Differential expression of two bc1complexes in the
strict acidophilic chemolithoautotrophic bacterium
Acidithiobacillus ferrooxidans suggests a model
for their respective roles in iron or sulfur oxidation
Patrice Bruscella,13 Corinne Appia-Ayme,1Gloria Levica ´n,2
Jeanine Ratouchniak,1Eugenia Jedlicki,2David S. Holmes3
and Violaine Bonnefoy1
1CNRS, Institut de Biologie Structurale et de Microbiologie, Laboratoire de Chimie Bacte ´rienne,
31 chemin Joseph Aiguier, 13402, Marseille Cedex 20, France
2Instituto de Ciencias BioMe ´dicas, Faculty of Medicine, University of Chile, Santiago, Chile
3Andres Bello University, Center for Bioinformatics and Genome Biology, Fundacio ´n Ciencias
para la Vida and Millennium Institute for Fundamental and Applied Biology, Santiago, Chile
Received 13 July 2006
6 October 2006
Accepted 9 October 2006
Three strains of the strict acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans,
including the type strain ATCC 23270, contain a petIIABC gene cluster that encodes the three
proteins, cytochrome c1, cytochrome b and a Rieske protein, that constitute a bc1electron-transfer
complex. RT-PCR and Northern blotting show that the petIIABC cluster is co-transcribed with
cycA, encoding a cytochrome c belonging to the c4family, sdrA, encoding a putative short-chain
dehydrogenase, and hip, encoding a high potential iron–sulfur protein, suggesting that the six
genes constitute anoperon, termedthepetIIoperon. Previousresults indicatedthatA.ferrooxidans
organized except that it lacks hip. Real-time PCR and Northern blot experiments demonstrate that
petI is transcribed mainly in cells grown in medium containing iron, whereas petII is transcribed
in cells grown in media containing sulfur or iron. Primer extension experiments revealed possible
transcription initiation sites for the petI and petII operons. A model is presented in which petI
is proposed to encode the bc1complex, functioning in the uphill flow of electrons from iron to
NAD(P), whereas petII is suggested to be involved in electron transfer from sulfur (or formate) to
oxygen (or ferric iron). A. ferrooxidans is the only organism, to date, to exhibit two functional bc1
The cytochrome bc1complex is a central component of
the energy transduction of the biosphere and is involved in
almost all respiratory (aerobic as well as anaerobic) and
photosynthetic electron-transfer chains (Hunte et al., 2003
and references therein). In prokaryotes, the bc1complex,
while common, is not universal. The complex is composed
of three subunits, a cytochrome c1, a cytochrome b
and a Rieske iron–sulfur protein, that transfer electrons
from a membrane-localized quinone to a small soluble
redox protein such as cytochrome c, plastocyanin, or a
high-potential iron–sulfur protein (HiPIP) (Menin et al.,
1998; Hunte et al., 2003 and references therein).
Reverse electron flow from cytochrome c through the
cytochrome bc1complex to quinone has also been reported
in a variety of chemolithoautotrophic organisms. During
autotrophic growth of these bacteria, the energetic substrate
has to provide electrons for reduction of NAD(P) to
NAD(P)H, which is required for CO2fixation and other
anabolic processes. When the midpoint potential of the
electron donor is more positive than that of the NAD(P)/
NAD(P)H couple, the reduction of NAD(P) requires
energy. Depending on the level at which the electrons
enter the respiratory chain, they have to be transported
cytochrome bc1 complex, the quinone pool and the
NAD(P)H dehydrogenase (Griesbeck et al., 2000). This
uphill (reverse) electron transport uses the proton-motive
3Present address: Department of Microbiology and Molecular Genetics,
University of Texas Medical School, 6431 Fannin St, Houston, TX
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are given in the text.
