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Applied and Environmental Microbiology, September 1998, p. 3188-3194, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Localization and Solubilization of the Iron(III)
Reductase of Geobacter sulfurreducens
Sarra
Gaspard,
Francisco
Vazquez, and
Christof
Holliger*
Limnological Research Center, Swiss Federal
Institute for Environmental Science and Technology (EAWAG), CH-6047
Kastanienbaum, Switzerland
Received 29 December 1997/Accepted 19 June 1998
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ABSTRACT |
The iron(III) reductase activity of Geobacter
sulfurreducens was determined with the electron donor NADH and
the artificial electron donor horse heart cytochrome c. The
highest reduction rates were obtained with Fe(III) complexed by
nitrilotriacetic acid as an electron acceptor. Fractionation
experiments indicated that no iron(III) reductase activity was present
in the cytoplasm, that approximately one-third was found in the
periplasmic fraction, and that two-thirds were associated with the
membrane fraction. Sucrose gradient separation of the outer and
cytoplasmic membranes showed that about 80% of the iron(III) reductase
was present in the outer membrane. The iron(III) reductase could be
solubilized from the membrane fraction with 0.5 M KCl
showing that the iron(III) reductase was weakly bound to the
membranes. In addition, solubilization of the iron(III) reductase from
whole cells with 0.5 M KCl, without disruption of cells, indicated that
the iron(III) reductase is a peripheral protein on the outside of the
outer membrane. Redox difference spectra of KCl extracts showed the
presence of c-type cytochromes which could be oxidized by
ferrihydrite. Only one activity band was observed in native
polyacrylamide gels stained for the iron(III) reductase activity.
Excision of the active band from a preparative gel followed by
extraction of the proteins and sodium dodecyl sulfate-polyacrylamide
gel electrophoresis revealed the presence of high-molecular-mass,
cytochrome-containing proteins in this iron(III) reductase
activity band. From these experimental data it can be hypothesized that
the iron(III) reductase of G. sulfurreducens is a
peripheral outer membrane protein that might contain a
c-type cytochrome.
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INTRODUCTION |
Dissimilatory microbial
iron(III) reduction has been proposed as the most important
chemical change that takes place in the development of certain
anaerobic soils and sediments (26). Iron(III) reduction may have been the first globally significant mechanism for
the oxidation of organic matter to carbon dioxide (17). Several studies have demonstrated that iron(III)
reduction accounts for the oxidation of up to 65% of organic
matter in anaerobic sediments (7, 17). Microbial
oxidation coupled with iron(III) reduction is an important natural
mechanism for contaminant removal from groundwater or shallow aquifers
polluted with landfill leachates (1). Furthermore,
iron(III) reduction can influence the biogeochemical cycles of
important nutrients, such as carbon, sulfur, and phosphorus (17, 28). Up to now, numerous iron(III)-reducing
bacteria have been described in the literature (8,
16). These bacteria are versatile towards electron donors and
acceptors which can be used for growth.
Although iron(III)-reducing bacteria have been studied for many
years, little is known about the biochemistry and the mechanism of
iron(III) reduction. Iron(III)-reducing bacteria are able to utilize Fe(III) either chelated or in insoluble inorganic minerals such as ferrihydrite, goethite, and others. The question arises whether
Fe(III) is taken up and reduced in the periplasm or cytoplasm or
whether electrons are transferred to Fe(III) minerals outside of
the cells. Evidence has been presented that a direct contact between
the cells and the metal oxide is necessary for the conservation of
energy (3). Iron(III) reductase activity was found to be predominantly in the membranes of the iron(III)-reducing bacteria (23). In the case of Shewanella putrefaciens, 54 to 56% of this activity was localized in the outer membrane
(23). The involvement of b- and c-type
cytochromes as electron carriers in the iron(III) respiration chain
has been reported previously (23). In addition, it has been
shown that the outer membranes of anaerobically grown cells of
S. putrefaciens have a high cytochrome content
(22). This suggests that those cytochromes localized in the
outer membrane may play a key role in iron(III) reduction. In
this study, we report investigations on the localization of the
iron(III) reductase activity of Geobacter sulfurreducens
with a new enzyme assay. Results obtained by isolating the outer
membrane and by treating whole cells with a high KCl
concentration to solubilize the iron(III) reductase indicated that
the iron(III) reductase of G. sulfurreducens is a
peripheral protein of the outer membrane. Evidence is presented that a c-type cytochrome is involved in iron(III)
reduction.
