FEMS Microbiology Letters 195 (2001) 253^258
www.fems-microbiology.org
Di¡erences in Fe(III) reduction in the hyperthermophilic archaeon,
Pyrobaculum islandicum, versus mesophilic Fe(III)-reducing bacteria
Susan E. Childers *, Derek R. Lovley
Department of Microbiology, Morrill Science Center, University of Massachusetts, Amherst, MA 01003, USA
Received 27 October 2000; received in revised form 27 December 2000; accepted 27 December 2000
Abstract
The discovery that all hyperthermophiles that have been evaluated have the capacity to reduce Fe(III) has raised the question of whether
mechanisms for dissimilatory Fe(III) reduction have been conserved throughout microbial evolution. Many studies have suggested that
c-type cytochromes are integral components in electron transport to Fe(III) in mesophilic dissimilatory Fe(III)-reducing microorganisms.
However, Pyrobaculum islandicum, the hyperthermophile in which Fe(III) reduction has been most intensively studied, did not contain
c-type cytochromes. NADPH was a better electron donor for the Fe(III) reductase activity in P. islandicum than NADH. This is the
opposite of what has been observed with mesophiles. Thus, if previous models for dissimilatory Fe(III) reduction by mesophilic bacteria are
correct, then it is unlikely that a single strategy for electron transport to Fe(III) is present in all dissimilatory Fe(III)-reducing
microorganisms. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Hyperthermophile ; Archaeon; Dissimilatory Fe(III) reduction ; Cytochrome
1. Introduction
Dissimilatory Fe(III) reduction is an environmentally
signi¢cant process in sediments, shallow aquifers and the
deep subsurface [1^3]. The capacity to conserve energy to
support growth from electron transport to Fe(III) is found
in discrete clades of microorganisms throughout the Bacteria and the Archaea [4^11]. The fact that the capacity for
Fe(III) reduction is phylogenetically widespread and is
found in deeply branching groups in the Bacteria and
the Archaea, has led to the suggestion that the Fe(III)
reduction was an early form of respiration and that the
mechanisms for electron transport to Fe(III) have been
conserved throughout microbial evolution [12^14]. However, this hypothesis has not been adequately evaluated
because of a lack of information on mechanisms for
Fe(III) reduction in a diversity of Fe(III)-reducing microorganisms. The biochemistry of dissimilatory Fe(III) reduction has only been studied intensively in species of
Shewanella and Geobacter which, respectively, are members of the Q and N subclasses of the Proteobacteria. If,
in fact, mechanisms for Fe(III) reduction have been con-
* Corresponding author. Tel. : +1 (413) 545-1048;
Fax: +1 (413) 545-1578; E-mail: [email protected]
served throughout microbial evolution, then it would be
expected that microorganisms outside the Proteobacteria
would reduce Fe(III) in a manner similar to that proposed
for Shewanella and Geobacter species. Pyrobaculum islandicum is a member of the Thermoproteales, which represent a slowly evolving lineage of the Archaea within the
universal phylogenetic tree [15]. Like Shewanella and Geobacter species, P. islandicum conserves energy to support
growth by coupling the oxidation of hydrogen or organic
compounds to the reduction of Fe(III) [14,16]. However,
the results presented here suggest that the mechanism for
Fe(III) reduction in P. islandicum is signi¢cantly di¡erent
from the models for electron transport to Fe(III) that have
been proposed for Shewanella and Geobacter species.
2. Materials and methods
2.1. Culture conditions and preparation of extracts
P. islandicum was cultivated anaerobically at 95³C using
a modi¢cation of DSM medium 390 containing peptone
(0.25%), yeast extract (0.02%) and thiosulfate (20 mM)
or Fe(III) citrate (20 mM) as the electron acceptor. Vitamins and trace minerals were provided from stock solutions [17]. Sodium sul¢de was replaced with L-cysteine
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 0 1 8 - 0
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(0.5 mM) and sodium tungstate (10 WM) was included.
