APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2006, p. 6980–6985
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 11
c-Type Cytochromes in Pelobacter carbinolicus?
Shelley A. Haveman,* Dawn E. Holmes, Yan-Huai R. Ding, Joy E. Ward,
Raymond J. DiDonato, Jr., and Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003
Received 15 May 2006/Accepted 14 August 2006
Previous studies failed to detect c-type cytochromes in Pelobacter species despite the fact that other close relatives
in the Geobacteraceae, such as Geobacter and Desulfuromonas species, have abundant c-type cytochromes. Analysis
of the recently completed genome sequence of Pelobacter carbinolicus revealed 14 open reading frames that could
encode c-type cytochromes. Transcripts for all but one of these open reading frames were detected in acetoin-
fermenting and/or Fe(III)-reducing cells. Three putative c-type cytochrome genes were expressed specifically during
Fe(III) reduction, suggesting that the encoded proteins may participate in electron transfer to Fe(III). One of these
proteins was a periplasmic triheme cytochrome with a high level of similarity to PpcA, which has a role in Fe(III)
reduction in Geobacter sulfurreducens. Genes for heme biosynthesis and system II cytochrome c biogenesis were
identified in the genome and shown to be expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels
of protein extracted from acetoin-fermenting P. carbinolicus cells contained three heme-staining bands which were
the predicted amount of heme c per protein, and the ratio of heme-stained protein to total protein were much
have indicated are required for optimal Fe(III) reduction in G. sulfurreducens were not present in the P. carbinolicus
genome. These results suggest that further evaluation of the functions of c-type cytochromes in the Geobacteraceae
Pelobacter species seem to be an anomaly within the family
Geobacteraceae. They are phylogenetically intertwined with
Geobacter and Desulfuromonas species and have the capacity
to use Fe(III) as an electron acceptor (29, 34), yet they were
previously found to lack c-type cytochromes (34, 43–47), which
are abundant in Geobacter and Desulfuromonas species and are
thought to be important in electron transfer to Fe(III) in these
organisms (8, 25, 28, 30, 35, 41). This has led to questions about
the evolution of the different genera within the Geobacteraceae
and about the true role of c-type cytochromes in Fe(III) re-
duction in this family. In fact, the apparent lack of c-type
cytochromes in Pelobacter but conservation of the structural
gene for electrically conductive pilin is one line of evidence
suggesting that pili serve as the electrical conduit between the
outer surface of Geobacteraceae cells and Fe(III) oxides (40).
Pelobacter species are common in anaerobic subsurface en-
vironments (16, 22, 37, 50, 52). Pelobacter carbinolicus, which
grows by fermentation of butanediol, acetoin, and ethylene
glycol to ethanol and acetate, was isolated from marine mud
(43). P. carbinolicus can also grow by oxidizing ethanol and
other alcohols (i) in coculture with H2-oxidizing methanogens
or acetogens (43) or (ii) with Fe(III) or Soas an electron
acceptor (34). However, these organic electron donors are only
incompletely oxidized to acetate, in contrast to the ability of
Geobacter and Desulfuromonas species to completely oxidize
acetate and other organic electron donors to carbon dioxide
(32). Yet P. carbinolicus appears to be capable of conserving
energy from electron transfer to Fe(III) because it can grow via
Fe(III) reduction with H2as the electron donor (34), and the
cell yields per unit of Fe(III) reduced with both organic elec-
tron donors and H2are equivalent to those for Geobacter
species (E. S. Shelobolina, unpublished data).
Analysis of the recently completed genome sequence of P.
carbinolicus DSM2380 (www.jgi.doe.gov) led to the surprising
finding that this organism contains genes predicted to encode
c-type cytochromes, as well as genes for heme biosynthesis and
cytochrome c biogenesis. Here we report that most of these
c-type cytochrome genes are expressed under one or more
growth conditions and that low levels of c-type cytochromes
can be detected biochemically.