1022006/000067 G 2007 SGMPrinted in Great Britain
Microbiology (2007), 153, 102–110
force generated by hydrolysis of ATP derived from electron
In the strictly acidophilic chemolithoautotrophic Gram-
negative bacterium Acidithiobacillus ferrooxidans, a cyto-
chromebc1complexwasshownby spectroscopic techniques
to function in reverse in ferrous-iron-grown cells, even in
(downhill) direction in sulfur-grown cells (Brasseur et al.,
2002, 2004). This raised questions regarding the mechanism
regulating the flow of electrons either uphill or downhill in
the same complex. A candidate operon, termed petI, was
identified in A. ferrooxidans ATCC 33020 and ATCC 19859
(Levica ´n et al., 2002) that could potentially encode a bc1
complex, and a second distinct operon, termed petII, was
experimentally validated in A. ferrooxidans ATCC 33020
(Bruscella et al., 2005) and bioinformatically detected in the
type strain A. ferrooxidans ATCC 23270 (Brasseur et al.,
2002), raising the possibility that one of the two distinct bc1
complexes might be involved in the uphill flow of electrons
and the other in the downhill flow (Brasseur et al., 2004).
The aim of the research described in this paper was to
deepen our understanding of the petII operon and to study
the expression of both the petI and petII operons in response
to ferrous iron and sulfur in order to gain further insight
into the respiratory chains in which these two cytochrome
bc1complexes areinvolved andhowtheir synthesis might be
Bacterial strains, plasmids and growth conditions. Acidi-
thiobacillus ferrooxidans ATCC 33020, ATCC 19859 and ATCC
23270 were obtained from the American Type Culture Collection. A.
ferrooxidans was grown at 30uC under oxic conditions in ferrous
iron or sulfur medium as described previously (Yarza ´bal et al.,
Escherichia coli strain TG1 (supE hsdD5 thi D(lac-proAB) F9: traD36
proAB lacIqlacZDM15) was used for phagemid propagation and was
grown on LB medium (Ausubel et al., 1992). The phagemid SK+
Bluescript was purchased from Stratagene.
Analytical methods. The ferrous iron concentrations were deter-
mined by the o-phenanthroline method (Muir & Anderson, 1977).
DNA manipulations. General DNA manipulations were performed
according to Ausubel et al. (1992). Before manipulations, A. ferrooxi-
dans cells were washed several times in basal salt solution correspond-
ing to the medium in which they were grown, in order to remove
ferrous iron precipitates or sulfur aggregates. Genomic DNA from A.
ferrooxidans was prepared using the NucleoSpin Tissue kit (Macherey-
Nagel), according to the manufacturer’s instructions for bacterial
DNA extraction. Taq polymerase purchased from Eppendorf was used
for PCR. The oligonucleotides were obtained from Sigma-Genosys
Corporation. The nucleotide sequences of the cloned fragments were
determined from both strands by GENOME Express.
Plasmid construction. In order to synthesize the hip and cycA1
RNA probes used for the Northern blot experiments, an internal
fragment of each gene was amplified by PCR (Table 1) and cloned
into the EcoRV restriction site of the SK+Bluescript vector, between
the T7 and T3 promoters.
RNA manipulations. Total RNA was extracted from 500 ml fer-
rous iron- or sulfur-cultures at different stages of growth (early
exponential, mid exponential, late exponential or stationary phase)
with the High Pure RNA isolation kit (Roche) as described pre-
viously (Guiliani et al., 1997). For RT-PCR and real-time PCR, total
RNA was treated twice with DNase I (Roche) and DNA contamina-
tion was checked by PCR.
Northern blotting. Formaldehyde gels were used for Northern
blotting, as described by Ausubel et al. (1992). RNA was transferred
by capillary action to positively charged nylon membranes pur-
chased from Roche. RNA was UV cross-linked to the membrane
with the Stratalinker from Stratagene. DIG-labelled hip and cycA1
RNA probes were obtained by in vitro transcription performed on
SK-hip or SK-cycA1 plasmids, linearized with EcoRI or HindIII
restriction enzymes, using T7 or T3 RNA polymerase and DIG-UTP
from the Strip-EZ kit (Ambion). Prehybridization and hybridization
steps were performed under high-stringency conditions with the
DIG-labelled hip and cycA1 RNA probes. Detection was performed
by chemiluminescense with CSPD (Roche).
