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J Bacteriol, July 1998, p. 3686-3691, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Periplasmic and Extracellular c-Type Cytochrome of
Geobacter sulfurreducens Acts as a Ferric Iron Reductase and
as an Electron Carrier to Other Acceptors or to Partner
Bacteria
Sabine
Seeliger,1
Ralf
Cord-Ruwisch,2 and
Bernhard
Schink1,*
Fakultät für Biologie,
Universität Konstanz, D-78457 Konstanz,
Germany,1 and
Biotechnology, Murdoch
University, Perth, Western Australia, 6150, Australia2
Received 9 January 1998/Accepted 4 May 1998
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ABSTRACT |
An extracellular electron carrier excreted into the growth medium
by cells of Geobacter sulfurreducens was identified as a c-type cytochrome. The cytochrome was found to be
distributed in about equal amounts in the membrane fraction, the
periplasmic space, and the surrounding medium during all phases of
growth with acetate plus fumarate. It was isolated from
periplasmic preparations and purified to homogeneity by cation-exchange
chromatography, gel filtration, and hydrophobic interaction
chromatography. The electrophoretically homogeneous cytochrome had
a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced
state absorption maxima at wavelengths of 552, 522, and 419 nm. The
midpoint redox potential determined by redox titration was 
0.167 V. With respect to molecular mass, redox properties, and molecular
features, this cytochrome exhibited its highest similarity to
the cytochromes c of Desulfovibrio salexigens
and Desulfuromonas acetoxidans. The
G. sulfurreducens cytochrome c reduced
ferrihydrite (Fe(OH)3), Fe(III) nitrilotriacetic acid,
Fe(III) citrate, and manganese dioxide at high rates. Elemental
sulfur, anthraquinone disulfonate, and humic acids were reduced more
slowly. G. sulfurreducens reduced the cytochrome with
acetate as an electron donor and oxidized it with
fumarate. Wolinella succinogenes was able to reduce
externally provided cytochrome c of G. sulfurreducens with molecular hydrogen or formate as an
electron donor and oxidized it with fumarate or nitrate as an electron
acceptor. A coculture could be established in which G. sulfurreducens reduced the cytochrome with acetate, and the
reduced cytochrome was reoxidized by W. succinogenes in the
presence of nitrate. We conclude that this cytochrome can act as
iron(III) reductase for electron transfer to insoluble iron hydroxides
or to sulfur, manganese dioxide, or other oxidized compounds, and it
can transfer electrons to partner bacteria.
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INTRODUCTION |
The reduction of ferric iron to
ferrous iron is an important process in sediments and hydromorphic
soils (38). The process and the involvement of bacteria in
it have been studied with defined bacterial cultures for more than 4 decades (4, 7, 12, 22). Iron reduction has gained interest
again recently with the discovery of specifically iron-reducing
bacteria which can couple this process to the oxidation of a broad
range of substrates (27-30). Several species of
specifically iron-reducing bacteria are known today, including
Shewanella (Alteromonas)
putrefaciens (32), Geobacter
metallireducens (30, 33), Geobacter
sulfurreducens (11), and several strains of unknown
taxonomic affiliation. Several nitrate-reducing or -fermenting bacteria
can reduce iron(III) facultatively as an additional method of electron
release.
Iron is a transition element that easily changes between the redox
states Fe(II) and Fe(III). The standard redox potential of the
Fe3+-Fe2+ couple (770 mV) is applicable only
in strongly acidic solution (pH <2.5), in which both ions
are well soluble. At neutral pH, the redox transition occurs
mainly between, e.g., Fe(OH)3 (ferrihydrite) and the
Fe2+ ion at a redox potential around +150 mV
(52). Thus, the redox potential at which neutrophilic iron
reducers release their electrons is in the same range as that of the
fumarate-succinate couple (+30 mV). The main problem with iron
reduction under these conditions is the low solubility of iron(III)
hydroxides. The free Fe3+ ion concentration in a
Fe(OH)3-saturated neutral solution is around
10
19 M; with other iron oxohydroxides (goethite,
hematite, and lepidocrocite), the free Fe3+ concentration
is even lower (47). Thus, iron-reducing bacteria have to
deliver their electrons to an essentially insoluble acceptor system,
and the question of how cells can accomplish this at sufficient rates
has not yet been answered convincingly. It was suggested recently
(35) that humic compounds could act as redox mediators between iron-reducing bacterial cells and iron hydroxides; however, the
efficiency of this coupling with natural humic compounds still needs to
be evaluated. In the present communication, we report on the
purification and characterization of a periplasmically localized
c-type cytochrome of G. sulfurreducens that
can act as an extracellular iron reductase and that can transfer
electrons as well to other acceptor systems.