MATERIALS AND METHODS
Bioinformatics. The predicted protein-encoding sequences in the P. carbinolicus
genome sequence (www.jgi.doe.gov) were searched for CXXCH heme c binding
Package, version 10.3 (Accelrys Inc., San Diego, CA). The subcellular location of the
CXXCH-containing putative proteins was predicted using several programs, includ-
ing PSORTb (12), SubLoc (19), TMPred (15), and SignalP (5). Conserved LXXC
lipoprotein binding motifs in signal sequences were used to predict outer membrane
cytochromes (14). Proteins that were predicted to be cytoplasmic membrane asso-
ciated, periplasmic, or outer membrane associated were further analyzed by
BLASTP (2) to predict the function and location based on similarity to other
proteins, with a cutoff E value of ?10?5. The predictions were based solely on
sequence analysis and were not confirmed experimentally. Proteins predicted to
catalyze heme biosynthesis and cytochrome c biogenesis were identified on the basis
of similarity to such proteins in other bacteria (4, 23, 26).
Media and culture conditions. P. carbinolicus DSM2380 was cultured at 30°C
under strictly anaerobic conditions in media containing (per liter) 9.0 g of NaCl,
2.7 g of MgCl2· 6H2O, 2.5 g of NaHCO3, 0.25 g of NH4Cl, 0.6 g of NaH2PO4·
H2O, 0.1g of KCl, and 0.14 g of CaCl2· 2H2O. Vitamins and minerals (10 ml
liter?1each) were added from stock solutions (31). Media were dispensed into
anaerobic pressure tubes or bottles, and the tubes or bottles were gassed with
80% N2–20% CO2, sealed with butyl rubber stoppers, and autoclaved. Media
were reduced with sterile Na2S at a final concentration of 0.02 mM. Electron
* Corresponding author. Mailing address: Department of Microbi-
ology, University of Massachusetts, Amherst, MA 01003. Phone: (413)
577-0217. Fax: (413) 545-1578. E-mail: email@example.com.
?Published ahead of print on 25 August 2006.
donors were added from sterile, anaerobic stock solutions at a final concentra-
tion of 10 mM (acetoin [3-hydroxy-2-butanone]) or 2 mM (ethanol). Fe(III) was
provided in ethanol-containing cultures in the form of Fe(III) nitrilotriacetic acid
(NTA) at a final concentration of 5 mM (41). Due to the salinity of the medium,
much of the Fe(III) was insoluble. Cell growth on acetoin was monitored by
measuring the optical density at 600 nm with a Genesys 2 spectrophotometer
(Spectronic Instruments, Rochester, NY). Fe(II) production in Fe(III) NTA
cultures was monitored with ferrozine (33).
Nucleic acid extraction. Cultures used for DNA or RNA extraction were
grown to mid-log phase, transferred to 50-ml conical tubes, and centrifuged at
3,150 ? g for 20 min at 4°C. Cell pellets used for RNA extraction were frozen in
liquid nitrogen and stored at ?80°C. Chromosomal DNA was extracted from
acetoin-grown P. carbinolicus cells using a FastDNA Spin kit for soil (QBiogene,
Irvine, CA) or a MasterPure DNA purification kit (EPICENTRE Biotechnolo-
gies, Madison, WI). Total RNA was isolated using protocols described previously
(17, 18). For acetoin-grown cells, cell pellets from 50 ml of culture were resus-
pended in 4 ml of Tris-EDTA-sucrose buffer (18), and RNA was extracted by the
protocol used for RNA extraction from the surfaces of current-harvesting elec-
trodes (18). For Fe(III) NTA-ethanol-grown cells, cell pellets from 50 ml of
culture were resuspended in 8 ml TPE buffer (17), and RNA was extracted by
using the protocol for RNA extraction from sediments (17), except that yeast
tRNA was omitted. To confirm that RNA was not contaminated with DNA,
PCRs were performed using RNA as the template.
PCR. Specific primers were designed for the genes for the 14 putative c-type
cytochromes, as well as for cytochrome c biogenesis genes and selected heme
biosynthesis genes (Table 1). Each 50-?l PCR mixture contained 10 ?l of 5? Q
solution (QIAGEN), 1.5 mM MgCl2, 5 ?l of 10? PCR buffer (QIAGEN), each
deoxynucleoside triphosphate (Sigma) at a concentration of 0.02 mM, 50 pmol of
each primer, 0.2 mg/ml bovine serum albumin (New England BioLabs), 1 U of
Taq DNA polymerase (QIAGEN), and 50 ng of chromosomal DNA. PCR
amplification was carried out with a PTC-200 thermal cycler (MJ Research,
Waltham, MA) as follows: denaturation at 95°C for 5 min, followed by 35 cycles of
at 72°C for 10 min. The annealing temperature used for each primer set is shown in
Table 1. PCR products were purified from agarose gels with a QIAquick gel extrac-
tion kit (QIAGEN) and were cloned with a TOPO TA cloning kit, version R
(Invitrogen). Plasmids were extracted from 8 to 12 colonies per PCR and sequenced
with the M13F primer to confirm that the correct gene was amplified.