Reverse transcriptase-PCR (RT-PCR). Coupled RT-PCR experi-
ments were performed with the Promega Access RT-PCR system.
RT-PCR was carried out in two steps: (i) the reverse transcription
was done on approximately 1 mg total RNA (DNA free) extracted
Omniscript RT kit purchased from Qiagen; (ii) routine PCR ampli-
fication, with the oligonucleotides of interest (Table 1), was done
using the cDNA obtained as matrix, as described above. For each
RT-PCR experiment, three controls were used: one without template
to detect potential contamination, one with genomic DNA as a posi-
tive control for PCR amplification and one with RNA not treated
with reverse transcriptase to check for DNA contamination during
of A.ferrooxidans, withthe
Real-time PCR. The rrs gene encoding the 16S rRNA has been
shown to be expressed at the same (constitutive) level under both
conditions of growth examined (on ferrous iron and sulfur media)
(Yarza ´bal et al., 2004) and was used as a reference standard. Equal
amounts of total RNA, extracted from ferrous iron- and sulfur-
grown cells at different stages of growth, were retrotranscribed with
the Superscript II reverse transcriptase (InVitro Life Technologies)
at 42uC for 50 min, followed by 15 min at 70uC to inactivate the
enzyme. Real-time PCR quantification was performed on the total
cDNA obtained,using theLightCycler
LightCycler Fast Start DNA master (plus) SYBR Green I kit, with
external standards, as described in Roche Molecular Biochemichals
technical note no. LC 11/2000 and Yarza ´bal et al. (2004). Real-time
PCR experiments were performed several times, using RNA samples
from at least two independent cultures. The sequences of the oligo-
nucleotide primers are given in Table 1.
Superscript II reverse transcriptase (InVitro Life Technologies) as
follows: extension at 42uC or 50uC for 50 min, followed by heating
at 70uC for 15 min to inactivate the enzyme. The oligonucleotides
used (Table 1) were [32P]ATP-labelled with T4 polynucleotide kinase
from Biolabs. The experiments were done in duplicate, using RNA
samples from independent cultures.
Primer extensionwas performed with
Nucleotide sequence accession numbers. The GenBank/EMBL
database accession numbers, for the petI operon sequences from A.
ferrooxidans ATCC 33020 are: AJ438314 (59 untranslated region
upstream from cycA1), AM261982 (cycA1 internal region), AJ318502
(intergenic region cycA1–sdrA1), AM261983 (sdrA1 internal region),
AJ318503 (intergenic region sdrA1–petA1), AJ318504 (petA1 internal
region), AJ318505 (intergenic region petA1–petB1), AJ318506 (petB1
internal region),AJ413191 (intergenicregionpetB1–petC1),
Two bc1operons in Acidithiobacillus ferrooxidans
AJ413192 (petC1 internal region), AJ413171 (intergenic region
petC1–resB), AJ413193 (resB internal region) and AJ413194 (inter-
genic region resB–resC).
The GenBank/EMBL database accession numbers for the petII operon
sequences from A. ferrooxidans ATCC 33020 are: AJ427631 (59
untranslated region upstream from cycA2), AM261984 (cycA2 internal
region), AJ318500 (intergenic region cycA2–sdrA2), AM261984 (sdrA2
internal region), AJ311888 (intergenic region sdrA2–petA2), AJ413195
(petA2 internal region), AJ311889 (intergenic region petA2–petB2),
AJ318501 (petB2 internal region), AJ413196 (intergenic region petB2–
petC2) and AJ320262 (petC2–hip region).
were detected using an HMM model trained on A. ferrooxidans
sigma-70-like promoters (M. Santa Ana, J. Valdes, M. Chacon, T.
techniques. Potential sigma-70-like promoters
Table 1. Oligonucleotides used in this study
Primer (names used in Figs 1 and 2)Sequence Gene
Expression plasmid construction
(for in vitro transcription for RNA probes)
II Ext RC4(26)
PCR, RT-PCR and real-time PCR*
petII 59 untranslated region
petI 59 untranslated region
*The superscript letters indicate the use(s) of the primer: a, PCR; b, RT-PCR; c, real-time PCR.