The headspace was N2 and the ¢nal pH was 6.0. For
growth on hydrogen and Fe(III) citrate, yeast extract
was decreased (0.01%), peptone omitted, sodium bicarbonate (23 mM) was added and the headspace was H2 :CO2
(80:20). P. aerophilum was cultivated using a modi¢ed
medium [18] with peptone (0.1%), yeast extract (0.1%)
and nitrate (0.1%) or Fe(III) citrate (20 mM) as the electron acceptor. The headspace was N2 and the ¢nal pH was
6.8. Cells were harvested and washed twice with 30 mM
bicarbonate bu¡er (pH 6.8) for whole cell assays or with
50 mM PIPES (pH 6.8), 10% glycerol for preparation of
cell extracts. Cells were lysed by sonication under a stream
of N2 gas and cellular debris removed by centrifugation.
The resulting extracts were subjected to ultracentrifugation
at 100 000Ug for 1 h at either 5 or 25³C. All fractions
were stored under N2 in glass serum bottles sealed with
butyl rubber stoppers. Protein was quanti¢ed with the
Bio-Rad Protein Assay using bovine serum albumin as a
standard.
2.2. Enzyme assays
Whole cell assays (5 ml) contained 30 mM bicarbonate
bu¡er, 5 mM Fe(III) citrate, cells (0.1^0.8 mg protein) and
either 10 mM pyruvate or H2 as electron donor. For H2 dependent reduction, the headspace was £ushed 5 min and
pressurized to 101 kPa with H2 :CO2 (80:20). Tubes were
preheated at 90³C for 5^10 min and reactions were initiated by addition of cells. At times, samples were removed
to determine Fe(II) formation using ferrozine [19]. Enzymatic assays (3 ml) were carried out in sealed anaerobic
pressure tubes and contained 50 mM MES (pH 6.5 at
room temperature), 1 mM ferrozine, 1.7 mM Fe(III) citrate, extracts (0.02^0.05 mg protein) and 0.2 mM
NADPH. Inhibitors were added at the concentrations indicated. Tubes were preheated at 75³C for 10 min and
reactions initiated with the addition of NADPH. Reduction of Fe(III) to Fe(II) was measured by monitoring the
change in absorbance at 562 nm. One unit is equal to
1 Wmol of Fe(III) reduced min31 mg31 protein.
Hydrogenase activity was determined at 90³C. Assays (3
ml) were done in sealed anaerobic tubes and contained
0.05^0.1 M HEPES (pH 8.4 at room temperature), 2 mM
benzyl viologen and extracts (0.01^0.03 mg protein). The
headspace was £ushed 5 min and pressurized to 101 kPa
with H2 :CO2 (80:20). Benzyl viologen reduction was
monitored at 600 nm (O = 7400 M31 cm31 ). One unit is
equal to 2 Wmol BV reduced per Wmol of hydrogen oxidized min31 mg31 protein.
brane fractions (0.2^1 mg ml31 protein) obtained by ultracentrifugation were analyzed. For evaluation of pyridine
hemochromes, 0.5 ml of membranes was mixed with 0.5
ml of 0.1 N NaOH containing 20% pyridine. SDS^PAGE
was done with membrane and soluble fractions and gels
stained for heme.
3. Results
3.1. Characterization of Fe(III) reductase activity
Washed cell suspensions of P. islandicum that had been
grown with peptone and 20 mM Fe(III) citrate reduced
Fe(III) at 90³C (Fig. 1). The average speci¢c activity of
Fe(III) reduction with hydrogen as an electron donor was
0.08 Wmol Fe(II) formed min31 mg31 cell protein. After a
rapid initial reduction, Fe(III) reduction could be maintained at a constant rate for at least 2.5 h. Cells preincubated with cupric chloride, an inhibitor of hydrogenases
[20], could not reduce Fe(III) indicating that hydrogendependent Fe(III) reduction required a functional hydrogenase. The speci¢c activity of Fe(III) reduction with pyruvate as electron donor was initially 5-fold faster (0.38
Wmol Fe(II) formed min31 mg31 cell protein) than it was
with hydrogen, but this rate was not maintained beyond
20^30 min (not shown). When the cells were lysed using
sonication, no hydrogen-dependent Fe(III) reduction
could be detected in cell free extracts. Extracts could reduce Fe(III) when pyruvate was provided as the electron
donor which indicated that the Fe(III) reductase was still
intact after cell lysis. The extracts had hydrogenase activ-
2.3. Cytochromes
Sodium dithionite reduced minus oxidized spectra were
recorded at room temperature using an UV-2401 PC spectrophotometer (Shimadzu). Both the soluble and mem-
Fig. 1. Fe(III) reduction by whole cells. At times, 0.1 ml was removed
for determination of Fe(II). For assays containing cupric chloride, an
aliquot of cells was preincubated with 1 mM cupric chloride 20 min prior to injection into the assay tubes.
