Microarray and bioinformatic analyses suggest models for carbon metabolism in the autotroph Acidithiobacillus ferrooxidans
ABSTRACT Acidithiobacillus ferrooxidans is a chemolithoautotrophic bacterium that uses iron or sulfur as an energy and electron source. Bioinformatic analysis of the A. ferrooxidans draft genome sequence was used to identify putative genes and potential metabolic pathways involved in CO2 fixation, 2P-glycolate detoxification, carboxysome formation and glycogen utilization. Microarray transcript profiling was carried out to compare the relative expression of the predicted genes of these pathways when the microorganism was grown in the presence of iron versus sulfur. Several gene expression patterns were confirmed by real-time PCR. Genes for each of the above-predicted pathways were found to be organized into discrete clusters. Clusters exhibited differential gene expression depending on the presence of iron or sulfur in the medium. Concordance of gene expression within each cluster suggested that they are operons. Most notably, clusters of genes predicted to be involved in CO2 fixation, carboxysome formation, 2P-glycolate detoxification and glycogen biosynthesis were upregulated in sulfur medium, whereas genes involved in glycogen utilization were preferentially expressed in iron medium. These results can be explained in terms of models of gene regulation that suggest how A. ferrooxidans can adjust its central carbon management to respond to changes in its environment.
Article: Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: case study with the extremophile Acidithiobacillus ferrooxidans.[show abstract] [hide abstract]
ABSTRACT: Normalization is a prerequisite for accurate real time PCR (qPCR) expression analysis and for the validation of microarray profiling data in microbial systems. The choice and use of reference genes that are stably expressed across samples, experimental conditions and designs is a key consideration for the accurate interpretation of gene expression data. Here, we evaluate a carefully selected set of reference genes derived from previous microarray-based transcriptional profiling experiments performed on Acidithiobacillus ferrooxidans and identify a set of genes with minimal variability under five different experimental conditions that are frequently used in Acidithiobacilli research. Suitability of these and other previously reported reference genes to monitor the expression of four selected target genes from A. ferrooxidans grown with different energy sources was investigated. Utilization of reference genes map, rpoC, alaS and era results in improved interpretation of gene expression profiles in A. ferrooxidans. This investigation provides a validated set of reference genes for studying A. ferrooxidans gene expression under typical biological conditions and an initial point of departure for exploring new experimental setups in this microorganism and eventually in other closely related Acidithiobacilli. The information could also be of value for future transcriptomic experiments in other bacterial systems.BMC Molecular Biology 07/2009; 10:63. · 2.86 Impact Factor
Microarray and bioinformatic analyses suggest models for carbon
metabolism in the autotroph Acidithiobacillus ferrooxidans
Corinne Appia-Aymea,1, Raquel Quatrinib,1, Yann Denisa, François Denizota,
Simon Silverc, Francisco Robertod, Felipe Velosob, Jorge Valdésb,
Juan Pablo Cárdenase, Mario Esparzaf, Omar Orellanaf,
Eugenia Jedlickif, Violaine Bonnefoya, David S. Holmesb,⁎
aLaboratoire de Chimie Bactérienne, IBSM, CNRS, Marseille, France
bAndrés Bello University and Millennium Institute for Fundamental and Applied Biology, Santiago, Chile
cUniversity of Illinois, Chicago, USA
dIdaho National Laboratory, Idaho Falls, USA
eUniversity of Santiago, Santiago, Chile
fICBM, Faculty of Medicine, University of Chile, Santiago, Chile
Acidithiobacillus ferrooxidans is a chemolithoautotrophic bacterium that uses iron or sulfur as an energy and electron source.
Bioinformatic analysis of the A. ferrooxidans draft genome sequence was used to identify putative genes and potential metabolic
pathways involved in CO2fixation, 2P-glycolate detoxification, carboxysome formation and glycogen utilization. Microarray
transcript profiling was carried out to compare the relative expression of the predicted genes of these pathways when the
microorganism was grown in the presence of iron versus sulfur. Several gene expression patterns were confirmed by real-time PCR.
