Anaerobic Sulfur Metabolism Coupled to Dissimilatory Iron Reduction in the Extremophile Acidithiobacillus ferrooxidans

The purity of the A. ferrooxidans culture (ATCC 23270^T) was confirmed by streaking onto a variety of overlay media (32). A. ferrooxidans was preadapted to growth on 1% (wt/vol) S⁰ in a shake flask culture (30�C; initial pH, 2.5) with a basal salts-trace element mix before inoculation into a 2.3-liter bioreactor fitted with pH, temperature, and aeration control (Electrolab, United Kingdom). The basal salts contained (final concentrations) MgSO₄ � 7H₂O (1 g/liter), (NH₄)₂SO₄ (0.9 g/liter), Na₂SO₄ � 10H₂O (0.3 g/liter), KH₂PO₄ (0.1 g/liter), KCl (0.1 g/liter), and Ca(NO₃)₂ � 4H₂O (0.03 g/liter) with a trace element solution of (final concentrations) ZnSO₄ � 7H₂O (20 mg/liter), CuSO₄ � 5H₂O (2 mg/liter), MnSO₄ � 4H₂O (2 mg/liter), CoSO₄ � 7H₂O (2 mg/liter), Cr₂(SO₄)₃ � 15H₂O (1 mg/liter), H₃BO₃ (1.2 mg/liter), Na₂MoO₄ � 2H₂O (1 mg/liter), NiSO₄ � 6H₂O (2 mg/liter), Na₂SeO₄ � 10H₂O (2 mg/liter), Na₂WO₄ 2H₂O (0.2 mg/liter), and NaVO₃ (0.2 mg/liter). The reactor contained 100 g S⁰ (sterilized by Tyndallization) and 2 liters of basal salts-trace element mix. The temperature was maintained at 30�C, the pH was maintained at 1.8 (by automated addition of 1 M H₂SO₄ or NaOH), and the bioreactor was aerated with sterile air at 1 liter/min and stirred at 150 rpm. Bacterial growth was monitored by using a Thoma counting chamber (Hawksley, United Kingdom). Culture agitation and aeration were stopped when cell numbers increased to ∼10⁹/ml, allowing the S⁰ particles to collect as a loose sediment. Approximately 1.5 liters of the surface liquor was aseptically withdrawn during exponential growth when nutrients were not depleted, and cells were harvested by centrifugation (10,000 � g for 20 min at 4�C) and washed three times in basal salts solution (pH 2). Part of the obtained cell pellet was frozen (−20�C) ahead of proteomic analysis, while the remainder was suspended in RNAlater (Qiagen, United Kingdom) and stored at −20�C prior to transcriptomic investigations. The bioreactor was topped up with 1.5 liters of sterile basal salts-trace elements, and two further cycles of aerobic growth were carried out under identical conditions.

S⁰ was also used for anaerobic growth, but the electron acceptor was switched from O₂ to Fe³⁺. Following harvesting of the third aerobic batch culture, the bioreactor was inoculated with A. ferrooxidans grown anaerobically in 25 mM ferric sulfate, 1% (wt/vol) S⁰, pH 2.0, at 30�C in an anaerobic jar (AnaeroGen system; Oxoid, United Kingdom). Basal salts-trace elements were added to bring the bioreactor culture volume to ∼2 liters, and ferric sulfate was added (from a filter-sterilized 0.5 M solution, pH 1.5) to give a final concentration of ∼20 mM. The culture was deaerated with a stream of oxygen-free N₂ (2 liters/min for 30 min), and then the N₂ flow was lowered to 1 liter/min. Carbon was provided by intermittently sparging the bioreactor (∼30 min/day) with a 10% CO₂–90% N₂ gas mix. Temperature and pH were maintained as for aerobic growth. Fe³⁺ reduction was monitored by measuring Fe²⁺ concentrations using the ferrozine assay (33), and when >90% of the iron had been reduced, additional ferric sulfate solution (equivalent to 20 mM Fe³⁺) was added to the bioreactor. Sequential addition of relatively small volumes of ferric sulfate ensured that no Fe³⁺ precipitates formed, and for each batch culture, the equivalent of ∼100 mM Fe³⁺ was added. Anaerobic cultures were harvested as described previously, and cell pellets from three separate batch cultures were obtained.

