL silicone oil PN200 (density
ferrichrome uptake assays At the times indicated, samples
of 50 or 100 lL were withdrawn and applied onto the
silicone oil layer in the microtest tube The tubes were
centrifuged immediately for 90 s in a Beckman Microfuge
E The cells passed the silicone oil layer according to their
layer The residual radiolabelled ferrichrome in the binding
medium remained on top of the silicone layer After the
centrifugation step, cells were stored in the test tube at
room temperature until the uptake assay was completed
(H Killmann and G Gestwa, unpublished data)
At the end of the assay, the microtest tubes were cut with
a scalpel in the middle of the silicone oil layer and the lower
part of the test tube containing the cells was placed upside
down in a fresh test tube and centrifuged for 10 min at
10 000 g The empty tube was removed, and the mixture of
completely transferred to a 20-mL polyethylene vial After adding 10 mL liquid scintillation counting cocktail, the radioactivity was determined by liquid scintillation counting
Purification of the FhuA proteins The E coli K-12 strain CH21 freshly transformed with the
of 0.5 before T7 RNA polymerase synthesis was induced by
(pH 8.0) that contained 1 mg deoxyribonuclease and two tablets of COMPLETE (Roche), cells were disrupted with a French press The disrupted cells were mixed with 15 mL
2% Triton X-100) and incubated for 10 min at room temperature The outer membrane fractions were isolated
by centrifugation for 1 h with 30 000 g To remove the added Triton X-100 the outer membranes were washed four
suspended in 12 mL solubilization buffer containing
N,N-dimethyl-dodecylamine-N-oxide (LDAO) To solubilize the FhuA
then centrifuged and 5 mL of the pooled supernatant fractions dialysed for 4h at room temperature in 500 vols
The protein solutions were concentrated to 1 mL by ultrafiltration (Centricon YM30, Millipore) before loading
binding buffer Chromatography was performed as des-cribed by the manufacturer (Qiagen) with the exception that all buffers contained 0.1% LDAO The elution buffer containing the purified proteins was replaced by a buffer
PD10 columns (Pharmacia)
Black lipid bilayer membrane experiments Membranes were formed from a 1% (w/v) solution of diphytanoyl PtdCho (Avanti Polar Lipids) in n-decane in a Teflon cell consisting of two aqueous compartments con-nected by a circular hole with an area of approximately
grade; Merck) were used unbuffered and had a pH of
throughout the experiments The single-channel measure-ments were performed with a pair of Ag/AgCl electrodes (with salt bridges) switched in series with a voltage source and a current amplifier (Keithley 427) The amplified signal was monitored with a storage oscilloscope and recorded with a strip chart recorder Stock solutions containing the FhuA deletion derivatives were added after the lipid membrane turned optically black to reflected light For determination of zero-current membrane potentials
Trang 5insertion of pores was observed until a conductance of at
least 0.1 nS was reached corresponding to the formation of
a sufficient number of channels Then the instrumentation
was switched to the measurement of the zero-current
potentials and a KCl gradient was established by adding
stirring The zero-current membrane voltage reached its
stationary value about 2–5 min after addition of the
concentrated KCl-solution and was analysed using the
Goldman–Hodgkin–Katz equation [29]
R E S U L T S
FhuAD5–160 does not form stable channels
in lipid bilayers
The FhuA b-barrel lacking the central N-terminal globular
domain was incorporated into lipid bilayer membranes to
determine the increase in conductance To purify the
protein, the fhuAD5–160 gene was cloned into plasmid
pBK7H (Table 1) downstream of the phage T7 gene 10
promoter and specifically transcribed by the T7 RNA
residue 405 [30] which is exposed at the cell surface [5,6]
FhuAD5–160 was contained in the outer membrane fraction
in lower amounts than wild-type FhuA (Fig 1A) It was
agarose column (Fig 1B) Control purifications were
per-formed with strain CH21 transper-formed with the pT7-6
expression vector
FhuAD5–160 was added to the aqueous phase on one or
both sides bathing a black lipid bilayer membrane formed
from diphytanoyl PtdCho/n-decane across a small circular
hole FhuAD5–160 increased the membrane conductance as
The conductance increase did not occur in a step-wise
fashion as found with porins of Gram-negative bacteria
[27,31] This means that FhuAD5–160 failed to form stable
channels The current recordings revealed a high degree of
current noise The most frequently observed conductance
was recorded when purified wild-type FhuA was added to
the lipid bilayer membranes This was also the case in the
control recordings with purified samples of the control strain CH21 transformed with pT7-6 Control measure-ments were carried out with a 100-fold concentrated protein solution compared to the measurements of the different FhuA deletion derivatives (data not shown)
FhuAD5–160 D322–336 increased the permeability
of lipid bilayer membranes The loop L4reduces the entrance of the surface cavity of FhuA to about half its diameter [6] To see whether loop 4 restricts the permeability of the open channel that was formed by removal of the globular domain, we constructed
Fig 1 (A) Stained proteins after SDS/PAGE
of outer membrane fractions of E coli HK97 fhuA transformed with plasmids (listed in Table 1) encoding the FhuA proteins indicated
in the figure Arrows denote wild-type FhuA and the various FhuA deletion derivatives The molecular masses of standard proteins in kDa are indicated (B) His-tagged proteins obtained by chromatography on Ni–agarose columns as they were used for the lipid bilayer experiments Ten-lL samples of a 0.5 mgÆmL)1protein solution were applied per lane.