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MATERIALS AND METHODS |
Sources of strains.
G. sulfurreducens PCA (ATCC
51573) was obtained from D. Lovley, Amherst, Mass. Wolinella
succinogenes DSM 1740 was obtained from A. Kröger,
Frankfurt, Germany. Pelobacter acetylenicus WoAcy1 (DSM
3246), Pelobacter propionicus DSM 2379, Clostridium
homopropionicum DSM 5847, and Methanospirillum hungatei
SK (DSM 3595), were from our own culture collection. All strains were
checked for purity at regular intervals by phase-contrast microscopy
after growth in mineral medium or in complex medium (AC-medium; Difco
Laboratories, Detroit, Mich.) (diluted 1:10).
Media and growth conditions.
G.
sulfurreducens and W. succinogenes were grown in
carbonate-buffered, cysteine-reduced mineral medium (50)
containing a seven-vitamin solution (50), selenite-tungstate
solution (49), and the trace element solution SL10
(51). The final pH of the medium was adjusted to 7.2 to 7.4. The growth temperature was 28°C. Substrates were added from sterile,
neutralized stock solutions. Mass cultures of G. sulfurreducens cells were grown in 5-liter carboys with 10 mM
acetate plus 40 mM fumarate as substrates. Cells were harvested by
centrifugation at 10,000 × g for 25 min.
Localization experiments.
Cells were harvested at the end of
the logarithmic-growth phase by 25 min of centrifugation at 10,000 × g, washed once, and resuspended in 50 mM Tris-HCl (pH
7.0). Sucrose (20% [wt/vol]), 2 mM EDTA, and 1 mg of lysozyme per ml
(21,500 U · mg of protein1) were added, and the
suspension was incubated for at least 1 h at 28°C. Spheroplast
formation was monitored microscopically. Spheroplasts were removed by
30 min of centrifugation at 5,000 × g, and the
supernatant was cleared of cell debris afterwards by 30 min of
centrifugation at 45,000 × g to give the periplasmic fraction. Malate dehydrogenase activity was monitored in the
periplasmic fraction and culture supernatant and served as a tracer of
the cytoplasmic fraction by measuring NADH oxidation with oxaloacetate (45).
Purification of cytochrome c.
Purification started
with the periplasmic fraction, which was applied to a cation-exchange
column (0.6 by 5 cm) (Mono S, prepacked; Pharmacia, Uppsala, Sweden)
preequilibrated with 25 mM sodium phosphate buffer (pH 6.3) as the
eluent. In a linear gradient up to 1 M NaCl, the cytochrome eluted at a
150 mM (±10 mM) concentration of NaCl. The fraction containing
cytochrome c (detected by its reduced absorption spectrum)
was loaded onto a gel filtration column (1.25 by 30 cm; Superose 12 prepacked; Pharmacia) run with 0.15 M ammonium acetate buffer (pH 6.3).
If necessary, the fraction containing the cytochrome was lyophilized
before further ammonium acetate was added, to give a 1.7 M ammonium
acetate concentration, and loaded onto a hydrophobic interaction column
(1.25 by 10.5 cm; phenyl Superose prepacked; Pharmacia). The cytochrome
eluted with 1.7 M ammonium acetate, whereas the remaining contaminating proteins were retained and eluted at lower ionic strength. The cytochrome fraction was lyophilized, diluted in two steps with 10 volumes of distilled water each, and lyophilized to give a concentrated
solution.
Characterization of cytochrome c.
For sodium dodecyl
sulfate (SDS) gel electrophoresis, the method of Laemmli
(24) was applied with 12 or 14% polyacrylamide resolving
gels and 4% stacking gels. Samples were diluted in sample buffer
containing 60 mM Tris-HCl, 2% (wt/vol) SDS, 10% (wt/vol) glycerol,
0.025% (wt/vol) bromophenol blue, and no mercaptoethanol. Electrophoresis was carried out in a dual slab cell (Mini-Protean II;
Bio-Rad, Richmond, Calif.) with Tris-glycine-SDS buffer (25 mM, 250 mM,
0.1% [wt/vol], respectively), starting at 30 mA until samples
entered the resolving gel and separating at 40 mA.