RT-PCR. Reverse transcription (RT) was performed with an Enhanced Avian
First Strand synthesis kit (Sigma) as described previously (17). Two negative
controls lacking reverse transcriptase or RNA were included for each gene.
PCRs were performed as described above, using 5 ?l of the RT reaction mixture
as the template for PCR. As described above for PCR products, RT-PCR
products were gel purified, TOPO cloned, and sequenced.
Proteomics. Late-log-phase acetoin-grown P. carbinolicus cells or fumarate-
acetate-grown Geobacter sulfurreducens cells were harvested by centrifugation at
3,150 ? g for 20 min at 4°C, and the cell pellets were stored at ?20°C. Cells were
washed in 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM MgCl2and Com-
plete protease inhibitor cocktail (Roche). Washed cells were suspended in the
same buffer at a concentration of 0.2 g (wet weight) of cells per ml of buffer. The
cells were lysed by sonication at 4°C. The cell debris was removed by centrifu-
gation at 9,000 ? g for 15 min, and the supernatant contained whole-cell protein.
Ten micrograms of protein from each organism was separated on a sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and c-type
cytochromes were heme stained as described previously (11, 51).
Heme-stained protein bands were excised from the gel and washed with 500 ?l
of MilliQ water for 30 min at 20°C to remove the chemical residue. The gel
pieces were dehydrated in 200 ?l of 50 mM ammonium bicarbonate in 50%
acetonitrile for 1 h, followed by acetonitrile for 30 min. Each gel piece was
digested in 20 to 40 ?l digestion buffer (20 mM ammonium bicarbonate con-
taining 75 ng trypsin) at 37°C overnight. The supernatant was recovered, and the
remaining peptides were extracted from the gel piece by washing it with 80%
acetonitrile–1% formic acid in water. The extracts were pooled, and the volume
was reduced to 5 to 10 ?l with a Speedvac (Vacufuge, Germany). The eluted
tryptic peptides were desalted and concentrated with a commercial ZipTip C18
pipette tip (Millipore). Peptides were detected by matrix-assisted laser desorp-
tion ionization–time of flight mass spectrometry as previously described (20).
Peptide mass fingerprints were analyzed by using the MS-FIT Protein Prospector
program (UCSF Mass Spectrometry Facility).
RESULTS AND DISCUSSION
c-Type cytochrome genes. The P. carbinolicus genome se-
quence contains 58 CXXCH motifs in 42 predicted proteins.
Individual predicted proteins contain between one and five
TABLE 1. Primers used for RT-PCR analysis of P. carbinolicus cytochromes and cytochrome c biosynthesis genes
GeneDescriptionForward primer (5?–3?) Reverse primer (5?–3?)
Glutamate synthase, large subunit
Cytochrome c family protein
caa3-type cytochrome c oxidase,
Cytochrome c family protein
Cytochrome c nitrite reductase, nrfH
Cytochrome c nitrite reductase nrfA
Cytochrome c7, ppcA
Cytochrome c family protein
Glutamyl tRNA synthetase, hemA
Heme transport and ligation, ccsB
Heme transport and ligation, ccsA
VOL. 72, 2006c-TYPE CYTOCHROMES IN P. CARBINOLICUS6981
CXXCH motifs. Twenty-eight of the putative c-type cyto-
chromes are predicted to be cytoplasmic and therefore not
expected to bind heme c. Of the remaining 14 protein se-
quences, 4 are predicted to be associated with the cytoplasmic
membrane, 6 are predicted to be periplasmic, and the other 4
may be outer membrane associated (Table 2).