Fig 2 Single-channel recording of a diphytanoyl PtdCho/n-decane membrane in the presence of the FhuAD5–160 mutant The aqueous phase contained 1 M KCl (pH 6) and 50 ng FhuAD5–160ÆmL)1 The applied membrane potential was 20 mV; T ¼ 20 C Note that the current did not increase in a step-wise fashion but showed a high current noise indicating rapid fluctuations of the channel-forming unit.
Trang 6His6-tagged FhuAD5–160 D322–336 The purified deletion
derivative (Fig 1) increased the conductance of lipid bilayer
membranes but did not show uniform single-channel
conductance or a step-wise increase (Fig 3) Higher time
resolution of the current than shown in Fig 2 revealed
frequent and rapid opening and closing of channels which
was also observed with FhuAD5–160 (Fig 2) but had a
much higher amplitude The conductance of FhuAD5–160 D322–336 was higher than that of FhuAD5–160 (Table 2) which indicated that removal of half of the L4loop increased the conductance of the b-barrel
FhuAD5–160 D335–355 forms stable channels Because FhuAD5–160 did not form stable channels in the reconstitution experiments we attempted to combine the D5–160 excision with the 322–355 deletion which we have previously shown converts FhuA into a stable channel [17]
We failed to observe transformants which expressed FhuAD5–160 D322–355, presumably because the protein was toxic to cells We then constructed FhuAD5–160 D335–
355 As reported previously [18] FhuAD335–355 increased the membrane conductance only slightly and did not form stable channels in lipid bilayer membranes SDS/PAGE of isolated outer membrane fractions identified the FhuAD5–
160 D335–355 protein The amounts were lower than those
of wild-type FhuA or FhuAD5–160 (Fig 1A)
deriv-ative on nickel agarose (Fig 1B) inserted readily into planar lipid bilayers and produced discrete step-wise current increase at a transmembrane potential of 20 mV (Fig 4)
If each step corresponded to a single channel, the unitary
steps were fairly homogeneous as shown by the histogram (Fig 5) Only a small number of small steps were observed, which probably represent smaller substates of the open channel (see Table 2 for a summary of the results of the lipid bilayer experiments) The conductance of the FhuAD5–160 D335–355 mutant was smaller than that of FhuAD322–355 (3 nS) determined previously under otherwise identical conditions [17]
Single-channel analysis of the FhuA deletion mutants Table 2 shows the average single-channel conductance G of the FhuA mutant proteins as a function of the KCl concentration in the aqueous phase Measurements were
KAc Only FhuAD5–160 D335–355 displayed a linear relationship between single-channel conductance and KCl
Table 2 Single-channel properties of the various FhuA deletion mutants in different salt solutions The membranes were formed of diphytanoyl PtdCho dissolved in n-decane The aqueous solutions were used unbuffered and had a pH of 6 unless otherwise indicated The applied voltage was 20 mV, and the temperature was 20 C The average single-channel conductance, G, was calculated from at least 80 single events The selectivity
of the different mutants in KCl was derived from zero-current membrane potential measurements ND, not determined.
FhuA mutants
Single-channel conductance G [nS]
Fig 3 Single-channel recording of a diphytanoyl PtdCho/n-decane
membrane in the presence of FhuAD5–160 D322–336 The aqueous
phase contained 1 M KCl (pH 6) and 50 ng FhuAD5–160 D322–
336ÆmL)1 The applied membrane potential was 20 mV; T ¼ 20 C.
Note that the time resolution of the current recording is higher than
that of Figs 2 and 4.