Heme staining in SDS gels was performed as described earlier
(48) with modifications by Goodhew et al. (20).
Silver staining of proteins was performed at room temperature according
to the following procedure: gels were incubated in an aqueous solution of 50% (vol/vol) methanol, 12% (vol/vol) acetic acid, and 0.5 ml of
37% formaldehyde liter1 for 1 h and washed three
times in 50% (vol/vol) ethanol-water for 20 min. The next incubation
was in an aqueous solution of 0.2 g of
Na2S2O3 · 5H2O
for exactly 1 min, followed by washing with distilled water three times
for 20 s each. Afterwards, gels were incubated in an aqueous
solution of 2 g of AgNO3 liter1 plus
0.75 ml of 37% formaldehyde liter1 for exactly 20 min
and washed with distilled water twice for exactly 20 each. Color
development in an aqueous solution of 20 g of sodium carbonate
liter1 0.5 ml of 37% formaldehyde liter1,
and 4 mg of Na2S2O3 · 5H2O liter1 was stopped immediately when the
first bands appeared. Gels were washed twice with distilled water for 2 min and incubated in an aqueous solution of 50% (vol/vol) methanol
plus 12% (vol/vol) acetic acid for 10 min. The last washing step was
in 50% methanol-water for 20 min, and gels were stored afterwards in
the same solution at 4°C.
The molecular mass of the purified cytochrome
c was
estimated by SDS-polyacrylamide gel electrophoresis (PAGE)
(
24) and
by matrix-assisted laser ionization desorption mass
spectroscopy
(MALDI) in a biflex linear time of flight setup (Bruker,
Billerica,
Mass.). The midpoint redox potential (E
0') was
determined by titration
with 100 µM flavin mononucleotide (FMN)
(E
0' =
190 mV) and 100
µM indigodisulfonate
(E
0' =
125 mV) as redox indicators, dithionite
as
reductant, and air as oxidant. Absorptions of dyes and cytochrome
were
recorded with a double-beam UV-visible light (UV/VIS) spectrophotometer
(Uvikon 860; Kontron, Zurich, Switzerland).
Analytical methods.
Cytochrome c was quantified
by determining the redox difference absorption spectra of
dithionite-reduced minus air- or hydrogen peroxide-oxidized
preparations. Spectra were recorded with double-beam UV/VIS
spetrophotometers (Uvikon 860 or 930; Kontron) at room temperature. The
glass and quartz cuvettes used had a 1-ml total volume and a 1-cm light
path and were sealed with rubber stoppers and gassed with nitrogen for
anoxic measurements. Peak heights were measured relative to a baseline
drawn between the troughs at 530 to 535 and 565 to 570 nm. Protein was
quantified according to the method of Bradford (6).
Chemicals.
All chemicals were analytical or reagent grade
and were obtained from Biomol (Ilvesheim, Germany), Boehringer
(Mannheim, Germany), Eastman Kodak (Rochester, N.Y.), Fluka (Neu-Ulm,
Germany), Merck (Darmstadt, Germany), Pharmacia (Freiburg, Germany),
Serva (Heidelberg, Germany), and Sigma (Deisenhofen, Germany). Gases
were purchased from Messer-Griesheim (Darmstadt, Germany) and
Sauerstoffwerke Friedrichshafen (Friedrichshafen, Germany).
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RESULTS |
Cytochrome contents of cells and cell subfractions.
Cells of
G. sulfurreducens contained cytochromes which
stained colonies in deep-agar cultures pinkish-red, as observed earlier (11). Redox difference spectra of cell extracts exhibited
absorption bands at wavelengths of 552, 522, and 419 nm typical of
those of a c-type cytochrome. During growth with acetate
plus fumarate in liquid medium, 29% of this cytochrome was found in
the culture supernatant, 28% was found in the periplasmic space, and
21% was associated with the membrane fraction (Table
1). No cytochrome was detected in the
cytoplasm. The membrane-associated cytochrome c could not be
solubilized with 0.2 M KCl, but was resolved from the membranes nearly
quantitatively with 1% Triton X-100. The comparably high proportion of
cytochrome found in the extracellular space was not caused by excessive
aging of the culture in the stationary phase: the proportion of
cytochrome distribution in the various compartments was stable
throughout the entire period of growth with acetate plus fumarate (Fig.