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255
Table 1
P. islandicum Fe(III) reductase and hydrogenase activities in extracts of cells grown under di¡erent culture conditions
Culture conditions
Fe(III) reductase (U mg31 )
Hydrogenase (U mg31 )
Peptone+Fe(III) citrate
Peptone+thiosulfate
H2 :CO2 +Fe(III) citrate
1.50 þ 0.6 (n = 5)
1.25 þ 0.2 (n = 2)
1.25 þ 0.03 (n = 1)
0.84 þ 0.1 (n = 3)
0.68 þ 0.1 (n = 2)
1.13 þ 0.3 (n = 2)
Assays were done in triplicate as described in Section 2. The results shown are averages of `n' di¡erent extract preparations.
ity that was sensitive to cupric chloride suggesting that the
inability to detect reduction of Fe(III) with hydrogen was
not due to inactivation of hydrogenase by the cell lysis
procedure. Further attempts to detect hydrogen-dependent
Fe(III) reduction using di¡erent extract preparations and
di¡erent bu¡er and pH conditions were unsuccessful.
Fe(III) was reduced by cell extracts when NADPH was
provided as the electron donor. However, the assay temperature was lowered from 90³C to 75³C because of high
abiotic reduction of Fe(III) by NADPH in the absence of
cell extracts at 90³C. Cell extracts exhibited no Fe(III)
reductase activity at room temperature or when NADPH
was omitted. NADPH was preferred over NADH as an
electron donor for Fe(III) reduction. The Km values for
NADPH and NADH were 0.04 (Vmax 0.071 Wmol min31 )
and 3.33 mM (Vmax 0.434 Wmol min31 ), respectively. With
NADPH as the donor, the Km for Fe(III) citrate was 0.37
mM (Vmax 0.050 Wmol min31 ). NADPH-dependent reduction of Fe(III) was stimulated 1.8^2-fold by the inclusion
of 5 WM FAD or FMN. A similar NADPH-dependent
Fe(III) reductase activity was observed in extracts of P.
aerophilum (0.7 U mg31 ), but the rates were approximately
half of those in P. islandicum (1.5 U mg31 ). Proteins of P.
islandicum were separated with PAGE under non-denaturing conditions and were stained for NADPH-dependent
Fe(III) reductase activity, but an in-gel activity similar to
the NADH-dependent Fe(III) reductase activity recovered
in PAGE gels of Geobacter sulfurreducens extracts [21]
could not be detected. Fe(III) reductase activity in extracts
was not e¡ected by a 1 h exposure to air at room temperature and was stable after storage at 4³C for up to
1 month.
The Fe(III) reductase activity recovered in cell extracts
of P. islandicum was constitutively expressed under di¡erent growth conditions. Cells grown on peptone and Fe(III)
citrate had similar activity as cells grown on peptone and
thiosulfate (Table 1). The electron donor for growth on
Fe(III) had no e¡ect on the Fe(III) reductase activity as
hydrogen-grown cells had the same activity as cells grown
on peptone. Hydrogenase activity was also constitutive
under all the growth conditions tested.
3.2. Localization and inhibition of Fe(III) reductase
When the soluble and membrane fractions of cell free
extracts were separated via ultracentrifugation, 86% of the
Fe(III) reductase activity was recovered in the soluble fraction (Table 2). In contrast, 93% of hydrogenase activity
was recovered in the membrane fraction (Table 2). The
e¡ect of potential inhibitors on Fe(III) reductase activity
was evaluated (Table 3). Sodium cyanide, a metalloenzyme
inhibitor, completely inhibited Fe(III) reductase activity.
A 50% decrease in activity was seen using the thiol inhibitor, p-chloromercuriphenylsulfonic acid (pCMBS). Fe(III)
activity was partially inhibited using 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), an inhibitor of electron
transfer between quinone and cytochrome b. Neither quinacrine nor rotenone inhibited activity at the concentration tested.