Evidence for production of hydrogen sulfide (H₂S) by A. ferrooxidans maintained under anaerobic conditions in the presence of S⁰ was obtained by incubating cells in the presence of Cu²⁺, which reacts with H₂S to form insoluble CuS in acidic liquors (34). Six 25-ml universal bottles, each containing 10 mM ferrous sulfate, 10 mM copper(II) sulfate, 100 mg of S⁰, and basal salts-trace elements (total volume, 20 ml), were inoculated with 1 ml of an A. ferrooxidans culture (approximately 1 � 10⁸ cells/ml) grown aerobically on S⁰. Two noninoculated cultures were also prepared. Ferrous iron, rather than Fe³⁺, was used in this experiment to preclude the reaction 2Fe³⁺ + H₂S → 2Fe²⁺ + S⁰ + 2H⁺. All eight bottles were incubated under anaerobic conditions (AnaeroGen system; Oxoid, United Kingdom) for up to 6 weeks, and concentrations of soluble Cu²⁺ were measured using ion chromatography (34).

Proteomic analysis of total soluble protein and outer membrane-enriched fractions was carried out as described previously (35). This method enriches for outer membrane proteins but is not compatible with inner membrane proteins. Protein samples were extracted from triplicate bacterial samples. First-dimension isoelectric focusing was performed using Immobiline DryStrip immobilized pH gradient gels (18 cm; nonlinear pH range, pH 3 to 10; GE Healthcare), followed by second-dimension SDS-polyacrylamide gel electrophoresis. The Coomassie-stained gels were analyzed with image analysis software (Melanie, version 7.03; Genebio). The criteria used to identify protein spot intensities were defined by Mangold et al. (35). For analysis of the cytoplasmic proteins, all spots detected were taken into account. However, five predominant spots in gels from the anaerobic outer membrane-enriched fraction were excluded to avoid a skewed analysis which would otherwise misleadingly identify many proteins as upregulated under aerobic conditions (see Fig. S2 in the supplemental material). Proteins were identified by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (35). Peptide mass fingerprints were searched against a local database containing the genome sequence of A. ferrooxidans ATCC 23270^T using the Mascot search engine, allowing 2 missed cleavages and a peptide tolerance of 50 ppm (36). Database hits were significant, with a score of 47 at a 0.05 significance level.

Cells were pelleted from suspensions in RNAlater and washed in 10 mM H₂SO₄. Total RNA was extracted using a modified acid-phenol extraction method, including an initial step with TRIzol reagent, as described by the manufacturer (Invitrogen). RNA was further purified using a High Pure RNA isolation kit (Roche Applied Science) and treated with DNase I (Roche Applied Science) according to the manufacturer's instructions. The absence of DNA was verified by PCR for each sample. RNA was used for the synthesis of cDNA fluorescently labeled with Cy3 and Cy5 (20, 37). Microarray transcript profiling was carried out as described previously (20, 37). Arrays were scanned for fluorescent signals using a ScanArray 4400A scanner (MDS Analytical Technologies). The processed aerobic (S⁰/O₂) and anaerobic (S⁰/Fe³⁺) signal intensities for each spot were used for calculating the aerobic/anaerobic mRNA concentration ratio (37). Each gene expression ratio was calculated from 40 values calculated from 4 biological and 10 technical replicates and normalized by the nonlinear Lowess method using the Acuity (version 4.0) program (Molecular Devices). A 0.7-fold deviation from the 1:1 hybridization (log₂) ratio was taken as indicative of differential levels of RNA. Hierarchical cluster analysis (Pearson correlation, average linkage) was performed using the Genesis software suite (38).