1). Malate dehydrogenase as a tracer
enzyme of cytoplasmic contaminations was distributed at a ratio of
91%/4%/5% between the cytoplasmic, membrane, and periplasmic
fractions. No malate dehydrogenase activity was found in the culture
supernatant, indicating that there was no major spill of cytoplasmic
proteins into the growth medium due to cell lysis. Similar amounts of
cytochrome were also excreted in cultures grown with acetate plus
ferrihydrite.
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FIG. 1.
Growth and simultaneous release of cytochrome
c into the growth medium by cells of G. sulfurreducens.  , optical density at 570 nm;  , extracellular
cytochrome c.
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The cytochrome was most easily accessible at a high concentration in
the periplasmic fraction, and therefore enrichment and
isolation
started from this fraction. Part of the contaminating
proteins was
bound to an anion exchanger, DE 52, in 50 mM Tris-HCl
buffer (pH 7.0),
which did not bind the cytochrome. A Mono S cation
exchanger
equilibrated with 25 mM sodium phosphate (pH 7.0) bound
83% of the
cytochrome, which was eluted subsequently with a linear
NaCl gradient
at a 0.17 M concentration. Gel filtration on Sepharose
12 and
hydrophobic interaction chromatography yielded a homogeneous
cytochrome
preparation which exhibited only one protein band after
SDS-PAGE and
silver staining (Table
2 and Fig.
2). This band
was also stained with high
sensitivity by specific heme staining.
If the cytochrome of the crude
cell extract was subjected to the
same enrichment procedure, about 40%
of the total cytochrome present
bound to the anion exchanger.
Obviously, there were other cytochromes
present in the cells that
exhibited binding properties different
from those of the
periplasmic cytochrome
c, and the appearance
of more
than one heme-staining band in gel electrophoresis (Table
2) indicated
the presence of more than one cytochrome in this
bacterium. However, in
the extracellular and the periplasmic fractions,
only one type
of cytochrome was found which exhibited identical
properties with
respect to binding, spectral absorption, and electrophoretic
mobility.
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FIG. 2.
SDS-PAGE of purified cytochrome c.
Lanes 1 to 4 were silver stained; lanes 5 to 8 were identical runs
stained for hemes. Lanes 1, 3, and 6, commercial protein standards
(lane 1, lactalbumin, 14.4 kDa, trypsin inhibitor, 20.1 kDa, carbonic
anhydrase, 30.0 kDa [higher masses not distinguishable]; lane 3, insulin  chain, 3.5 kDa; lane 6, aprotinin, 6.5 kDa [higher masses
not distinguishable]), lane 2; commercial horse heart cytochrome
c; lanes 4, 5, 7, and 8, periplasmic cytochrome
c of G. sulfurreducens. Lanes 1, 2, 4, and 5 contained 1.2 to 1.5 µg of protein per band; lanes 3, 6, 7, and
8 contained 2 to 3 µg of protein per band. Bands with higher
molecular masses appearing in heme-stained gels loaded with large
amounts of protein (lanes 7 and 8) were not detected in protein
sequencing or MALDI measurements, which are very sensitive to
impurities.
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Since the periplasmic cytochrome tended to bind to
ultrafiltration membranes, we avoided such steps in the purification
procedure.
If necessary, cytochrome solutions were concentrated by
lyophilization
in ammonium acetate buffer (pH 6.2) to avoid excessive
buffer
salt concentrations.
Characterization of the periplasmic cytochrome
c.
A first estimation of the molecular mass of the
electrophoretically homogeneous cytochrome c of
G. sulfurreducens by SDS-PAGE in
Tris-glycine or Tris-tricine buffer indicated a molecular mass of
11.2 ± 0.3 kDa in comparison with standard proteins with
molecular masses of 1.4 to 26.6 kDa. A more exact determination
by MALDI revealed a molecular mass of 9.57 ± 0.02 kDa. Absorption
spectra of the cytochrome c in its oxidized or reduced form
are shown in Fig. 3. The reduced
cytochrome had absorption maxima at 552, 522, and 418 nm, and the
oxidized form had a maxim um at 407 nm. The specific absorption of the
band of the reduced form of the cytochrome at a wavelength of 552 nm was 32.5 mM1 · cm1, calculated on
the basis of the MALDI-determined molecular mass.