104 Microbiology 153
P. Bruscella and others
L’Heureaux, E. Jedlicki, & D. S. Holmes, unpublished results).
Potential transcription factor binding sites were searched for using
MAT inspector (Cartharius et al., 2005) and information theory
(Schneider, 1999). Potential rho-independent translational stop sites
were detected according to de Hoon et al. (2005).
Genetic organization and conservation of the
petII operon in multiple strains of A.
of the type strain A. ferrooxidans ATCC 23270 (http://
www.tigr.org/), DNA primers were designed for each gene
and intergenic region of the petII operon. The sizes of the
resulting amplified fragments suggest that the organization
of the petII operon previously described in the type strain of
A. ferrooxidans ATCC 23270 (Brasseur et al., 2002) is
conserved in A.ferrooxidans ATCC33020(Fig. 1a,b) andA.
ferrooxidans ATCC 19859 (data not shown). The sequences
of the PCR fragments obtained from the ATCC 33020 strain
indicate that, as in ATCC 23270, there are two pet loci in
ATCC 33020 and ATCC 19859.
The petII gene cluster constitutes an operon
RT-PCR experiments were performed with convergent
oligonucleotide primers designed from two adjacent genes
of the ATCC 23270 petII locus (see Table 1 and Fig. 1) on
total RNA extracted from ATCC 33020 grown with ferrous
iron (Fig. 1c). RT-PCR products of the expected size were
generated between each gene pair of the petII cluster,
suggesting that it is an operon.
A candidate transcriptional start site of the petII operon was
detected by reverse-transcriptase-mediated primer exten-
sion experiments (Fig. 2a). This was compared to the
predicted start site of the petI operon using similar
techniques (Fig. 2b and data not shown). In both cases
bioinformatic techniques upstream of the respective
transcriptional start sites (Fig. 2c). Transcription from the
petI promoter was detected only in iron-grown cells and
transcription from thepetIIpromoter in iron- and insulfur-
grown cells (Fig. 2a, b), which was in agreement with
Fig. 1. Characterization of the petII operon in A. ferrooxidans ATCC 33020. (a, b) PCR amplification of (a) intergenic
fragments and (b) gene internal fragments from the petII locus; (c) RT-PCR amplification of the intergenic regions of the petII
locus. The locations of the oligonucleotides used for PCR and RT-PCR experiments are shown above and below the map of
the petII operon. The sequences of the oligonucleotides are given in Table 1. LR, DNA molecular mass marker X from Roche;
LI, 1 kb Plus DNA ladder from Invitrogen.
Two bc1operons in Acidithiobacillus ferrooxidans
Regulation of the petI and petII operons
Northern blotting and real-time PCR experiments were
carried out to determine the relative levels of expression of
the petI and petII operons in cells grown in either iron or
Antisense RNA probes complementary to the cycA1 and hip
genes were hybridized to total RNA extracted from A.
ferrooxidans ATCC 33020 grown in iron or sulfur medium.
The largest transcript detected with the cycA1 probe was
4000 nt long (Fig. 3a), which suggested that the petI operon
includes transcripts that correspond to the full length
operon, confirming results obtained from RT-PCR experi-
ments (Levica ´n et al., 2002). Smaller transcripts were also
observed. Because the cycA1 probe corresponds to the first
gene of the petI operon, these data suggest that the largest
transcript is processed. With the hip probe, the largest
transcript detected was 4600 nt long (Fig. 3b), again
potentially corresponding to a full-length transcript of the
operon. Smaller transcripts were also observed, perhaps
resulting from RNA processing or transcription from
internal promoters. However, no internal promoters were
detected upstream of hip by reverse transcriptase-mediated
primer extension experiments (data not shown), reducing
the likelihood of this explanation.