Trang 7concentration, which is expected for wide water-filled chan-nels similar to those formed by Gram-negative bacterial porins [32,33] The single-channel conductance showed a minor restriction as it increased 21-fold while the KCl concentration was increased 30-fold FhuAD5–160 D335–
355 showed a higher conductance in KAc as compared with LiCl which suggested some preference for cations over anions For the other mutant proteins no clear dependence
on the aqueous salt concentrations was recorded which may
be caused by the rapid transition of the FhuA deletion channels between different conductance states as Figs 2 and
3 indicate Furthermore, with the latter mutant proteins no clear selectivity for ions was observed
Selectivity of the FhuA mutant proteins Zero-current membrane potential measurements were per-formed to further determine the selectivity of the FhuA mutant proteins After the incorporation of 100–1000 channels into the membranes, the KCl concentration on
the addition of concentrated KCl The more dilute side of
preferential movement of potassium ions through the mutant channels These data demonstrate selectivity for cations and support the data obtained from the single-channel experiments (Table 2) The zero-current membrane potentials for KCl were on average between 14and 32 mV
at a fivefold KCl gradient across the membranes Analysis
of the potential using the Goldman–Hodgkin–Katz equa-tion [29] suggested that anions also move through the
of the mutant proteins with higher single-channel conduct-ance was lower than that of FhuAD5–160 with a smaller conductance
Active transport of ferrichrome by FhuA deletion derivatives
Previously, we have shown that FhuAD5–160 exhibits all TonB-dependent FhuA activities [19] except uptake of microcin J25 [20] Therefore, we tested whether FhuAD5–
160 retained ferrichrome transport activities when addi-tional deletions were introduced E coli HK97 with a chromosomal fhuA mutation was transformed with the plasmids carrying fhuA deletion genes The amounts of the FhuA mutant proteins present in cells used to determine the properties of the mutants are shown in Fig 1A Cells
with a rate that amounted to 59% of wild-type FhuA This comparison has to take into account that the cells contained less FhuAD5–160 than wild-type FhuA There
is no linear increase of the ferrichrome transport rates with increasing concentrations of FhuA above a certain FhuA concentration The ferrichrome transport rate of FhuAD5–
160 D322–336 amounted to 45% and that of FhuAD322–
336 to 75% of the rate of wild-type FhuA (Fig 6A and Table 3) In contrast, FhuAD5–160 D335–355 did not transport ferrichrome (Fig 6A and Table 3) Since FhuAD335–355 was also transport inactive, removal of the globular domain did not convert FhuAD335–355 into
an active transporter
Fig 4 Single-channel recording of a diphytanoyl PtdCho/n-decane
membrane in the presence of FhuAD5–160 D335–355 The aqueous
phase contained 1 M KCl (pH 6) and 50 ng FhuAD5–160 D335–
355ÆmL)1 The applied membrane potential was 20 mV, T ¼ 20 C.
Fig 5 Histogram of the probability P(G) of the occurrence of a given
conductivity unit observed with membranes formed of diphytanoyl
PtdCho/n-decane in the presence of FhuAD5–160 D335–355 mutant.
P(G) is the probability that a given conductance increment G is
ob-served in the single-channel experiments It was calculated by dividing
the number of fluctuations similar to those of Fig 3 with a given
conductance increment by the total number of conductance
fluctua-tions The aqueous phase contained 1 M KCl (pH 6) and 50 ng
FhuAD5–160 D335–355ÆmL)1 The applied membrane potential was
20 mV; T ¼ 20 C The average single-channel conductance was
2.5 nS for 94single-channel events (right-hand maximum).
Trang 8Increase of outer membrane permeability
by the FhuA deletion derivatives
As FhuAD5–160 D335–355 formed , in lipid bilayer
formed open channels in the outer membrane of E coli cells To study diffusion of ferrichrome into the periplasm,
we used the fhuA tonB double mutant HK99 that was devoid of active transport across the outer membrane but actively transported ferrichrome via the FhuBCD proteins from the periplasm across the cytoplasmic membrane into
cyto-plasm when cells are washed on filters to remove excess
only the periplasm is washed away As HK99 is devoid of
membrane via the FhuA deletion derivatives synthesized after transformation of HK99 with plasmids carrying the
derivatives tested, only FhuAD5–160 D335–355 supported
membrane and subsequent active transport across the cytoplasmic membrane (Fig 6B) Addition of excess
which was probably bound to periplasmic FhuD and some may have been unspecifically bound to cells and the filters
from the periplasm into the cytoplasm from where it was no longer released during the chase
Wild-type FhuA and the FhuA deletion mutants other than FhuAD5–160 D335–355 did not support diffusion of
synthesizing plasmid-encoded wild-type FhuA remained
Ferrichrome was released by the chase with nonradioactive ferrichrome which is considered to be the amount of
Table 3 Ferrichrome binding and transport rates of wild-type FhuA and various FhuA deletion derivatives The E coli strains CH1857 fhuACDB tonB and HK97 fhuA were transformed with the plasmids listed in Table 1 that encoded the FhuA proteins listed in the left panel.