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FIG. 3.
Absorption spectra of purified cytochrome
c. Dashed line, air oxidized; solid line, dithionite
reduced.
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The midpoint redox potential of the isolated cytochrome
c
was determined by redox titration with FMN (E
0' =
0.190
V) and
indigodisulfonate (E
0' =
0.125 V) as redox
indicators. The titration
was performed in both directions, either with
dithionite as reductant
or with oxygen as oxidant. The titration curve
(Fig.
4) exhibited
only one inflection
point, and a midpoint redox potential of
0.167
V ± 1 mV was
calculated.
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FIG. 4.
Redox titration of purified cytochrome
c with dithionite and air. Indicator dyes were
indigodisulfonate ( ) (E0' = 0.125 V) and FMN ()
(E0' = 0.190 V). The line was fit to the curve
according to the Nernst equation. The midpoint potential of the
cytochrome for assumed independent electron transfers was calculated to
be 0.167 V.
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Electron donors and acceptors.
In the absence of additional
mediators, the periplasmic cytochrome c was reduced
with dithionite, H2S, or FeSO4 at high rates. It was oxidized quickly (>12 µmol · min1
· mg of protein1) by O2,
Fe(OH)3, or MnO2, and more slowly by colloidal
sulfur (S0), anthraquinone disulfonate, and humic acids
(Sigma). In the presence of phenazine methosulfate, the
cytochrome could also be reduced by NADH or be oxidized by FMN. Flavin
adenine dinucleotide did not oxidize the cytochrome, even in the
presence of phenazine methosulfate.
The cytochrome could also be reduced with hydrogen in the presence of a
palladium charcoal catalyst or with hydrogen or formate
in the presence
of crude cell extracts of
W. succinogenes (0.1
nmol · min
1 · mg of
protein
1).
W. succinogenes cell extracts
also oxidized the reduced cytochrome
in the presence of fumarate (0.5 nmol · min
1 · mg of
protein
1).
Coupling of electron transfer from G. sulfurreducens to external electron acceptors.
G.
sulfurreducens cells grown with acetate plus fumarate reduced or
oxidized the extracellular cytochrome, depending on the relative
electron availability. With excess acetate over fumarate, the
cytochrome was quantitatively reduced; with excess fumarate, it was
quantitatively oxidized (Fig. 5). The
reduced or oxidized state of the culture could even be recognized by
the naked eye, changing between pink-orange (reduced) and yellow
(oxidized). The reduction and oxidation of G. sulfurreducens cytochrome c could also be catalyzed by
cell suspensions of other bacteria, such as W. succinogenes, in the presence of excess hydrogen or formate or
excess fumarate, as documented in Fig. 6.
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FIG. 5.
Absorption spectra of G. sulfurreducens cultures grown under different substrate supply
situations. Dashed line, 10 mM acetate and 60 mM fumarate; solid line,
10 mM acetate and 30 mM fumarate.
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FIG. 6.
Reduction and oxidation of G. sulfurreducens cytochrome c by washed intact cells of
G. sulfurreducens and W. succinogenes (each at about 0.02 mg of protein per ml) in the
presence of excess nitrate (2 mM [added after 20 min]) or excess
acetate (3 mM [added after 117 min]).
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G. sulfurreducens and
W. succinogenes could be coupled in an interspecies electron transfer
in which acetate was oxidized
by
Geobacter and the
electrons were transferred to cytochrome
c; the latter was
reoxidized by
W. succinogenes with nitrate as
the
terminal electron acceptor. Such a syntrophic coculture has
been
described in a separate paper (
13). We checked various
bacteria described previously to be active in interspecies electron
transfer for possible interaction with the cytochrome preparation.
P. acetylenicus,
P. propionicus,
Clostridium homopropionicum,
and
M. hungatei
neither reduced nor oxidized the
G. sulfurreducens periplasmic cytochrome.
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DISCUSSION |
Comparison of the G. sulfurreducens
periplasmic cytochrome with other known cytochromes.