One gene (PCAR1628) is predicted to encode a cytochrome
c7that belongs to a family of well-conserved cytochromes in
the Geobacteraceae, which includes cytochrome c7in Desulfu-
romonas acetoxidans (3, 6) and Geobacter metallireducens (1)
and PpcA in G. sulfurreducens (28). In G. sulfurreducens this
cytochrome appears to function as a periplasmic intermediary
electron carrier between cytoplasmic electron donors and
outer membrane-associated Fe(III) reductase (28).
PCAR2984 encodes a putative outer membrane diheme cy-
tochrome c and is conserved in Geobacteraceae genomes up-
stream of a gene for pyruvate kinase. A mutant lacking the
homolog in G. sulfurreducens (GSU3332) was incapable of
reducing poorly crystalline Fe(III) oxides and was deficient in
reduction of U(VI), suggesting that this cytochrome may play
a role in electron transfer to extracellular electron acceptors
(E. S. Shelobolina, unpublished data).
PCAR2867 and PCAR2866 encode proteins that are homol-
ogous to the two subunits of cytochrome c nitrite reductase,
which catalyze nitrite reduction to ammonia in many bacteria
(48) and are conserved in the Geobacteraceae. In P. carbinoli-
cus PCAR2866 the unique CXXCK heme binding motif (48) is
replaced by a CXXCH motif. The role of cytochrome c nitrite
reductase in P. carbinolicus, which is not known to use nitrite as
an electron acceptor, could be similar to that in Desulfovibrio
vulgaris, in which nitrite reduction is not coupled to growth and
cytochrome c nitrite reductase is used for nitrite detoxification
The periplasmic pentaheme cytochrome c encoded by
PCAR2550 is part of a conserved operon (PCAR2550 to
PCAR2553) that is predicted to encode a cytoplasmic mem-
brane-bound quinol:cytochrome c oxidoreductase. A homolo-
gous complex was purified from Chloroflexus aurantiacus, and
homologous operons are found in individual members of seven
bacterial phyla, suggesting that the complex has been laterally
transferred (53). A similar but distinct complex has been found
in the genome sequences of G. metallireducens, D. vulgaris, and
Desulfovibrio desulfuricans but not in the G. sulfurreducens
sequence (53) or in the draft sequence of Desulfuromonas
acetoxidans (www.jgi.doe.gov). The gene encoding cytoplasmic
membrane-bound monoheme cytochrome c, PCAR2549, is im-
mediately upstream of PCAR2550, but this cytochrome has no
significant similarity to other proteins.
TABLE 2. Predicted c-type cytochromes in P. carbinolicus and mRNA expression determined by RT-PCR
Glutamate synthase, large subunit
Cytochrome c family protein
In operon with another
In operon with
PCAR2549 Hypothetical protein1 PCAR2550 to
PCAR2529 caa3-type cytochrome c oxidase,
1 PCAR2526 to
Cytochrome c family protein
Cytochrome c nitrite reductase, nrfH
Electron transfer from
PCAR2866Cytochrome c nitrite reductase, nrfA5 PCAR2867
PCAR2550 Cytochrome c5PCAR2549 to
PCAR1628 Cytochrome c7, ppcA
PCAR0152Hypothetical protein 1PCAR0153 to
PCAR2984Cytochrome c family protein2PCAR2983
Protein disulfide isomerase
6982 HAVEMAN ET AL.APPL. ENVIRON. MICROBIOL.
PCAR2529 encodes subunit II of a caa3-type cytochrome c
oxidase (encoded by PCAR2526 to PCAR2529), which is ho-
mologous to the oxidases in other members of the Geobacter-
aceae, as well as the characterized oxidase in Rhodothermus
marinus (42). G. sulfurreducens has a cytochrome c oxidase (36)
and has been shown to grow with low levels of oxygen as a
terminal electron acceptor (27).