The results of Northern blotting experiments indicated that
the petI and petII operons are differentially expressed. Using
the same amount of total RNA, the hybridization signal is
clearly much more intense for cycA1 when cells are grown in
iron versus sulfur medium (Fig. 3a). However, hip appears
to be transcribed under both conditions, with slightly more
transcripts in sulfur medium (Fig. 3b). This suggests that
the petI and petII operons are regulated in response to the
To corroborate the Northern blot results, the amount of
transcripts corresponding to petI and petII was quantified at
all stages of the cell growth by real-time PCR. The results are
shown in Table 2.The petI andpetIIpattern of expressionin
response to the energetic substrate was similar to that
detected by Northern blotting, with much higher expression
of petI in iron- than in sulfur-grown cells and expression of
petII in sulfur- and in iron-grown cells. In addition, several
interesting points were noticed: (i) as shown for the rus
operon (Yarza ´bal et al., 2004), the amounts of all the petI
transcripts decreased significantly after 3 days of growth in
ferrous-iron-grown cells when complete oxidation of
Fig. 2. (a, b) Reverse-transcriptase-mediated primer-extension experiments. Reverse transcription experiments were
performed with specific primers from (a) the petII (23 and 24) and (b) the petI (25 and 26) operons of A. ferrooxidans
ATCC 33020: one internal to the first gene of the operon (23 and 25) and one located in the 59 untranslated region of the
operon (24 and 26). Total RNA was extracted from early-exponential iron-grown cells (lane F), or mid-exponential sulfur-grown
cells (lane S). RT products are indicated with arrows. A sequence ladder, produced with primers 24 for the petII operon and
26 for the petI operon, is shown to the left of the RT products (lanes T, G, C and A). The DNA sequences are shown on the
right. Possible transcriptional start sites are indicated with an asterisk (*). (c) The 59 untranslated regions of the petII and petI
operons are shown with the putative ”35 and ”10 regions in bold underlined lower case, and transcriptional start sites in
bold upper case.
106 Microbiology 153
P. Bruscella and others
ferrous iron to ferric iron had occurred; (ii) the petII operon
was expressed in ferrous iron-grown cells, mainly in the
early exponential phase; (iii) hip transcripts were clearly
more abundant in sulfur- than in iron-grown cells. hip
may have an additional regulatory mechanism, which we
propose is at a post-transcriptional level because no internal
promoter could be detected upstream of hip by primer
extension (data not shown).
Table 2. Quantification of the petI and petII transcripts by real-time PCR in ferrous iron
(F)- and sulfur (S)-grown cells at different stages of growth
Total RNA was extracted from ferrous-iron-grown cells at 17 h (early-exponential phase), 1 day (mid-
exponential phase), 2 days (late-exponential phase) and 3 days (stationary phase). Total RNA was
extracted from sulfur-grown cells at 1 day (early-exponential phase), 2, 3 and 4 days (mid-exponential
phase), and 5 days (late-exponential phase). All values (except for rrs, encoding the 16SrRNA) are
expressed as n-fold relative to 16S rRNA (6106). The data are representative of real-time PCR experi-
ments performed on at least two independent cultures. The experiment shown used single cultures and
the values are the mean of duplicate assays that almost always varied by less than 25%.
Gene Growth conditions:
17 h1 d2 d 3 d 1 d2 d 3 d 4 d5 d
Fig. 3. Northern blot hybridization analysis. Total ATCC 33020 A. ferrooxidans RNA from early-exponential iron-grown cells
or mid-exponential sulfur-grown cells probed with DIG-labelled cycA1 (a) or hip (b) RNA. Arrows show the largest transcripts.
The positions and sizes of the RNA ladder (Invitrogen) are indicated on the left of each blot.