FhuA proteins
Fc transport rates per min into HK97 a
(% wild-type)
Fc binding to CH1857 Iron ions/cell b
(% wild-type)
a
[55Fe3+]Ferrichrome transport rates per minute were calculated from the linear region between 5 and 13 min of Fig 6A The rate was related to the transport rate of wild-type FhuA (100%) b The mean values of [ 55 Fe 3+ ]ferrichrome (Fc) bound to the FhuA derivatives minus the mean values after addition of 150 l M nonradioactive ferrichrome (chase) was taken as the fraction that is bound to FhuA The percentage is related to ferrichrome bound to wild-type FhuA (100%).cFhuAD5–160 D335–355 did not take up ferrichrome by active transport but by TonB independent diffusion The high concentration of iron ions measured during the binding assay (17597 iron ions per cell) was due to the diffusion of ferrichrome into the periplasm and does not reflect the binding capacitiy of FhuAD5–160 D335–355 for ferrichrome.
Fig 6 (A) Time-dependent transport of [55Fe3+]ferrichrome (1 l M )
into E coli HK97, (B) Time-dependent uptake of [55Fe3+]ferrichrome
(10 l M ) into E coli HK99 After 21 min a 150-fold surplus of
non-radioactive ferrichrome was added as a chase The E coli strains
HK97 fhuA aroB and HK99 fhuA tonB aroB were transformed with
plasmids (listed in Table 1) encoding the FhuA deletion derivatives
indicated in the figure.
Trang 9FhuAD322–336 bound similar amounts of [55Fe3+
]ferri-chrome as HK99 wild-type FhuA Cells of the other FhuA
deletion derivatives contained only very small amounts of
To support the conclusion of a diffusive entry of
FhuAD5–160 D335–355, we determined the
com-pared it with the uptake into HK99 transformed with
plasmids that encoded wild-type FhuA and the other FhuA
deletion derivatives Only HK99 FhuAD5–160 D335–355
showed a linear increase of uptake with increasing
FhuAD5–160 D335–355 was no longer linear, presumably
because transport across the cytoplasmic membrane became
rate limiting The FhuA deletion mutants showed only a
associ-ated with the cells which in the case of HK99 FhuAD322–
336 and FhuA wild-type reflected mostly binding to FhuA
somewhat through the other FhuA deletion derivatives into
the periplasm (Fig 7)
synthesized wild-type FhuA and FhuAD322–336 transport
into HK97 (Fig 6A) correlated qualitatively with binding
to HK99 (Fig 6B) However, although HK97 FhuAD5–
160 and HK97 FhuAD5–160 D322–336 transported
very low Because during the transport assay cells on filters
washed through the filter Therefore, binding was deter-mined by a recently devised method in which cells are centrifuged through silicone oil and collected above a layer
of a NaI solution During this procedure cells remain viable (H Killmann and G Gestwa, unpublished data) This
]ferri-chrome binding to FhuA and the FhuA deletion derivatives The used E coli CH1857 is a tonB mutant deficient in ferrichrome transport across the outer membrane and it lacks the fhuABCD genes for transport across the
left is that of plasmid encoded FhuA and its derivatives In
ferrichrome to 30% of the level of the wild-type FhuA
from the loss of four binding sites delivered by the cork domain out of a total of 10 ferrichrome binding sites
in FhuA This result demonstrated that centrifugation through silicone oil was a much milder procedure than washing of cells on filters, and indicates weak binding of
FhuAD322–336 88% and FhuAD335-335 showed 21% binding compared to cells with wild-type FhuA (Table 3) Binding to FhuAD5–160 D335–355 could not be determined
as ferrichrome diffused into the periplasm and remained there during centrifugation through silicone oil (determined value was 17 597 iron ions per cell)
Sensitivity of cells synthesizing FhuA deletion derivatives to antibiotics
Another approach to identify water-filled protein channels
in the outer membrane is the determination of the sensitivity
of cells to antibiotics which are prevented from entering cells
by the permeability barrier of the outer membrane Novo-biocin, erythromycin, rifamycin and vancomycin are anti-biotics which are too large to diffuse rapidly through the pores formed by the porins (Table 4) Cells of HK97 wild-type FhuA were resistant to the indicated antibiotics except rifamycin (Table 4) All FhuA deletion derivatives increased sensitivity to the antibiotics The highest sensitivity to the antibiotics was conferred by FhuAD335–355 and FhuAD5–