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MATERIALS AND METHODS |
Growth conditions.
G. sulfurreducens was cultivated
anaerobically in 1-liter bottles filled with 0.7 liter of defined
medium (5). One liter of basal medium contained the
following: 2.5 g of NaHCO3, 1.5 g of
NH4Cl, 0.68 g of NaH2PO4,
0.1 g of KCl, 6.8 g of CH3COONa, 13.7 g of
Fe(III)-citrate, 10 ml of vitamin solution, and 10 ml of trace
element solution, whose composition was as described previously
(19). The pH was adjusted to 6.8. The bottles were sealed
with butyl rubber stoppers, and the headspace was replaced with
N2-CO2 (80%-20% [vol/vol]). Cultures were
incubated in the dark for 4 days without shaking at 30°C.
Cell harvest and preparation of subcellular fractions.
All
steps were carried out in an anaerobic glove box (Coy Laboratory, Ann
Arbor, Mich.). If not mentioned otherwise, N2-saturated Tris buffer (100 mM, pH 8.1), containing 0.1 mM dithiothreitol, was
used. G. sulfurreducens cultures in the late-growth phase were acidified to pH 5 to dissolve iron(II) precipitates, harvested by
centrifugation (10,000 × g, 20 min, 4°C), and washed
three times with Tris buffer. To prepare cellular fractions, a protocol adapted from a method described by Myers and Myers (22) was used. For the preparation of spheroplasts, cells (110 mg of protein) were resuspended in 30 ml of the Tris buffer containing 25% (wt/vol) sucrose. To accomplish cell wall lysis, a 1/10 volume of a lysozyme solution (6.4 mg · ml
1) and a 1/10 volume of a
Na2-EDTA solution (20 mg · ml1) were
added with constant stirring at 15-min intervals. Finally, MgCl2 (0.1 M) was added to a final concentration of 12.8 mM. Separation of spheroplasts from the periplasmic fraction
was obtained by centrifugation at 20,000 × g for 30 min. Spheroplasts (pellet) were resuspended in Tris buffer to a final
protein concentration of 9 mg/ml.
To obtain the membrane and soluble fraction, a few crystals of DNase
were added to an EDTA-lysozyme-treated cell suspension, and the cells
were broken by ultrasonic treatment (10 times, 100 W, 30 s) with a
sonifier (model 250; Branson Ultrasonics, Danbury, Conn.) in an
ice-water bath. Cell debris was removed by centrifugation (4,000 × g, 10 min, 4°C). The crude extract (supernatant) was centrifuged at 200,000 × g (1 h, 4°C), yielding the
soluble fraction containing the cytoplasmic and periplasmic
fractions and the membrane fraction (pellet). The latter was
resuspended in Tris buffer to a final concentration of 15 mg/ml. The
protein concentration was determined by using bicinchoninic acid as
described previously (30), with bovine serum albumin as a
standard.
Separation of outer and cytoplasmic membranes.
Separation of
outer and cytoplasmic membranes was achieved according to a procedure
adapted from Myers and Myers (22). An aliquot of 1 ml of the
membrane fraction containing 1,585 mg of protein was layered onto 10.5 ml of a 50 to 80% (wt/wt) sucrose gradient and centrifuged for 17 h at 82,500 × g. Two colored discrete bands were
observed, one between 5 and 8 cm from the bottom of the tube
(low-density band) and one on the bottom of the tube (high-density
band). The bands were retrieved from the tube by removing fractions of
0.5 to 1 ml with a syringe. The fractions were resuspended in and
subsequently dialyzed for 5 h at 4°C against a 10 mM HEPES
buffer containing 0.1 mM dithioerythreitol (DTE). To identify the
fraction containing the outer membrane, the content of KDO
(2-keto-3-deoxyoctonate), a specific constituent of the lipopolysaccharide in the outer membrane, was determined
(13).
Enzyme assay.