Genesfor eachof theabove-predicted pathwayswere foundtobe organized intodiscreteclusters. Clusters exhibiteddifferential gene
expression depending on the presence of iron or sulfur in the medium. Concordance of gene expression within each cluster suggested
that they are operons. Most notably, clusters of genes predicted to be involved in CO2fixation, carboxysome formation, 2P-glycolate
detoxification and glycogen biosynthesis were upregulated in sulfur medium, whereas genes involved in glycogen utilization were
preferentially expressed in iron medium. These results can be explained in terms of models of gene regulation that suggest how A.
ferrooxidans can adjust its central carbon management to respond to changes in its environment.
Keywords: Calvin cycle; Glycogen; Glycolate; Carboxysome; Genome of Acidithiobacillus ferrooxidans
The acidophilic bacterium Acidithiobacillus ferroox-
idans is an obligate chemolithoautotroph. It fixes CO2
using energy and reducing power derived from the
oxidation of iron or sulfur. CO2fixation in both chemo-
lithoautotrophs and photoautotrophs occurs via the
E-mail address: firstname.lastname@example.org (D.S. Holmes).
1These authors contributed equally to the work.
Calvin-Benson-Bassham reductive pentose phosphate
cycle (Calvin cycle). The genes and biochemical
reactions of the Calvin cycle are highly conserved
between organisms facilitating their discovery and
prediction in novel organisms both by DNA sequence
analysis and by experimentation.
Early studies showed a relationship between the rate
of iron and sulfur oxidation and the rate of CO2
fixation in A. ferrooxidans [1,2]. Several enzymes of
the Calvin cycle have been detected in A. ferrooxidans
including the key enzyme D-ribulose-1,5-bisphosphate
carboxylase/oxygenase (RuBisCO) . RuBisCO cat-
alyzes the formation of two molecules of 3-phospho-
glyceric acid (PGA) from ribulose bisphosphate and
CO2. A plant-type hexadecameric form of the enzyme,
consisting of eight copies each of the small (CbbS) and
large (CbbL) subunits, has been purified from A.
ferrooxidans. This enzyme was shown to have Km
values for CO2 and ribulose bisphosphate that are
similar to those of RuBisCO from plants and green
algae but are four- to fivefold lower than those typical
of bacteria .
Two structurally distinct forms of RuBisCO (I and
II), with different catalytic properties, are present in
autotrophs . Form I is composed of both large
(catalytic) and small subunits in a hexadecameric
structure, and form II is composed exclusively of
multiples of the large subunit. It has been suggested
that form I provides the cell with fixed carbon whereas
form II functions primarily as a terminal electron
acceptor, assisting in the maintenance of the redox
balance of the cell . In A. ferrooxidans (strain Fe1),
two sets of identical genes, originally termed rbcLS1
and rbcLS2 but more correctly known as cbbLS1 and
cbbLS2, encoding the large and small subunits of form I
RuBisCO have been cloned and characterized [7–9].
Co-transcription of the large and small subunit genes
was shown to occur when A. ferrooxidans was grown on
iron, but sequence identity between the two copies does
not allow to distinguish between their expression.
Located upstream of cbbLS1, and divergently tran-
scribed from this gene cluster, is a well conserved LysR-
type transcriptional regulator gene (cbbR) known to
bind specifically to overlapping promoter elements in
the intergenic sequence between cbbR and cbbL1. In
addition, the presence of a putative CbbR binding site
upstream of the second set of the cbb genes (cbbLS2)
suggests that both RuBisCO form I gene clusters are
under the control of the CbbR regulator .
Many CO2-fixing microorganisms, including che-
molithoautotrophic bacteria and cyanobacteria, contain
polyhedral inclusion bodies known as carboxysomes
. These structures were originally isolated from
Halothiobacillus neapolitanus (previously Thiobacillus
neapolitanus)  and found to contain substantial
amounts of RuBisCO type I . In addition to
RuBisCO, up to seven additional polypeptides are
associated with the carboxysome; five of these have
been identified as shell proteins . These polypep-
tides are encoded by genes within the cso gene cluster,
an apparent operon that also includes cbbL and cbbS
RuBisCO subunits in several Thiobacillus species 
and in cyanobacteria . Additionally, low levels of
carbonic anhydrase (CA) appear to co-purify with the
particles from Synechococcus  and Synechocystis
. Recently, the carboxysomal shell protein, CsoS3
from H. neapolitanus, was shown to constitute a novel
evolutionary lineage of CAs (ε class) . The location
of the carboxysomal CA in the shell suggests that it
could supply the active sites of RuBisCO in the
carboxysome with the high concentrations of CO2
necessary for optimal RuBisCO activity.