PCAR0152 encodes a putative outer membrane cytochrome
with no significant similarity to other proteins. This gene is
located between the genes for an ABC-type transporter with
homology to the Escherichia coli Fep ferric enterobactin trans-
porter (7) and therefore may play a role in the uptake of
The functions of the remaining six putative cytochromes
cannot be predicted by sequence homology, either because
they have no significant BLASTP hits or because they are
similar to proteins that are not known to bind heme c. These
cytochromes include two that are predicted to be cytoplasmic
membrane bound, the cytochrome encoded by PCAR2570 and
the glutamate synthase homolog encoded by PCAR2944; two
periplasmic cytochromes, one encoded by PCAR0558 and one
encoded by PCAR0192, the latter of which is homologous to
molybdopterin oxidoreductase in the N terminus and pre-
corrin-4 methylase in the C terminus; and two that are pre-
dicted to be outer membrane bound, the triheme cytochrome
encoded by PCAR2745 and the protein disulfide isomerase
encoded by PCAR2069.
Heme biosynthesis and cytochrome c biogenesis genes. In
order for the putative cytochromes to be functional, heme c
must be covalently bound to the proteins in the periplasm.
Formation of c-type cytochromes requires heme biosynthesis,
transport of heme and apoprotein to the periplasm, and cova-
lent attachment of heme to CXXCH motifs. P. carbinolicus
possesses all of the genes required for heme biosynthesis in
four regions of the genome (Table 3). Consistent with its an-
aerobic physiology, P. carbinolicus contains a homolog of the
oxygen-independent protoporphyrinogen oxidase HemG (en-
coded by PCAR0772), which catalyzes the penultimate step in
heme biosynthesis, but not HemY, which catalyzes the same
reaction in an oxygen-dependent manner (10). Likewise, it
contains a homolog of the oxygen-independent coproporphy-
rinogen III oxidase HemN (encoded by PCAR0110), but it
lacks the oxygen-dependent form, HemF (10). hemACD are
located in an operon with genes encoding phosphoheptose
isomerase, siroheme synthase, and a cytochrome biogenesis
protein homolog (PCAR3062 to PCAR3067). hemB is located
downstream of this operon in a dicistronic operon containing a
gene encoding a conserved hypothetical protein (PCAR3060
The steps after heme biosynthesis are collectively referred to
as the cytochrome c biogenesis pathway. There are three dif-
ferent systems for cytochrome c biogenesis, called systems I, II,
and III (24, 38). System II is found in gram-positive bacteria, ?-
and ε-Proteobacteria, and chloroplasts (23, 24). P. carbinolicus,
along with other members of the Geobacteraceae, has genes for
the system II cytochrome c biogenesis pathway (49). The four
genes fall into two operons (Table 3) encoding four integral
membrane proteins. PCAR2228 and PCAR2229 encode CcsA
and CcsB homologs, which transport heme to the periplasm
and may catalyze the covalent linkage of heme to apocyto-
chrome c (24). ResA (encoded by PCAR1954) is a thioredoxin
that reduces the cysteines of the apocytochrome c so that heme
can be attached, and CcdA (encoded by PCAR1953) rereduces
mRNA expression. Most of the putative c-type cytochrome
genes were expressed during fermentation and/or during
Fe(III) reduction; the only exception was PCAR2570 (Table
2). PCAR0152, encoding the cytochrome associated with genes
for the Fe3?-siderophore ABC transporter, was expressed dur-
ing fermentation but not during Fe(III) reduction, supporting
the hypothesis that this cytochrome has a role in iron uptake
during iron limitation. Three cytochrome genes were expressed
during Fe(III) reduction but not during fermentation, includ-
ing PCAR1628 encoding the periplasmic cytochrome c7and
PCAR2550 encoding the periplasmic pentaheme cytochrome
in the quinol:cytochrome c oxidoreductase. Together, these
two cytochromes could transport electrons from the quinone
pool to the outer membrane. The gene encoding a cytoplasmic
TABLE 3. Predicted P. carbinolicus heme biosynthesis and cytochrome c biogenesis genes and mRNA expression determined by RT-PCR
Glutamyl tRNA synthase
Glutamate 1-semialdehyde aminotransferase
Cytochrome c biogenesis
Heme transport and ligation
Heme transport and ligation
Thioredoxin for reduction of CXXCH cysteines
Thioredoxin for reduction of ResA
aND, not determined.
VOL. 72, 2006c-TYPE CYTOCHROMES IN P. CARBINOLICUS 6983
membrane-bound cytochrome related to glutamate synthase
was also expressed only during Fe(III) reduction, but the role
of this cytochrome is not clear.