Quantitative real-time PCRs (qPCRs) were performed with an iCycler thermal cycler (Bio-Rad) and a KAPA SYBR FAST qPCR kit (Kapabiosystems). Twenty-microliter PCR mixtures contained 2 μl of a 1:100-diluted cDNA sample, 200 nM each primer, and 1� KAPA SYBR FAST qPCR master mix. The reference dye carboxy-X-rhodamine was included at a final concentration of 5 nM. The cycling protocol was as follows: initial denaturation for 10 min at 95�C, followed by 40 cycles of 30 s each at 95�C, 56�C, and 72�C. Fluorescence was measured after the extension phase at 72�C. Specific amplification was confirmed by a single peak in the melting curve. For each experimental condition, total RNA was extracted from two independent A. ferrooxidans cultures. Each RNA sample was retrotranscribed to evaluate concentration levels relative to the concentration of the constitutive reference gene rpoC (39). The reactions for each target gene were performed in triplicate, and each target gene was simultaneously amplified in the same PCR run. Thus, data sets consist of 6 values per gene per experimental setup generated under standardized PCR cycling conditions. Tenfold dilutions of stationary-phase genomic DNA (range, 10 ng to 1 pg) were used to generate a 5-point standard curve for each gene by using the cycle threshold (C_T) value versus the logarithm of each dilution factor. The reaction efficiency (E), which was equal to [10(−1/slope)]⁻¹, for every gene was derived from the slope of the corresponding standard curves. The genes tested by qPCR and the respective primers used in the reactions are listed in Table S1 in the supplemental material.

The sequence and annotation of the complete A. ferrooxidans ATCC 23270^T genome were retrieved from Gen-Bank/EMBL/DDBJ (CP001219) (15). The annotated genome was displayed in the Artemis interactive format (www.sanger.ac.uk/Software/Artemis). The bioinformatics programs used to characterize candidate genes and their predicted protein products included blastp, tblastn, blastx, and psiblast (www.ncbi.nlm.nih.gov) and the suite of protein characterization programs available in InterproScan (www.ebi.ac.uk/interpro). Metabolic reconstructions were carried out on the basis of comparisons with the model metabolic pathways obtained from the BIOCYC (www.biocyc.org), KEGG (www.genome.ad.jp/kegg), and ERGO (Integrated Genomics) databases. Comparative genomic analyses were performed using the Microbesonline (www.microbesonline.org) and String (http://string.embl.de/) programs.

The data for all hybridizations were submitted to the MIAMExpress database under accession number E-MEXP-3361.

This study compared protein levels and RNA synthesis during aerobic and anaerobic growth of A. ferrooxidans on S⁰ (Table 1; details are given in Tables S2 and S3 in the supplemental material). A total of 78 upregulated and 13 unique protein spots were identified from the soluble- and outer membrane-enriched aerobic cultures, while 11 upregulated and 6 unique protein spots were identified from the anaerobic cultures (see Fig. S1 and S2 in the supplemental material). Comparative transcriptomic microarrays of A. ferrooxidans grown aerobically and under anaerobic conditions on sulfur identified a total of 42 and 44 upregulated genes, respectively.

Some discrepancies between protein levels measured by proteomics and levels of RNA determined by transcriptomics were noted. Such discrepancies have been observed in many instances of transcriptomic/proteomic analyses and have been variously attributed to differences in stability/degradation, posttranscriptional regulation, and posttranslational modification (40–43). In this study, levels of RNA have been validated by qPCR for various genes. A concordance between the results has been observed (Table 1).

Genes and proteins that could potentially be ascribed to S⁰ energy metabolism, Fe³⁺ reduction, electron transfer and energy conservation, and chemoautotrophy and carbon metabolism are shown in Table 1. Other genes and proteins that display differential regulation are listed in Table S2 in the supplemental material and are grouped according to Gene Ontology predictions: (i) cell wall, membrane, and envelope biogenesis; (ii) chaperone functions; (iii) translation transcription and cell cycle; (iv) amino acid metabolism; (vi) nucleotide metabolism and transport; and (vii) gene regulation.