Iron(III) reductase activity was assayed
anaerobically in 1-cm cuvettes by photometric monitoring of the
appearance of Fe(II) over time as adapted from the method of Lascelles
and Burke (15). The reaction mixture contained either 1 mM
NADH or 4 µM dithionite-reduced horse heart cytochrome c
as the electron donor, 0.5 mM ferrozine as the Fe(II) chelating agent,
and either 0.15 mM Fe(III)-nitriloacetic acid (NTA),
Fe(III)-citrate, or Fe(III)-EDTA as electron acceptor in
N2-saturated 50 mM HEPES buffer (pH 7). Horse heart
cytochrome c was added from a stock solution that was
prepared by adding 50 µl of a 4.6 mM sodium dithionite solution to 1 ml of 50 mM HEPES buffer (pH 7) containing 2 mg of horse heart
cytochrome c. The concentration of reduced horse heart
cytochrome c in the sample cuvette was calculated by
measuring the A552 (A552 = 29,500 M1 · cm1). The enzyme assay
was started by adding whole cells or cell extracts of G. sulfurreducens (0.03 to 0.5 mg of protein) to both sample and
reference cuvettes. The activity was monitored by measuring the
increase in A562 (32) due to the
formation of a Fe(II)-ferrozine complex (A562 = 28,000 M1 · cm1) against a reference
where no electron donor was added. An activity of 1 mU corresponded to
1 nmol of Fe(II) formed per min. In the absence of ferrozine, the
enzyme activity could be also monitored by measuring the decrease in
A552 due to the reoxidation of the reduced horse
heart cytochrome c as previously described
(A552 = 29,500 M1 · cm1) (12).
UV-visible spectroscopy.
UV-visible spectra of KCl-extracted
proteins were recorded on a U-2000 spectrophotometer (Hitachi, Tokyo,
Japan) in a 1-cm quartz cuvette containing 0.1 to 0.3 mg of protein/ml.
To record the redox difference spectra of KCl extracts, the protein
solution was reduced with sodium dithionite and subsequently reoxidized by adding increasing amounts of an anaerobic solution of ferrihydrite to the reaction mixture in order to obtain final concentrations ranging
from 5.4 to 13.7 mM.
Extraction of iron(III) reductase.
Membrane fractions,
crude extracts or whole cells were stirred for 1 h at 4°C in a
high-ionic-strength salt buffer (100 mM Tris, 0.1 mM DTE, 0.5 M KCl
[pH 7.6]) in order to release the iron(III) reductase.
Native polyacrylamide gel electrophoresis (PAGE) stained for the
iron(III) reductase.
For detection of enzymatic reduction of
Fe(III) in the presence of ferrozine in native polyacrylamide gels,
a procedure adapted from Moody and Dailey (21) was used. An
aliquot of 40 µl of the KCl-extracted proteins containing 950 µg of
protein after dialysis and concentration (Amicon ultrafiltration cell
with PM-10 filter) was applied to a 1-mm, 12% Ready gel (Bio-Rad). The
gel was run for 3 h at 10 mA. The gel was removed and washed for 1 min in 200 ml of 0.1 mM Tris buffer (pH 7.6). After the washing buffer
was removed, the gel was placed in 170 ml of the same buffer containing
30 mg of NADH, 2 mg of reduced horse heart cytochrome c, and
107 mg of ferrozine. The reaction was started by adding 150 µl of a
300 mM Fe(III)-NTA solution. A purple band was observed where
iron(III) reduction took place after 30 min of incubation at
ambient temperature. Photographs could then be taken.
To isolate the protein of the activity band, a preparative gel was
performed according to the following procedure. The dialyzed
and
concentrated KCl-extracted proteins were applied on the 8
lanes of the
1-mm, 12% Ready gel. To avoid disturbance due to
iron for further
processing, one lane was separated from the others
by cutting the gel
and then was stained for the iron(III) reductase
activity. With
this procedure, the position of the active band
of the other lanes
could be estimated and the bands could be excised.
These bands were
suspended in 5 ml of 50 mM HEPES (pH 7) containing
0.1 mM DTE and
homogenized on ice with a potter. Sodium dodecyl
sulfate (SDS) was
added to a final concentration of 1%. The solution
was incubated for
30 min at 50°C and then for 90 min at 37°C.
The sample was
centrifuged for 5 min at 12,000 ×
g. The proteins
of
the supernatant were precipitated by adding 11 ml of ethanol
to 4 ml of
supernatant followed by incubation at
20°C for 2 days.