3.3. Cytochrome content
Di¡erence absorption spectra of extracts of P. islandicum and P. aerophilum were compared with the spectra
from extracts of G. sulfurreducens, a bacterium known to
contain c-type cytochromes. Cells were grown with Fe(III)
citrate and extracts of P. islandicum exhibited a small K
peak at 559 nm (Fig. 2a) while P. aerophilum extracts
showed a small peak at 552 nm (not shown) which has
previously been classi¢ed as a cytochrome bo species [22].
In comparison, extracts from G. sulfurreducens had a
much larger K peak at 552 nm (Fig. 2a). To di¡erentiate
the heme group of the P. islandicum cytochrome, membranes were treated with pyridine. P. islandicum ferro-
Table 2
Distribution of Fe(III) reductase and hydrogenase activities
Fraction
Protein (mg)
Fe(III) reductase
Hydrogenase
31
Speci¢c activity (U mg )
Cell extracts
Soluble fraction after ultracentrifugation
Particulate fraction after ultracentrifugation
4.7
3.1
1.1
0.83
1.35
0.64
Recovered (%)
Speci¢c activity (U mg31 )
Recovered (%)
86
14
2.78
0.17
6.42
7
93
The experiment was performed four times with di¡erent extract preparations from cells grown on peptone and Fe(III) citrate. The results shown are
from one representative experiment.
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Table 3
E¡ect of inhibitors on Fe(III) reductase activity in cell extracts
Inhibitor
Site of inhibition
Concentration (mM)
Inhibition (%)a
Quinacrine
£avin proteins
Rotenone
NADH dehydrogenase
Sodium cyanide
metalloenzymes
HOQNO
quinone transfer to cytochrome b
pCMBS
thiol groups
0.5
1
2.5
0.002
0.01
0.04
1
5
10
0.01
0.10
0.01
0.05
0.10
0
0
0
0
0
0
20
50
100
28
41
50
67
51
Assays were done in triplicate as described in Section 2.
Inhibition (%) determined as the decrease in the rate of activity versus a control with no inhibitor present.
a
Fig. 2. Dithionite reduced minus air oxidized spectra. a: Cell free extracts (0.25 mg ml31 protein) from P. islandicum (A) and G. sulfurreducens (B).
b: Membranes from P. islandicum (0.21 mg ml31 ) (A) and G. sulfurreducens (0.12 mg ml31 ) (B) treated with pyridine.
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257
4. Discussion
[26^28]. Furthermore an Fe(III)-reducing complex puri¢ed
from G. sulfurreducens contained a c-type cytochrome
whose absence from the complex prevented Fe(III) reduction [21].
In summary, the Fe(III) reductase activity in cell extracts of P. islandicum is signi¢cantly di¡erent than the
Fe(III) reductase activity that has been observed in extracts of dissimilatory, Fe(III)-reducing bacteria. This suggests that phylogenetically diverse Fe(III)-reducing microorganisms may have di¡erent mechanisms for Fe(III)
reduction. Thus, further investigations into this process
in P. islandicum and other Fe(III)-reducing organisms de¢cient in c-type cytochromes, such as Pelobacter carbinolicus [29], are warranted to better understand the biochemical diversity of Fe(III) reduction.
4.1. Di¡erences in Fe(III) reductase activity of
P. islandicum
References
chromes exhibited a peak at 555 nm indicative of heme b
whereas G. sulfurreducens ferrochromes exhibited a peak
at 550 nm characteristic for heme c (Fig. 2b).
Membranes of P. islandicum obtained by ultracentrifugation of cell extracts contained the majority of the cytochrome. Di¡erence spectra of the soluble portion of extracts showed no detectable cytochrome peaks.
Additionally, SDS^PAGE of the soluble and membrane
fractions was performed and proteins were stained for
heme. Only the membrane fraction contained a protein
that stained positive for heme con¢rming the absence of
cytochromes in the soluble fraction.
Several features of the P. islandicum Fe(III) reductase
are di¡erent from those reported in Fe(III)-reducing bacteria. A preference for NADPH as an electron donor to
Fe(III) reduction was found in P. islandicum. The other
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of Fe(III), however their role in vivo is currently unde¢ned
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