Oxidation of S⁰ by A. ferrooxidans under aerobic conditions is thought to be mediated by heterodisulfide reductase (Hdr), which is predicted to be an inner membrane-anchored protein with its active site in the cytoplasm (20, 35). S⁰ is poorly soluble in water, so the likely substrate for Hdr is the sulfane-sulfur compound glutathione persulfide (GSSH), which contains a disulfide bond that has been proposed to be cleaved by Hdr to produce SO₃²⁻ and glutathione (GSH) (44, 45). There are three predicted copies of the gene for the HdrB catalytic subunit on the A. ferrooxidans genome (20), one of which is found outside the hdr locus (AFE_2586). While products of this gene were detected under both aerobic and anaerobic conditions, suggesting that Hdr mediates S⁰ oxidation in both situations (Fig. 1), protein levels were greater under aerobic conditions. A hypothetical protein (AFE_2552) and HdrA (AFE_2553) were also found at higher levels in the aerobic culture (see Fig. S3 in the supplemental material). In contrast, both dsrE (AFE_2556) and tusA (AFE_2557) were upregulated during anaerobic growth on S⁰. Although it has not been experimentally verified in A. ferrooxidans, the products of these genes are predicted to be involved in a sulfur relay system (46–48). All subunits of the sulfur reductase (SreABCD [AFE_2178 to AFE_2181]) were expressed only under anaerobic conditions. The sre operon in Aquifex aeolicus encodes a sulfur-reducing multiprotein complex that has been biochemically confirmed to be a sulfur reductase (49). The product of the Sre complex is H₂S, and the upregulation of Sre would explain why both DsrE and TusA were also upregulated (assuming an H₂S transfer role rather than a sulfane-sulfur transfer role under these conditions), as it would be important for toxic H₂S to be expelled from the bacterial cells. Taken together, these observations suggest that disproportionation of S⁰ occurs when A. ferrooxidans is grown under anaerobic conditions. Oxidation of S⁰ to SO₃²⁻ generates 4 electrons and 6 protons (assuming that oxygen is derived from water, as would necessarily be the case in anaerobic cultures; S⁰ + 3H₂O → SO₃²⁻ + 4e⁻ + 6H⁺), and oxidation of SO₃²⁻ to SO₄²⁻ generates 2 electrons and 2 protons (SO₃²⁻ + H₂O → SO₄²⁻ + 2e⁻ + 2H⁺). The reduction of S⁰ to H₂S consumes 2 electrons and 2 protons (S⁰ + 2e⁻ + 2H⁺ → H₂S). Therefore, more S⁰ would need to be reduced than oxidized to maintain the balance of electrons if sulfur disproportionation was the exclusive energy-transducing metabolism occurring in anaerobic cultures. However, Fe³⁺ respiration (discussed below), where electrons are transferred directly to Fe³⁺, would modify the stoichiometry of S⁰ oxidation and reduction. Disproportionation of S⁰ was initially thought of as surprising, as the process is endergonic under standard conditions, except where sulfide is removed by reaction with metals (50), as is shown here for A. ferrooxidans cultured in the presence of Fe³⁺.

Support for the hypothesis that A. ferrooxidans produces H₂S when grown anaerobically on S⁰ came from anoxic cultures incubated in the presence of Cu²⁺ and Fe²⁺. Concentrations of soluble Cu²⁺ fell by 35 � 7.7 mg/liter (from 10 mM to ∼9.45 mM, balancing the consumption of approximately 0.5 mM S⁰ and the production of CuS) under these conditions, while noninoculated controls showed no decrease, and black precipitates (presumed to be CuS) formed in inoculated cultures (Cu²⁺ + H₂S → CuS + 2H⁺) but not in sterile controls.

The SO₃²⁻ produced from S⁰ oxidation may be used for energy production by converting SO₃²⁻ to adenosine 5′-phosphosulfate (APS), which is then converted to SO₄²⁻ using ATP sulfurylase (Sat; which was not differentially regulated) to generate SO₄²⁻ and ATP (Fig. 1) (20). A minor portion of the SO₃²⁻ may also be reduced to H₂S by the sulfite reductase (CysI [AFE_3122]) and assimilated as required for cellular growth (51). Interestingly, cysI and cysH (APS reductase [AFE_3123]) were also upregulated under anaerobic conditions, even though the cellular growth requirement would not be expected to be greater under such conditions.