Precipitated
proteins were collected by centrifugation at 12,000
×
g for 10 min and then resuspended in 100 µl of
H
2O.
Denaturing SDS-PAGE analysis of KCl-extracted proteins.
SDS-PAGE was carried out as described by Laemmli (14) after
the samples were desalted by dialysis. Proteins were visualized by a
silver-staining procedure (27), and the cytochromes were stained by a method with o-dianisidine and hydrogen peroxide
(10).
Chemicals.
Ferrozine, DNase I, horse heart cytochrome
c, NADPH, safranin T, phenosafranin, DTE, and
o-dianisidine-HCl tablets were purchased from Fluka (Buchs,
Switzerland); NADH, neutral red, and bromophenol blue were obtained
from Merck (Darmstadt, Germany); and benzyl viologen, methyl viologen,
rotenone, and 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) were from Sigma (Munich, Germany).
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RESULTS |
Iron(III) reductase activity.
The iron(III) reductase
activity of G. sulfurreducens could be measured with NADH
but not with NADPH (Table 1). If
Fe(III) NTA was used as an electron acceptor, the highest
Fe(III)-reducing activities were measured (10.6 mU/mg of
protein), whereas with Fe(III)-citrate and Fe(III)-EDTA,
lower (2.1 mU/mg of protein) and no activities were detected,
respectively. Cell extracts that were heated at 100°C for 5 min
showed no iron(III) reductase activity. Exposure to air for 2 h decreased the activity by 80%, indicating an oxygen lability of the
iron(III) reductase activity. The activity was highest at pH 7 (Fig. 1). In order to develop an
NADH-independent assay, different artificial electron donors were
tested (Table 1). The major difficulty was the rapid chemical reduction
of Fe(III) by these artificial electron donors, which did not allow us to distinguish between an enzymatic and chemical reduction. Also,
reducing agents such as cysteine, dithionite, and Ti(III) citrate could
not be used to reduce the iron(III) reductase since they also
reduced Fe(III) chemically. Only horse heart cytochrome c reduced by dithionite could be used as an alternative
electron donor (Table 1). The iron(III) reductase activity
increased with increasing concentrations of reduced horse heart
cytochrome c (Fig. 2).
However, at concentrations above 35 µM, activity measurements with
ferrozine were not possible due to a background absorption by reduced
horse heart cytochrome c at 562 nm that was too high. The
iron(III) reductase activity could alternatively be determined by
measuring the oxidation of reduced horse heart cytochrome c at 552 nm. In this case, due to the high absorbance of the reduced horse heart cytochrome c, it was not possible to use
concentrations above 55 µM, and the highest iron(III) reductase
activity measured was of 75 mU/mg of protein.
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FIG. 1.
Iron(III) reductase activity as a function of pH.
The assay was done in a solution containing HEPES, MES
(morpholineethane sulfonic acid), PIPES
[piperazine-N,N'-bis(2-ethanesulfonic acid)],
TAPS {N-[Tris-(hydroxymethyl)-methyl]-3-amino-propane
sulfonic acid}, and KH2PO4 buffer, each
constituent at a concentration of 20 mM. In addition, the reaction
mixture contained 0.3 mM Fe(III)-NTA, 0.5 mM ferrozine, and 1 mM
NADH.
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FIG. 2.
Iron(III) reductase activity as a function of
reduced horse heart cytochrome c concentration. The assay
was done in 50 mM HEPES buffer (pH 7) containing 0.3 mM Fe(III)-NTA
and 0.5 mM ferrozine.
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Iron(III) reductase activity was not affected by 50 µM HOQNO and was partially inhibited (50 to 80%) by
cyanide (KCN; 200
µM) with both NADH and horse heart cytochrome
c as the electron
donor. Rotenone completely inhibited the
iron(III) reductase activity
only with NADH.
Localization of the iron(III) reductase.
Breakage of the
cells of G. sulfurreducens by EDTA-lysozyme treatment
followed by sonication led to a 1.5-fold increase of iron(III)
reductase activity in comparison with unbroken cells when NADH was used
as an electron donor (Table 2). No
significant increase caused by breaking the cells was observed when
horse heart cytochrome c was used as the electron donor.