In the obligate, chemolithoautotroph T. neapolitanus
carboxysome synthesis appears to be regulated by the
concentration of CO2in the growth medium, with large
quantities appearing under CO2-limiting conditions
. In the facultative organism T. intermedius, the
observed number of carboxysomes under mixotrophic
growth conditions varied in approximate proportion to
the specific activity of RuBisCO, suggesting that the
synthesis of these structures is under metabolic control
. Whether the regulation of carboxysome formation
is linked to the control of RuBisCO synthesis is yet to be
A deleterious side product of the Calvin cycle is 2-
phosphoglycolate (2P-glycolate) which is typically
metabolized to innocuous compounds in CO2fixing
organisms. No information regarding this pathway has
been published for A. ferrooxidans. Information regard-
ing other aspects of CO2fixation in A. ferrooxidans is
also lacking. For example, nothing is known regarding
the reduction of Calvin cycle intermediates, nor how
ribulose 1,5-bisphosphate (RuBP) is regenerated. In
addition, there is a lack of information as to how carbon
derived from the Calvin cycle is channelled into
subsequent pathways for intermediate metabolism.
Challenged by these deficiencies, we undertook a
bioinformatic analysis of potential pathways involved
in CO2fixation, carboxysome formation, 2P-glycolate
detoxification and in the synthesis and utilization of
glycogen in A. ferrooxidans. In addition, microarray
transcript profiling of genes in these pathways was
carried out in order to advance our understanding of
C. Appia-Ayme et al.
how these pathways might be regulated when cells are
grown in either iron or sulfur.
2. Materials and methods
Bioinformatic analysis of candidate genes, from the
TIGR draft genome, and metabolic reconstruction of A.
ferrooxidans ATCC 23270 (type strain) were carried out
as previously described . Microarray transcript
profiling was carried out as described in the accompa-
nying paper . Sequences deposited in GenBank:
cbbRa: B49698; cbbL1b: RKBCLT, S18315; cbbS1c:
3. Results and discussion
Putative genes were identified in the genome of A.
ferrooxidans predicted to be involved in the Calvin
cycle, 2P-glycolate detoxification, the formation of
carboxysomes and glycogen metabolism (Table 1).
Several of these candidate genes potentially encode
proteins that exhibit conserved motifs and predicted
folds characteristic of the proposed function (Table 1).
The relative level of expression of these genes in cells
grown in either iron (Fe) or sulfur (S) medium was
evaluated by microarray analysis and confirmed in
several cases by real-time PCR (Table 1).
3.1. Calvin cycle
Two enzymes that are unique to the Calvin cycle
are ribulose 1,5-bisphosphate carboxylase/oxygenase
(RuBisCO) andphosphoribulokinase (PRK).
RuBisCO catalyzes the first step of the Calvin
cycle, the carboxylation of ribulose 1,5-bisphosphate
(RuBP) with CO2. Phosphoribulokinase (PRK) cata-
lyzes the last step of the cycle, which is the re-
generation of the CO2acceptor molecule, RuBP, via
the phosphorylation of ribulose 5-phosphate with ATP.
All other steps of the Calvin cycle are catalyzed by
enzymes common to other pathways of intermediary
metabolism. Briefly, these steps fulfill (i) the reduc-
tion of 3-phosphoglyceric acid, (ii) the formation of
fructose-6-phosphate and (iii) the regeneration of the
CO2acceptor RuBP. For convenience, the reactions of
the Calvin cycle can be grouped into four main
events: CO2 uptake, CO2 fixation, intermediate
reduction and regeneration of the CO2 acceptor
molecule. We have identified candidate genes in A.
ferrooxidans for each of these functions (Table 1).
The key genes and reactions are depicted in the
metabolic scheme presented in Fig. 1. Also included
in Fig. 1 is an indication of whether the expression of
each gene is enhanced when cells are grown in media
containing iron versus sulfur.