Expression of selected genes in the heme biosynthesis path-
way was determined by RT-PCR. These genes included the
genes encoding the enzymes for first two steps in the pathway
(hemA and hemL) and the final step (hemH), all of which were
expressed during fermentation (Table 3). A ccsA homolog in
the same operon as hemA was also expressed. The four cyto-
chrome c biogenesis genes were expressed as well (Table 3).
Therefore, all of the genes necessary for heme biosynthesis and
cytochrome c biogenesis are present and expressed in P.
carbinolicus, which allows functional c-type cytochromes to be
Protein expression. Previous attempts to detect cytochromes
in P. carbinolicus by difference spectroscopy of crude cell ex-
tracts and membrane fractions of fermentatively grown cells
(43) or of intact washed cells (34) were unsuccessful. Likewise,
no cytochromes were detected by difference spectroscopy of
fermentatively grown cells, but when whole-cell protein from
these cells was electrophoresed on SDS-PAGE gels and heme
stained, three bands were detected (Fig. 1). The same amount
of protein from G. sulfurreducens produced many more bands
and contained much more heme-containing protein (Fig. 1).
Proteins in the three heme-stained P. carbinolicus bands were
identified by matrix-assisted laser desorption ionization–time
of flight mass spectrometry, and the results were in agreement
with the RT-PCR results. The following three identified cyto-
chromes were all predicted to be soluble: cytochrome c nitrite
reductase encoded by PCAR2866, molybdopterin oxidoreduc-
tase/precorrin-4 methylase encoded by PCAR0192, and the
cytochrome c family protein encoded by PCAR0558.
Implications for Pelobacter physiology. Our results demon-
strate for the first time that P. carbinolicus contains c-type
cytochromes. As noted above, the functions of some of these
cytochromes can be inferred from homology with cytochrome
genes encoding known functions in other organisms. However,
definitive elucidation of the functions of the genes in P. carbi-
nolicus with genetic approaches has not been possible yet
because techniques for generating specific mutations via ho-
mologous recombination that have been successful in G.
sulfurreducens (8, 9, 20, 25, 28, 35) have not worked well in P.
The detection of transcripts for three c-type cytochrome
genes during growth on Fe(III) but not under fermentative
conditions suggests that the cytochromes may be specifically
involved in Fe(III) reduction. It is notable that one of these
differentially expressed cytochrome genes encodes a triheme,
periplasmic cytochrome that is highly conserved in the
Geobacteraceae and is essential for optimal Fe(III) reduction
in G. sulfurreducens (28). However, the triheme cytochrome is
much less abundant in P. carbinolicus than in G. sulfurreducens,
and G. sulfurreducens contains five homologs of this protein
(36) compared to just one homolog in P. carbinolicus. Further-
more, P. carbinolicus lacks genes for many c-type cytochromes
that have been found to be required for optimal Fe(III) re-
duction in G. sulfurreducens. These cytochromes include the
inner membrane cytochrome MacA (8) and the outer mem-
brane cytochromes OmcB (25), OmcS (35), and OmcE (35)
that are thought to be involved in electron transfer to Fe(III).
Also missing are OmcF, OmcG, and OmcH, which are outer
membrane cytochromes that may play a regulatory role during
Fe(III) reduction (20, 21). Not only is the overall number of
c-type cytochrome genes in P. carbinolicus much lower than
that in G. sulfurreducens, 14 versus 111 (36), but also the
number of hemes in P. carbinolicus cytochromes, 5 or less, is
generally lower than the number found in many G. sulfurredu-
cens cytochromes, which can have as many as 27 hemes.
The differences between the c-type cytochrome contents of
P. carbinolicus and G. sulfurreducens could conceivably be
linked to factors related to metabolism of acetate, which G.
sulfurreducens can use as a sole electron donor for Fe(III)
reduction but P. carbinolicus cannot use (32). Alternatively,
they could be related to differences in the environmental con-
ditions in the preferred habitats of the organisms. However,
further studies to definitively determine the functions of the
c-type cytochromes in both organisms are necessary before
substantive conclusions can be made.
This research was supported by grant DE-FC02-02ER63446 from
the Genomics: GTL Program of the Office of Science (BER), U.S.
Department of Energy.
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