The spheroplast fraction contained 77 and 68% of iron(III)
reductase activity, respectively, when horse heart cytochrome
c and NADH were used as electron donors, whereas the
periplasmic
fractions contained 23 and 32% of the
iron(III) reductase activity,
respectively (Table
2). The soluble
fraction of crude extracts
accounted for approximately the same
percentage of iron(III) reductase
activity as the
periplasmic fraction of the spheroplast preparation
(17 and
33%, respectively). The membrane fraction contained 83
and 67% of the
iron(III) reductase activity, respectively.
Separation of the outer and cytoplasmic membranes was achieved by
using a highly concentrated sucrose gradient of 50 to 80%.
The
outer membrane, identified by its high KDO content of 77 ±
5 ng/mg of protein, contained 75 and 79% of the iron(III)
reductase
measured with horse heart cytochrome
c and NADH,
respectively
(Table
3). The cytoplasmic
fraction had a low KDO content of
2.2 ± 0.2 ng/mg of protein and
contained 25 and 21% of the iron(III)
reductase activity,
respectively (Table
3).
Solubilization of the iron(III) reductase.
The activity
present in the soluble fractions might have been due to
iron(III) reductase that was only loosely associated with the
membranes. This hypothesis was substantiated by the finding that
the iron(III) reductase activity can be solubilized by
incubation of the membrane fraction with KCl 0.5 M (Table
4). When horse heart cytochrome
c was used as the electron donor, an iron(III) reductase
activity of 45.2 ± 5 mU was found in the soluble fraction that
corresponds to 188% activity compared to the membrane fraction before
treatment (Table 4). With NADH as the electron donor, only 14% of the
iron(III) reductase activity was present in the soluble fraction.
The results obtained after solubilization with KCl also showed that
with NADH as electron donor 59% less iron(III) reductase activity
was recovered compared with untreated membrane fraction, whereas with
horse heart cytochrome c an increase of total activity by a
factor of 2.6 was found.
The incubation of whole cells in Tris buffer containing 0.5 M KCl did
not disrupt the cells, as verified by microscopy, and
led to a
solubilization of the iron(III) reductase activity. The
soluble
fraction obtained with the KCl treatment contained 54%
of the
iron(III) reductase activity (measured with horse heart
cytochrome
c), whereas no iron(III) reductase activity was present
in the supernatant in the absence of KCl (Table
4). With NADH
as
electron donor, 34% of the iron(III) reductase activity was
solubilized by KCl. The chelating agent EDTA, known to destabilize
the
outer membrane by complexing Ca
2+ and Mg
2+ ions
present in the outer membrane, caused in combination with
KCl a
solubilization of up to 81% of the iron(III) reductase activity
(Table
5).
Cytochromes and iron(III) reductase activity in KCl
extracts.
The UV-visible spectrum of the KCl extract
obtained from whole cells exhibited a strong peak at 410 nm and
shoulders centered at 520 and 580 nm (data not shown). These features
are characteristic of hemoproteins. Redox difference spectra of this
sample exhibited a Soret band at 420 nm and 
and 
bands at 522 and 552 nm, respectively, all of which are characteristic of
c-type cytochromes. The addition of ferrihydrite
to the cuvette containing the dithionite-reduced cytochrome
c led to the disappearance of this spectrum (Fig.
3). This oxidation suggested that
c-type cytochromes are involved in iron(III) reduction.
Silver- and heme-stained SDS gels of the KCl fraction containing the
iron(III) reductase revealed the presence of numerous proteins and
several cytochromes (Fig. 4). Native PAGE
of the KCl extract stained for the iron(III) reductase activity exhibited one purple band in the upper part of the gel, indicating that
the iron(III) reductase could be a high-molecular-mass protein (data not shown). Analyzing the proteins obtained from a native preparative gel by SDS-PAGE revealed the presence of three
cytochrome-containing proteins with molecular masses ranging from 67 to
97 kDa (Fig. 4).
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FIG. 3.
UV-visible spectra of KCl extracts of whole cells.  , redox difference spectrum, after addition of ferrihydrite;
---, 8.3 mM;
-.-.-,
10.8 mM; ----, 13.7 mM.
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FIG. 4.