No definitive iron reductase in A. ferrooxidans was identified in this study. TetH (28) and ArsH (29) have been suggested to mediate Fe³⁺ reduction by A. ferrooxidans. However, in the present study, tetH (AFE_0029; Table 1) is the most highly upregulated gene under oxic conditions, and neither the RNA nor the protein product of arsH was detected above background levels (data not shown), diminishing the likelihood that either TetH or ArsH plays a major role in Fe³⁺ reduction.

We propose that an important functional coupling of S⁰ oxidation to Fe³⁺ reduction occurs via an indirect mechanism involving reduction of Fe³⁺ by H₂S generated by disproportionation of S⁰ under anaerobic conditions (2Fe³⁺ + H₂S → 2Fe²⁺ + S⁰ + 2H⁺) (Fig. 1B). An alternate hypothesis is based on the observation that the genes encoding the bc₁ complex (petA2 [AFE_2729] and petB2 [AFE_2730]) and the cytochrome c (cycA2 [AFE_2727]) were upregulated under anaerobic conditions (Table 1). In this model, electrons could flow from S⁰ to quinone and onwards to the bc₁ complex and then to CycA2, as shown in Fig. 1B. We do not know whether Cyc2 is the final protein of this electron transport chain reducing Fe³⁺ or whether there are additional members before the final acquisition of electrons by Fe³⁺, such as rusticyanin, as proposed by Kucera et al. (30). These mechanisms are not mutually exclusive and could potentially operate simultaneously as branched electron pathways or as alternate pathways. In either case, it would be important to understand how electron flow is regulated. An important consideration is that some strains of iron-oxidizing acidithiobacilli can grow by coupling the oxidation of hydrogen to the reduction of Fe³⁺ (7, 12). In such cases, S⁰ reduction to H₂S would not be expected to occur and an alternative mechanism for iron reduction other than the indirect mechanism must exist.

The aerobic electron transfer pathway and energy conservation have been investigated previously (see reference 20 and references therein), and the data in this study support the published model (Fig. 1). A proportion of the electrons generated from the anaerobic disproportionation of S⁰ could be transported by reverse electron flow to the NADH dehydrogenase. The energy required for this process could come from reverse proton motive force (24, 52).

Energy conservation under aerobic conditions is suggested to be via the F₀F₁ ATPase (53), and 3 of the 5 F₁ subunits (α-atpA [AFE_3205; identified in a mixture; see Table 1 for details], ε-atpC [AFE_3202], and the β subunit of atpD [AFE_3203]) were upregulated. The higher levels of the catalytic F₁ portion of the ATPase during aerobic growth suggest enhanced synthesis of ATP, which correlates with the observed higher growth rates under such conditions. In addition, a MotA/TolQ/ExbB proton channel family protein (AFE_0768) was upregulated; this has been shown in other organisms to be involved in electron transfer from NADH to the quinone pool, generating proton motive force (54). In contrast, under anaerobic conditions, the A (atpB [AFE_3209]), C (atpE [AFE_3208]), and B (atpF [AFE_3207]) F₀ subunits were upregulated (Table 1). The F₀ subunit does not require the F₁ catalytic portion to translocate protons, and therefore, a greater proportion of the proton transfer under anaerobic conditions may be used to generate a proton motive force rather than ATP. In addition to energy derived from generating a proton motive force, ATP can be generated from substrate-level phosphorylation (oxidation of SO₃²⁻ to SO₄²⁻) under both aerobic and anaerobic conditions, as described above. Also, the ubiquinone/menaquinone biosynthesis methyltransferase gene (ubiE [AFE_0289]) that is part of the ubiquinone biosynthesis pathway (55) was upregulated under anaerobic conditions.

A. ferrooxidans fixes CO₂ for growth via the Calvin-Benson-Bassham (CBB) cycle, and its genome contains a total of 5 cbb gene clusters with multiple copies of form I and a copy of form II ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO) (56). The second RubisCO copy (cbbL2 [AFE_2155]) from the cbb₂ operon was upregulated under aerobic conditions (Table 1; see Fig. S3 in the supplemental material), in agreement with earlier predictions (57). Under anaerobic conditions, several genes and proteins linked to CO₂ fixation (AFE_0536, AFE_1683, AFE_1685, AFE_1690, and AFE_3252) were upregulated (Table 1), probably due to the CO₂ supply being intermittent (56). Differences in carbon fixation in the presence of various levels of CO₂ are important for optimizing bioleaching (58).