The first step of carbon fixation is carboxylation of
the acceptor molecule, ribulose-1,5-bisphosphate
(RuBP), by ribulose-1,5-bisphosphate carboxylase/oxy-
genase (RuBisCO; EC 126.96.36.199). Since the substrate for
RuBisCO is CO2, a carbonic anhydrase (CA; EC
188.8.131.52) that catalyzes the reversible hydration of CO2
is needed to ensure rapid conversion of cytosolic HCO3
to CO2at concentrations that support optimal RuBisCO
operation of a number of active CO2 and HCO3
−is accumulated in the cytosol by the
3.2. 2P-Glycolate detoxification
RuBisCO is a bifunctional carboxylase/oxygenase
able to utilize both CO2and O2as substrates depending
on their availability. While the carboxylase reaction
initiates CO2fixation, the oxygenase reaction initiates
the C2 oxidative carbon cycle that results in the
poisonous intermediary, 2-phosphoglycolate (2P-glyco-
late) . Unless this product is dephosphorylated by a
2-phosphoglycolate phosphatase (PGP; EC 184.108.40.206) to
yield glycolate, the accumulation of the compound as a
dead-end metabolite results in the inhibition of triose-
phosphate isomerase . Further breakdown of
glycolate by a peroxisomal glycolate oxidase (GOX;
EC 220.127.116.11) allows carbon from 2-phosphoglycolate to
be recycled into the Calvin cycle.
Candidate genes predicted to encode enzymes that
participate in the detoxification of 2P-glycolate were
detected in the genome of A. ferrooxidans (Table 1) and
a scheme illustrating the possible carbon-salvaging 2P-
glycolate of this organism is shown in Fig. 1. All the
genes in this pathway were upregulated in cells grown in
3.3. Carboxysome formation
Obligate chemolithotrophic sulfur-oxidizing bacte-
ria, nitrifying bacteria and cyanobacteria, have a
polyhedral protein microcompartment that contains
RuBisCO together with a carboxysomal carbonic
anhydrase (CA). CA converts an accumulated cytosol-
ic pool of HCO3into CO2within the carboxysome,
elevating the CO2around the active site of RuBisCO.
Carboxysomes are not present in facultative auto-
trophs, despite the fact that when these organisms grow
as photoautotrophs they use the Calvin cycle to fix
CO2. Although the detailed biochemical mechanism by
C. Appia-Ayme et al.
Candidate genes of A. ferrooxidans suggested to be involved in carbon metabolism and the log2relative level of expression in cells grown in iron (Fe)
or sulfur (S) medium
Gene EC No.Assigned function Conserved domains Gene expression
(log2) in S or Fe
RuBisCO transcriptional regulator
Fructose bisphosphate aldolase
Ribose 5-P isomerase A
RuBisCO (large subunit)
RuBisCO (small subunit)
RuBisCO (large subunit)
RuBisCO (small subunit)
RuBisCO (large subunit)
RuBisCO (large subunit), form II
Carboxysome shell, carbonic anhydrase
1,4-α-Glucan branching enzyme
1,4-α-Glucan branching enzyme
C. Appia-Ayme et al.
which carboxysomes enhance autotrophic CO2 fixa-
tion is not well understood, collective evidence sug-
gests that the unique structural organization and the
bounding shell of carboxysomes provide a distinct
catalytic advantage for this process. Seven candidate
genes potentially involved in carboxysome formation
have been discovered in the genome of A. ferrooxidans
The existence of three forms of CAs, α, β and γ has
been known for some time. Candidate genes for the β
and γ forms have been detected in the genome A.
ferrooxidans (data not shown). It was demonstrated
recently that the carboxysomal shell protein, CsoS3,
from H. neapolitanus is a novel form of CA . This
shell-localized CA is proposed to supply CO2to the
active sites of RuBisCO in the carboxysome to support
optimal CO2 fixation activity by catalyzing the
reversible hydration of CO2. A. ferrooxidans has a
candidate gene for this form of CA (Table 1).
All seven predicted genes for carboxysome forma-
tion are upregulated when A. ferrooxidans is grown in S
medium (Table 1).
3.4. Glycogen metabolism
Glycogen is produced and accumulates in many
bacteria where it is thought to be used as a stored
source of energy and carbon [25,26]. Little is known
about glycogen synthesis and breakdown in A.