SDS-PAGE of different fractions of G. sulfurreducens extracts. The gels were stained for protein with
silver (A) and heme (B). Lanes 1, whole cells after KCl extraction;
lanes 2, KCl extract from whole cells; lanes 3, isolated band from a
preparative iron(III) reductase activity gel; lanes 4, molecular
mass standards including phosphorylase b (97.4 kDa), bovine
serum albumin (67 kDa), egg albumin (45 kDa), carbonic hydrase (29 kDa), soybean trypsin inhibitor (21 kDa), horse heart cytochrome
c (12.5 kDa), and trypsin inhibitor (6.5 kDa).
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DISCUSSION |
The iron(III) reductase activity assay with ferrozine
and Fe(III) complexed with NTA and citrate, respectively,
showed that the reduction of Fe(III)-NTA occurred five times faster
than the reduction of Fe(III)-citrate. This result is in
agreement with previous findings obtained with S. putrefaciens (2). The Fe(II) species formed with
citrate may be kinetically less labile than those formed with NTA due
to steric effects (9). Additional evidence for the effect of
kinetic lability was obtained with Fe(III) complexed with the
hexadentate ligand EDTA. In this case, no iron(III) reductase
activity could be measured in the assay, which was probably caused by
the inability of ferrozine to unbind Fe(II) from the Fe(II)-EDTA
complex. A similar finding has been described by Dobbin et al.
(9). However, Fe(III)-EDTA can act as an electron
acceptor for the iron(III)-reducing bacteria as shown by the
stimulation of benzene degradation by iron(III) reducers (20).
The iron(III) reductase activity of G. sulfurreducens could be measured with NADH as a physiological
electron donor but not with NADPH. The same was also reported for
S. putrefaciens (23). Since iron(III)
reduction by G. sulfurreducens is a respiratory process, electrons derived from NADH oxidation are probably not directly transferred to Fe(III) but rather are transferred via a
respiratory chain. This assumption is supported by the inhibition of
NADH-dependent iron(III) reduction by rotenone that was also found
for Geobacter metallireducens (11). In addition,
the increase of activity after the cells were broken that gave NADH
better access to the NADH dehydrogenase and the loss of activity upon solubilization of the iron(III) reductase by KCl that disrupted the
respiratory chain also provided evidence for this assumption.
Here, for the first time, an artificial iron(III) reductase
activity assay could be set up by using reduced horse heart cytochrome c as the electron donor. The electrons of the latter
probably enter the respiratory chain to the iron(III) reductase on
a different site than the electrons of NADH for two reasons. First, in
contrast to NADH, there was no increase in activity with horse heart
cytochrome c after the cells were broken. Second, disruption
of the respiratory chain by solubilizing the iron(III) reductase
did not result in a loss of activity as was found with NADH but rather
an increased iron(III) reductase activity. No indications for a
different site of electron transfer were obtained by the
inhibitor experiments. The reactions with either NADH or horse heart
cytochrome c showed the same inhibition pattern.
Interestingly, the inhibition pattern of G. sulfurreducens was different from the patterns described for
either S. putrefaciens (9, 23) or
G. metallireducens (11). HOQNO had
no effect on the iron(III) reductase activity of the two
Geobacter species, but it did inhibit the iron(III) reduction by S. putrefaciens. Cyanide, on the other
hand, inhibited the iron(III) reduction by S. putrefaciens and G. sulfurreducens, whereas no
effect was observed for G. metallireducens.
Similar to other iron(III) reducers, such as S. putrefaciens and G. metallireducens, where the
total iron(III) reductase activity was found exclusively in
the membrane fraction (9, 11, 23), the major part of the
iron(III) reductase activity of G. sulfurreducens was also found in the membrane fraction. The presence of iron(III) reductase activity in the outer membrane fraction has also been found
for S. putrefaciens. For G. sulfurreducens, 75 to 79% of the iron(III) reductase
activity of the membrane fraction was present in the outer membrane,
whereas for S. putrefaciens it was 54 to 56%. The
iron(III) reductase activity found in the cytoplasmic membrane
fraction of G. sulfurreducens could be an artifact due to the sonication treatment since cell breakage by sonication can cause
an extensive redistribution of membrane proteins between membranes
(31). The minor part of the iron(III) reductase activity recovered in the soluble fractions after ultracentrifugation or preparation of spheroplasts is probably due to the release of the
loosely membrane-associated iron(III) reductase during treatment. The solubilization of iron(III) reductase by KCl actually showed that this enzyme activity is loosely membrane associated since a high
salt concentration treatment is a well-known method for detaching
peripheral membrane proteins (25).