Two bc1operons in Acidithiobacillus ferrooxidans
To explore possible mechanisms involved in the differential
expression of the petI and petII operons depending on
energy source, the 59 untranslated regions of both operons
were compared using MAT inspector. No obvious con-
served regulatory motifs were detected.
Two differentially expressed operons encoding
bc1complexes in A. ferrooxidans
sequence analysis indicate that the genomes of three strains
of A. ferrooxidans, ATCC 19859, ATCC 33030 and the type
strain ATCC 23270, contain two operons, petIABC and
petIIABC, encoding two complete cytochrome bc1com-
plexes. Both are cotranscribed with cycA and sdrA, which
encode a c4-type cytochrome and a short-chain dehydro-
analysis,PCR experiments andDNA
Data from primer extension, Northern blotting and real-
time PCR experiments (Figs 2 and 3, Table 2) and
preliminary transcriptome analysis (Quatrini et al., 2006)
demonstrate that petI and petII are differentially transcribed
depending on the growth conditions. The petI operon is
the growth medium as an energy and electron source. In
contrast, the petII operon is transcribed in sulfur- or iron-
grown cells. Transient transcription is observed from the
petII operon when cells are first placed in ferrous iron
medium but at later stages of cell growth significantly less
expression is detectable (Table 2), a situation that is
reminiscent of the transient expression of the rus operon
(Yarza ´bal et al., 2003, 2004). Although no explanation for
this low amount of transient expression has been experi-
mentally validated, it has been speculated that, in the case of
the rus operon, it could be a response to an increase in
particularnutrientstoallow quick adaptationtothe environ-
ment, perhaps mediated by a Fis-like protein (Yarza ´bal et al.,
particularly degenerate (Hengen et al., 1997), was detected in
the proposed regulatory region of petII.
Given that petI is induced in ferrous iron medium, a
computational search was carried out for known iron-
regulated transcription factor binding sites in the region
upstream of cycA1 where a sigma-70-like promoter is
predicted (Fig. 2c). No obvious Fur-binding site (Lavrrar &
McIntosh, 2003) could be detected, although the A.
ferrooxidans genome is known to encode Fur and has
been shown experimentally to have Fur-binding sites
upstream of known iron-regulated genes (Quatrini et al.,
2005). Computational analysis also failed to detect
significant similarity in the A. ferrooxidans genome with
the two-component system PmrA–PmrB (Wosten et al.,
the RirA regulatory system (Todd et al., 2005) could be
detected upstream of either the petI or the petII operons,
leaving open, for the moment, the question of how these
operons are differentially regulated.
Proposed role for the redox proteins encoded
by the petI and petII operons
A. ferrooxidans is the first organism described so far to have
two complete and functional bc1complexes, raising the
Fig. 4. Proposed model of ferrous iron [Fe(II)] and sulfur (S0) energetic metabolism of A. ferrooxidans. The transcriptional
units and the corresponding redox proteins are represented with the same background patterns. Transcriptional start sites and
putative rho-independent transcriptional termination sites are represented by bent arrows and stem–loops, respectively.
108 Microbiology 153
P. Bruscella and others
question of why this is necessary. Genetic evidence has been
presented for the existence of a cytochrome-containing
complex functioning exclusively during iron oxidation
(Cabrejos et al., 1999; Levica ´n et al., 2002) and it has been
reported that a bc1complex functions only in reverse in
iron-grown cells (Elbehti et al., 2000), even in the presence
of an appropriate substrate (Brasseur et al., 2002). On the
other hand theexistenceof abc1complexhasbeenproposed
to be involved in the aerobic and anaerobic oxidation of
sulfur and formate processes (Corbett & Ingledew, 1987;
Pronk et al., 1991) and a bc1complex has been shown
recently to function in direct mode in sulfur-grown cells
(Brasseur et al., 2004). This raises the possibility that two
with iron and sulfur oxidation, respectively: one for uphill
flow during iron oxidation and the other for downhill flow
during sulfur oxidation. A corollary of this is that neither of
the two complexes can switch the direction of electron flow.