Candidate genes and predicted enzymes and path-
ways for the glycogen biosynthesis and breakdown have
been detected in the genome of A. ferrooxidans (Table
1). Metabolic reconstruction (Fig. 2) suggests that ADP-
glucose provides the donor sugar nucleotide, whose
synthesis is catalyzed by the enzyme glucose-1-
phosphate adenylyltransferase (glgC; EC 18.104.22.168).
The glucosyl moiety of ADP-glucose is transferred, in
Table 1 (continued)
Gene EC No.Assigned functionConserved domainsGene expression
(log2) in S or Fe
Enzyme commission (EC) numbers and conserved domains and motifs are shown for predicted protein products where appropriate.
⁎Microarray data validated by real-time PCR.
Fig. 1. Representation of the candidate genes and predicted enzymes and pathways for the Calvin cycle and for 2P-glycolate detoxification in A.
C. Appia-Ayme et al.
a reaction catalyzed by a specific ADP-glucose-
glycogen synthetase (glgA; EC 22.214.171.124), to a glycogen
primer to form a new α-1,4-glucosidic bond. Sub-
sequently, a branching enzyme (glgB1, glgB2; EC
126.96.36.199) catalyzes the formation of branched α-1,6-
glucosidic linkages. The release of energy and carbon
stored in glycogen is initiated by the enzyme glucan
phosphorylase (glgP, glgP2; EC 188.8.131.52), which releases
glucose-1-phosphate from the nonreducing terminus of
the α-1,4 chain .
3.5. Organization and expression of candidate genes
involved in the Calvin cycle, carboxysome formation,
2P-glycolate detoxification and glycogen metabolism
The proposed organization of selected candidate
genes predicted to be involved in the Calvin cycle,
carboxysome formation, 2P-glycolate detoxification
and glycogen metabolism in A. ferrooxidans is shown
in Fig. 3. Not all genes listed in Table 1 or indicated in
Figs. 1 and 2 have been included in this figure for
simplicity. Superimposed on the gene clusters is the
microarray expression data for cells grown in iron
versus sulfur (Fig. 3). Each experiment included
appropriate controls and statistical validation. In some
cases, microarray data was validated by real-time PCR
analysis (Table 1).
Several general observations can be made.
• Genes with related functions tend to be clustered
together, for example, those encoding carboxysome
formation and glycolate detoxification (clusters I and
III, respectively, Fig. 3).
• Given that the putative genes are densely packed
within the clusters, allowing little space for the
presence of individual promoters between genes, and
since each cluster contains at least some genes with a
common function, it is proposed that the clusters
correspond to operons. The observation that genes
within individual clusters are co-expressed in either
iron or sulfur supports the contention that each
cluster is an individual transcription unit.
• There are several instances where genes involved in
the same function are found in different clusters, for
example, those involved in the Calvin cycle are
distributed in at least five clusters, two of which are
shown as clusters I and II in Fig. 3. Since the majority
of genes involved in the Calvin cycle also carry out
other metabolic functions, having multiple clusters
allows their regulation to be uncoupled from the
Calvin cycle when required. There are several
instances of duplicate genes. For example, several
genes of the Calvin cycle are duplicated including
glpX encoding fructose-1,6-bisphosphatase 1 and 2,
cbbLS encoding the duplicated RuBisCO form I
complex, dnhA 1 and 2 encoding fructose-1,6-
bisphosphate aldolase, and tkt 1 and 2 encoding
transketolase. Except for RuBisCO, these genes are
also required for other metabolic functions. Dupli-
cated genes are probably controlled by different
promoters and regulatory signals. This would permit
differential expression of the two gene copies in
response to distinct triggering signals, extending the
response capacities of the microorganism or even
bypassing conditions of full repression of one of the
isozyme forms. The importance of redundancy
Fig. 2. Representation of the candidate genes and predicted enzymes and pathways for the biosynthesis and degradation of glycogen in A.