160 D335–355 Sensitivity mediated by FhuAD5–160 was increased by the additional deletion 335–355, whereas the sensitivity of FhuAD335–355 against SDS and Novobiocin was increased only slightly when the cork domain had been removed Another indicator for outer membrane permeabi-lity is sensitivity to SDS to which E coli K-12 is resistant
as long as the outer membrane barrier is intact Only FhuAD335–355 and FhuAD5–160 D335–355 rendered cells sensitive to SDS
Sensitivity to the antibiotics was also determined in the
ferrichrome and the concomitant structural changes in FhuA affected the permeability of the FhuA deletion derivatives We observed only slight effects if any (data not shown) which might be caused by the low binding of ferrichrome to the FhuA deletion derivatives This conclu-sion was supported by the decrease of the sensitivity of
Fig 7 Concentration-dependent uptake of [ 55 Fe 3+ ]ferrichrome into
E coli HK99 fhuA tonB aroB expressing the plasmid-encoded FhuA
proteins indicated in the figure.
Trang 10HK97 FhuAD322–336 to rifamycin by ferrichrome which binds to FhuAD322–336 (Table 3)
Growth of FhuA deletion mutants on maltodextrins LamB is the maltoporin through which maltodextrins diffuse across the outer membrane into the periplasm of E coli [31]
If lamB is deleted maltodextrins larger than maltotriose diffuse too slowly into the periplasm to support growth on maltodextrins as the sole carbon source We used E coli KB419 which is a lamB mutant with no polar effect on transport genes required for maltose transport across the cytoplasmic membrane Among the FhuA mutants tested
growth zone on maltotetraose and could grow on malto-pentaose (Table 4) In contrast, the growth zone of E coli KB419 FhuAD335–355 on maltotetraose was smaller and no growth occurred on maltopentaose The other FhuA dele-tion derivatives did not support growth on maltotetraose and maltopentaose except FhuAD5–160 (Table 4) Deletion 322–336 in FhuAD5–160 D322–336 did not increase but somewhat reduced growth of KB419 on the maltodextrins,
as compared with strains expressing only FhuAD5–160
FhuA activities of the FhuA deletion mutants FhuAD5–160 confers to E coli HK97 devoid of wild-type FhuA sensitivity to the FhuA-specific phages T1, T5, /80,
to colicin M and albomycin to the same or similar degree as wild-type FhuA We examined sensitivities of HK97 that synthesized the various FhuA deletion derivatives HK97 FhuAD5–160 D335–355 was resistant to all FhuA ligands as was HK97 FhuAD335–355 In contrast, HK97 FhuAD5–
160 D322–335 displayed sensitivity to T5, colicin M and albomycin which, however, was lower than the sensitivity of HK97 FhuAD322–335 The sensitivity to T5 was reduced 100-fold and the growth inhibition zones caused by colicin
M and albomycin were turbid indicating partial inhibition, whereas those of HK97 FhuAD322–335 were clear (data not shown)
D I S C U S S I O N
The globular domain of FhuA tightly closes the channel formed by the b-barrel and for this reason was designated cork [5] or plug [6] Although binding of ferrichrome causes
a large structural change in the crystal structure of FhuA it does not open the channel Excision of the entire globular domain resulted in FhuAD5–160 that showed TonB-dependent active ferrichrome transport The b-barrel domain alone functioned as an active transporter With higher concentrations of ferrichrome than are required for transport, tonB mutant cells grew on ferrichrome as sole iron source, indicating an open channel [19] Apparently, ferri-chrome diffused through FhuAD5–160 in contrast to wild-type FhuA that failed to support growth of a tonB mutant FhuAD5–160 also somewhat increased sensitivity of cells to antibiotics that are prevented from access to their target site
by the permeability barrier of the outer membrane [19] Maltodextrins that pass through the outer membrane via the LamB maltoporin entered the periplasm via FhuAD5–160 in
a lamB mutant These results indicated that FhuAD5–160 increased unspecifically the permeability of the outer
SDS 288
634Da 30
734Da 15
Maltotriose 504Da 4mg