Compared to known tri- and tetraheme class III cytochromes
(39) from mostly sulfate- and sulfur-reducing bacteria, the
cytochrome c described here has a rather low molecular mass
and a comparably high midpoint redox potential. Cytochromes of
Desulfovibrio vulgaris Miyazaki (43, 54),
D. vulgaris Hildenborough (1, 10, 14-16),
Desulfovibrio salexigens Benghazi (17),
D. gigas (3, 25, 53), D. desulfuricans Norway (8, 9), D. desulfuricans Essex 6 (15), and S. putrefaciens (36) have molecular masses of 13 kDa or
higher and standard redox potentials below 0.2 V. Desulfobulbus elongatus has a cytochrome with a
standard redox potential (0.165 V) similar to that of
G. sulfurreducens, but which also is of high
molecular mass (13.7 kDa) (42). All cytochromes mentioned have nearly identical absorption maxima (±2-nm wavelength) in both their reduced and oxidized forms. The cytochrome most similar
to the periplasmic cytochrome c of
G. sulfurreducens is the one isolated from
Desulfuromonas acetoxidans, with standard redox potentials
of the three hemes reported to be 0.102, 0.177, and 0.177 V
(18) or 0.140, 0.210, and 0.240 V (10) and with a molecular mass of 9.8 kDa (40). This cytochrome
c has been characterized as a triheme cytochrome
(2). The specific absorption coefficient of the
band of the reduced G. sulfurreducens cytochrome was 32.5 mM1 · cm1,
similar to that of the three-heme cytochrome c of
D. acetoxidans (30.8 mM1 · cm1) (40), and 2.5 times as high as that of
the monoheme horse heart cytochrome c. These findings,
together with the low molecular mass, suggest that the
G. sulfurreducens cytochrome contains three hemes,
and further biochemical and sequence data obtained recently confirm
this assumption (18a). The structural similarities
between these two c-type cytochromes are in accordance with
the apparent 16S rRNA sequence similarities between D. acetoxidans and G. sulfurreducens (11) and with the observation that D. acetoxidans can reduce ferric iron as well (41).
Physiological function and ecological significance of the
periplasmic cytochrome c.
The cytochrome described
here can be oxidized by an unusually broad variety of electron
acceptors, such as oxygen, Fe(OH)3, Fe(III) citrate,
Fe(III) nitrilotriacetic acid, MnO2, and sulfur, as
well as by humic acids and anthraquinone. The midpoint redox potential (0.167 V) is in the same range as that of the
sulfur-sulfide couple (0.24 V) and is slightly lower than those of
the various iron(III) hydroxide reduction reactions (0 to +0.2 V)
(47). It can accept electrons, e.g., from an nNADH-oxidizing
redox system described recently (19), and transfer them to
the various acceptor systems mentioned. With its rather unspecific
reactivity, this cytochrome lends itself as a carrier for electron
transfer to extracellular electron acceptors, especially since it
appears to be excreted at a significant amount into the surrounding
medium. Its comparably small size may be a prerequisite for this
excretion across the outer cell membrane. In none of the
above-mentioned publications was a significant release of cytochrome
into the growth medium mentioned. This phenomenon had been reported so far only for the fermenting bacterium Malonomonas rubra
(23). The cytochrome c3 of
D. vulgaris was reported to also reduce chromate or
uranium(VI) compounds (31, 34), but these acceptors are highly soluble, and their reduction does not depend on an extracellular electron carrier.
The possibility of electron transfer to insoluble acceptors such as
iron(III) oxohydroxides was discussed for
S. putrefaciens MR-1 (
37). According to this hypothesis, electrons are
transferred
by a cytochrome which is present in large amounts and which
is
tightly bound to the outer membrane. Accordingly, iron-reducing
bacteria would have to operate in immediate contact with the iron(III)
mineral in order to secure an effective electron transfer. In
the
present study, we found a cytochrome
c in the
periplasmic
space as well as in the membrane fraction, which
after the preparation
procedure was applied, includes the cytoplasmic
and the outer
membrane (
44), as well as the surrounding
medium. Based on our
findings concerning the distribution and
reactivity of the periplasmic
cytochrome
c of
G. sulfurreducens, we propose that this cytochrome
may act as an iron(III) reductase and may also be involved in
electron
transfer to the other acceptor systems mentioned. At
first sight,
excretion of a cytochrome into the medium appears
to be an excessively
expensive mechanism of electron transfer,
taking into account that
approximately 750 to 800 mol of ATP is
needed for the synthesis of 1 mol of protein with a molecular
mass of 9.5 kDa (
46).