C. Appia-Ayme et al.
becomes obvious in the case of facultative hetero-
trophs where Calvin cycle genes are completely
turned off in the presence of a metabolizable carbon
• With the exception of cbbR which is divergently
expressed, clusters I and II are upregulated in sulfur
medium (Table 1 and Figs. 1 and 3). This also applies
to other genes of the Calvin cycle as shown in Table 1
(but not illustrated in Fig. 3). This suggests that the
Calvin cycle is more active when cells are grown in
sulfur versus iron. Since a more active Calvin cycle
would generate more poisonous 2P-glycolate, it is
consistent that the 2P-glycolate detoxification path-
way is also upregulated in sulfur medium (Table 1
and Figs. 1 and 3). These proposed models of gene
organization will help focus future efforts to detect
common regulatory elements in these clusters that
respond to environmental signal(s) when sulfur but
not iron is present in the medium.Although genes of
the Calvin cycle are upregulated in sulfur medium,
two genes tkt1 and rpe (Table 1 and Fig. 1) are
upregulated in iron medium. These genes classically
represent an alternate way to regenerate ribulose-5P
from glyceraldehyde-3P via xylulose-5P (xylulose-
5P shunt, Fig. 1). Why they are specifically upregu-
lated in iron medium remains to be investigated.
• Microarray data indicates that copies 1 and 2 of
RuBisCO form I and their cognate post-translational
modulator genes cbbOQ had higher ratios in cells
grown in sulfur compared to iron (Table 1). In
addition, real-time PCR experiments using primers
specifically designed to distinguish between cbbO
copies suggest that only the major cluster encoding
CbbLS1 is upregulated in S medium and that
CbbLS2 and the putative operon encoding the
CbbM RuBisCO form II are expressed approximate-
ly at the same level in both iron and sulfur medium.
These results are consistent with those reported for
facultative autotrophs where RuBisCO form I is
predominant under autotrophic growth conditions,
whereas form II is expressed under all growth
conditions . It is speculated that form II RuBisCO
functions primarily as a terminal electron acceptor,
assisting in the maintenance of the redox balance of
the cell whereas the function of the form I enzyme in
A. ferrooxidans growing in sulfur is to provide the
cell with fixed carbon.The observation that the major
gene cluster encoding for CbbLS1 is upregulated in S
medium as judged by real time PCR is in potential
conflict with proteomic data that suggest that both
subunits of RuBisCO encoded by the major cbb
operon, and the modulator CbbQ (P30) are down-
regulated in sulfur . It is possible that expression
at the level of RNA of the major cluster (cbbI1) is
enhanced in sulfur medium but that the levels of the
respective proteins increase in iron medium, suggest-
ing that important translation regulatory mechanisms
remain to be discovered.
Fig. 3. Proposed genetic organization of the candidate genes of A. ferrooxidans involved in the Calvin cycle, glycolate detoxification, carboxysome
formation and glycogen metabolism. Arrows indicate proposed transcription.
C. Appia-Ayme et al.
• Genes involved in glycogen biosynthesis and the
glycogen branching/debranching system tend to be
upregulated in sulfur medium (Table 1 and cluster III,
Fig. 3), whereas those proposed to be required for
glycogen breakdown are upregulated in iron medium
(Table 1 and cluster IV, Fig. 3). The other two genes,
pyk and eno, associated with the glycogen break-
down gene glgP1 in cluster IV are predicted to be
involved in sugar metabolism, suggesting that this
cluster is involved in the recovery of energy and
carbon from glycogen. Theoretically, sulfur should
yield more energy than iron. Therefore, perhaps, it is
metabolically favorable to fix CO2and to channel
some of the fixed carbon to stored glycogen when
sulfur is available as an energy source. The glycogen
can be broken down later to yield carbon and energy
in leaner times, for example, when only iron is
available as an energy source.
Note added in proof
The complete sequence of A. ferrooxidans has now
been released (TIGR. org).
This work was supported in part by Fondecyt
1010623 and 1050063, Conicyt/CNRS, “Geomex” and
“Puces à ADN” from the Centre National de la
Recherche Scientifique and NSF. RQ was the recipient
of an American Society for Microbiology Antorcha
Fellowship and was supported by scholarships from the
DAAD and from the Fundación Ciencia para la Vida,
Chile. CAA was supported by a CNRS post-doctoral
fellowship. We thank the Institute for Genome Research
(TIGR) for the use of their draft genome sequence of A.
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