Whether this imposition comes from intrinsic mechanistic
differences in the sequence and structure of the two bc1
complexes that specify unidirectional flow of electrons or
from the action of additional, as yet unknown, structural or
regulatory components that could channel electrons to the
correct bc1complex, remains to be determined.
one functioning in reverse and transfers the electrons from
ferrous iron to NAD(P), while the bc1complex encoded by
petII is the one functioning directly, transferring electrons
from sulfur to oxygen and possibly involved in the aerobic
and anaerobic oxidation of sulfur and formate described by
Pronk et al. (1991).
Together with the three subunits of the bc1complex, the petI
and petII operons encode other redox proteins. Because
these genes are in the same transcriptional unit, these
proteins are probably involved in the same electron-transfer
chain. The sdrA1 gene has been predicted to encode a short
chain dehydrogenase (Levica ´n et al., 2002) but its function
remains unknown. The cytochrome c4encoded by the cycA1
gene has been proposed to belong to the electron-transfer
chain between ferrous iron and oxygen, and more precisely
to receive the electrons directly from ferrous iron and to
transfer them to rusticyanin (Giudici-Orticoni et al., 2000).
However, according to the data presented here, this
cytochrome c4is more likely to be involved in the reverse
electron pathway betweenferrous ironand NAD(P)because
contains another cytochrome c4encoding gene (cyc1) that
has been suggested (Appia-Ayme et al., 1999) to assume the
role postulated by Giudici-Orticoni et al. (2000). In the
the electrons from a cytochrome c and transfers them to
the quinol pool. We suggest that this cytochrome c is the
cytochrome c4encoded by the cycA1 gene (Fig. 4). Oxygen
balance the reducing equivalent from ferrous iron between
the two pathways: the exergonic one, through the aa3-type
oxidase towards oxygen, and the endergonic one, through
a bc1 complex toward NAD(P). As previously reported
(Brasseur et al., 2004), we propose that the branching point
is at the level of rusticyanin, which can give electrons to two
different cytochromes c4: CycA1 encoded by the petI operon
or Cyc1 encoded by the rus operon. In the former case,
electrons are transferred to NAD(P), while in the latter case,
they are transferred to oxygen (Fig. 4).
When functioning in direct mode, the bc1complex receives
electrons fromthequinol poolandtransfersthemeither toa
membrane-bound cytochrome c, and/or to a soluble redox
protein such a cytochrome c, or a high potential iron–sulfur
et al., 2003 and references therein; Bonora et al., 1999;
(CycA2) and a soluble HiPIP are encoded by the petII
operon, we suggest that these two redox proteins transfer
electrons between integral membrane complexes: bc1and a
terminal reductase, in particular a quinol oxidase (Brasseur
et al., 2004). An interesting possibility is that the electrons
are transferred either to the cytochrome CycA2 or to the
HiPIP Hip depending on the environmental conditions
(Fig. 4). Since hip is transcribed more in sulfur- than in
ferrous iron-growth conditions it is proposed that Hip is
involved in the electron transfer from sulfur.
We are grateful to Y. Denis (IBSM, Transcriptome Unit, Marseille,
France) for much helpful advice regarding real-time PCR. Preliminary
sequence data were obtained from The Institute for Genomic Research
website at http://www.tigr.org. Sequencing of A. ferrooxidans was
accomplished with support from the US Department of Energy. C.A.-
A. acknowledges the support of a post-doctoral fellowship from the
EEC. This study was partly supported by a CNRS/CONYCIT program
(16774) award to D.S.H. and V.B., and project ‘Geomex’ from the
CNRS. Part of this work was supported by ‘BIOMINE’ European
project (sixth PCRD no. NM2.ct.2005.500329). Additional funding
was received from Fondecyt 1056003 and a Microsoft sponsored
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