Nonetheless, an up to 200 nM concentration
of this cytochrome, as
observed in our culture medium in the stationary
phase, is equivalent
to about 5% of the total protein content
of the culture, and therefore
is in an affordable range of investment
for the cells. This situation
is to some extent comparable to
that of, e.g., cellulose degradation by
extracellular cellulases.
These proteins are also excreted, and this
energy investment has
to be covered by a sufficient return of
oligosaccharides.
Obviously, the excretion of 200 nM cytochrome as we observed it in our
cultures could be afforded by the cells concomitant
with good growth.
With the following assumptions
a cell size of
0.5 by 2.5 µm, a cell
surface area of 4.3 µm
2, a concentration of
10
8 cells per ml, a diffusion constant (
D) for
the cytochrome of
1.2 × 10
6 cm
2
· s
1 (
26), and a cytochrome concentration of
200 nM
the electron
transport rate via diffusion of reduced cytochrome
(transporting
three electrons with three hemes) would be in the range
of 18
nmol · min
1 · mg of
protein
1 along a diffusion distance of 10 µm to the
acceptor mineral,
would be 10 times as high along a distance of 1 µm,
and thus would
be well within the range of the metabolic activity of
actively
growing cells. The efficiency of electron transport through
extracellular
cytochromes could even be enhanced by the known ability
of certain
c-type cytochromes to transport electrons by
intermolecular electron
transfer (
5,
21), and the
near-neutral isoelectric point
of our cytochrome would favor such a
transfer in neutral solution.
If we realize further that iron-reducing
bacteria in nature grow
mainly in colonies of several hundred or
thousand cells in close
association with iron mineral and that the
excreted cytochrome
molecules are used and returned by such a community
much more
efficiently than in our suspended culture, the assumption
appears
realistic that an extracellular cytochrome can act as a
dissolved
electron carrier and iron reductase in bacterial iron
reduction.
In a separate paper, we reported that
G. sulfurreducens can grow and oxidize acetate in syntrophic
association with
W. succinogenes when nitrate is used
as a terminal electron acceptor (
13). Our
original
assumption that this syntrophic cooperation was based
on interspecies
hydrogen transfer turned out to be unlikely because
of the extremely
small hydrogen concentrations measured in these
cultures (0.02 to 0.04 nM), which could not account for the observed
electron flux. Also,
these cocultures contained free cytochromes
at the same concentration
as that in pure cultures of
G. sulfurreducens.
Based on the calculations described above, and taking into account
that
the average distance between single cells in a suspension
of
10
8 cells per ml is around 20 µm, the cytochrome at the
observed
concentration could contribute significantly to this
interspecies
electron transfer, especially if convective transport in
the free
liquid and active swimming of both partners enhance the
exchange
of reduced and oxidized cytochrome.
We conclude that the periplasmic cytochrome of
G. sulfurreducens can act as an iron(III)
reductase and also as a redox carrier
between the cell and various
extracellular electron acceptor systems,
including humic acids or
partner bacteria. Whether this concept
of electron transfer can be
generalized as well for other iron-reducing
bacteria still needs to be
examined. Further experiments on the
impact of free cytochrome
c on electron transfer and interaction
between bacterial
cells and with insoluble electron acceptors
are in progress in our
laboratory.
| |
ACKNOWLEDGMENTS |
We are indebted to Claudio Luchinat, Florence, Italy, for helpful
comments on the biochemistry of cytochromes and to Michael Przybylski
and his coworkers, Konstanz, Germany, for determination of the
molecular mass of our cytochrome by MALDI.
Support through a grant of the Deutsche Forschungsgemeinschaft,
Bonn-Bad Godesberg, Germany, on "Energetics of syntrophic associations" is gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fakultät
für Biologie, Universität Konstonz, Postfach 55 60, D-78457
Konstanz, Germany. Phone: 07531-88-2140. Fax: 07531-88-2966. E-mail:
Bernhard.Schink{at}uni-konstanz.de.
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