Many proteins associated with central carbon metabolism were upregulated under aerobic conditions (Fig. 2 and Table 1), reflecting higher growth rates, with relatively few upregulated under anaerobic conditions. The former included the α and β subunits from the pyruvate dehydrogenase multienzyme complex (AFE_1813 and AFE_3069) that connects glycolysis and the tricarboxylic acid (TCA) cycle, a single protein (isocitrate dehydrogenase [AFE_0424]) from the incomplete TCA cycle present in A. ferrooxidans, pentose phosphate pathway proteins (Fig. 2) (including a 6-phosphogluconate dehydrogenase [AFE_2024] and glucose-6-phosphate 1-dehydrogenase [AFE_2025]), and several nonoxidative branch proteins (AFE_1843, AFE_3247, and AFE_0419), possibly to produce reducing power and ribulose-5-phosphate for carbon sequestration. Other proteins identified as being upregulated under aerobic conditions included those with roles in carbon/energy storage and others associated with the glyoxylate cycle (Fig. 2).

The most prominent membrane protein spots on the anaerobic gels were a reproducible train of 5 spots. All the spots were identified as an OMPP1/FadL/TodX family protein (AFE_2542; high levels of the respective gene products were detected under both conditions in the transcriptomics assay) and the major outer membrane protein 40 (AFE_0365; not upregulated between the conditions) (Table 1). The OMPP1/FadL/TodX family protein is suggested to transport hydrophobic compounds from outside the cell to the periplasm (59). The protein spots with acidic isoelectric point (pIs) had higher intensities under anaerobic conditions, while the spots with a more basic pI had higher intensities under aerobic conditions (see Fig. S2 in the supplemental material). OMPP1/FadL/TodX family proteins have been demonstrated to be located in the outer membrane (A. Yarz�bal and V. Bonnefoy, unpublished data) and to be upregulated during aerobic S⁰ metabolism compared to during Fe²⁺ oxidation (19, 60). The gene encoding survival protein A (surA [AFE_3035]), which is required for correct folding of outer membrane proteins (61), was also upregulated under anaerobic conditions.

Several oxidative stress and molecular chaperone proteins were upregulated under aerobic growth (Table 1), presumably due to response to production of oxygen free radicals and higher growth rates. Other genes and proteins observed to be differentially regulated during aerobic and anaerobic growth were those involved in translation, transcription, cell cycle, amino acid metabolism, and nucleotide metabolism and transport, which were upregulated under aerobic conditions, and ribosome subunit proteins and different nucleotide metabolism and transport genes/proteins, which were upregulated under anaerobic conditions (see Tables S2 and S3 in the supplemental material).

The results of this study suggest models for the coupling of S⁰ oxidation to Fe³⁺ reduction. It is posited that they provide useful frameworks for the design of future biochemical experiments.

Evidence from transcriptomic and proteomic analysis, supported by some physiological data, suggests that Fe³⁺ reduction by A. ferrooxidans grown anaerobically using S⁰ as electron donor is mediated, at least in part, by an indirect mechanism involving H₂S. A new model representing the proposed mechanism is shown in Fig. 1. The extent to which indirect iron reduction is complemented by a direct mechanism involving electron flow from S⁰ to Fe³⁺ via the bc₁ complex, a soluble cytochrome, and possibly other electron carriers is unknown. Since H₂S would not be generated as a waste product by A. ferrooxidans when grown on hydrogen (electron donor) and Fe³⁺ (electron acceptor), an additional mechanism to the indirect pathway in the present study presumably exists. Faster growth rates under aerobic conditions correlated with a number of proteins involved in central carbon pathways being upregulated.

Fe³⁺ reduction coupled to S⁰ oxidation results in the generation of Fe²⁺, which is an energy substrate for many bioleaching microorganisms. This could represent an important mechanism for recycling the Fe³⁺/Fe²⁺ couple and might be of considerable significance for heap bioleaching of copper and other minerals where gradients of air and nutrients occur.