Because the major part of Fe(III) exists in the environment as
insoluble Fe(III) oxides, a localization of the terminal reductase in the outer membrane is certainly conceivable. It has been proposed that direct contact of the cell surface with the iron oxides is needed
to enable the bacteria to reduce Fe(III) (3, 4, 23). This would require an outer membrane iron(III) reductase facing the
outside of the cells. Indications for such a localization of the
iron(III) reductase in G. sulfurreducens were
obtained with the solubilization of iron(III) reductase activity
from whole cells by the KCl treatment. This treatment probably only
detached peripheral outer membrane proteins located on the outside of
the cells and did not detach proteins facing the periplasm. A recent study, however, showed that cell contact is not a requisite for reduction of solid Fe(III) oxides (6). In this study, an
adhesion-deficient Shewanella alga strain reduced amorphous
Fe(III) oxide at the same rates as a strain that strongly adhered
to amorphous Fe(III) oxide. If the electrons are not directly
transferred from the iron(III) reducers to the Fe(III) oxides,
the electron transfer could be accomplished by diffusion of electron
mediators or of free or complexed Fe(III). It has been shown that
G. sulfurreducens excretes c-type
cytochromes in the medium and that this cytochrome has iron(III)
reductase activity (29). This suggested that this cytochrome
could act as an electron mediator between the cells and the iron
oxides. Indications for the production of a cell surface protein that
efficiently chelate Fe(III) have been obtained for S. alga (6).
The presence of heme proteins in the protein fraction isolated from the
iron(III) reductase activity band of a native gel, the
solubilization of several c-type cytochromes by KCl from
whole cells of G. sulfurreducens, and the possible
involvement of these cytochromes in iron(III) reduction added
further evidence for the hypothesized key role that periplasmic
and outer membrane c-type cytochromes play in iron(III)
reduction (18, 22, 24, 29). For S. putrefaciens it has been shown that the outer membrane of
anaerobically grown cells contained, in addition to 54 to 56% iron(III) reductase activity (23), 80% of the
membrane-bound cytochromes (22). Recently, the gene of a
tetraheme cytochrome c of S. putrefaciens
has been cloned and sequenced (24). This c-type
cytochrome has been found in the cytoplasmic membrane as well as in the
soluble fraction. It has been proposed that the tetraheme cytochrome
c is involved in the transfer of electrons from the
cytoplasmic membrane to acceptor proteins such as fumarate reductase
located in the periplasm and iron(III) reductase located in the
outer membrane (24). Finally, a c-type cytochrome
that has been excreted into the medium by G. sulfurreducens and that has been purified is similar to the
cytochrome c3 of other bacteria (29).
This c-type cytochrome was also present in the membrane and
soluble fraction and was able to reduce different electron acceptors
including iron oxides. In contrast to the heme proteins present in the
iron(III) reductase activity band that had a high molecular mass of
70 to 100 kDa, the excreted c-type cytochrome had a
molecular mass of only 9.6 kDa (29). High-molecular-mass cytochromes were present in the outer membrane of Desulfovibrio vulgaris (Hildenborough) when cultivated in a medium with a high Fe2+ concentration (100 ppm) (33). A role in
anaerobic biocorrosion has been postulated for these
high-molecular-mass cytochromes. Whether the iron(III) reductase of
G. sulfurreducens is indeed a high-molecular-mass
cytochrome will be investigated in the near future.
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ACKNOWLEDGMENTS |
This work was financially supported by the European Environmental
Research Organization (EERO) and the Swiss Federal Institute for
Environmental Science and Technology (EAWAG).
We thank Derek Lovley for providing G. sulfurreducens;
Wolfram Schumacher for helpful comments and discussions; Birgit Krause, Kornelia Zepp, and Mario Snozzi for critically reviewing the
manuscript; and Bernhard Schink for giving us access to his data prior
to publication.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Swiss Federal
Institute for Environmental Science and Technology (EAWAG),
Limnological Research Center, Seestr. 79, CH-6047 Kastanienbaum,
Switzerland. Phone: 41-41-3492145. Fax: 41-41-3492168. E-mail:
holliger{at}eawag.ch.
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Abstract
Introduction
