Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli

Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases.

Variations in size of this enzyme are due to that ATP sulfurylase domain (ATS) can be bound to the APS kinase domain or similar to APS kinase (ASK). Exceptionally, ATP sulfurylase has been found to bind to other enzymes such as pyrophosphatase and APS reductase [5]. ATPS is involved in the assimilatory and dissimilatory reduction of sulfate catalyzing the activation of inorganic sulfate by ATP to form adenosine-5'-phosphosulfate (APS) and pyrophosphate [3]. The assimilation pathway is performed by aerobic organisms for amino acid synthesis such as cysteine, and the dissimilation pathway is carried out by prokaryotes, which in the absence of molecular oxygen they use sulfate as a terminal electron acceptor for cellular respiration, releasing hydrogen sulfide (H 2 S) [6]. Moreover, ATPS is involved in the indirect oxidation of sulfite, which could be present in a variety of chemolithoautotrophic and phototrophic bacteria, allowing for energy conservation by a chemiosmotic mechanism or by a substrate-level mechanism [7] where sulphite would be oxidized via a reverse adenosine phosphosulfate reductase (APR) activity according to: H 2 SO 3 + AMP ↔ APS + 2e -+ 2H + , with participation of ATPS (sulfate adenylyltransferase) in the final step according to: APS + PP i ↔ ATP + SO 4 2-.
Chemolithoautotrophic bacteria such as A. ferrooxidans would be responsible for the oxidation of sulphite in mining environments, where there is a large quantity of reduced inorganic sulfur compounds (RISC). In previous research the genomic sequence of A. ferrooxidans has been analyzed by bioinformatics software [8] and there are potential gene sequences that would encode the ATP sulfurylase enzyme. It is postulated that ATP sulfurylase is a monomer or homooligomer in organisms that oxidize reduced sulfur compounds and presents a subunit molecular weight (MW) ranging from 41 to 69 kDa, in a similar fashion to the ATPS sequence involved in sulfate assimilation found in Penicillium chrysogenum and Saccharomyces cerevisiae (58 kDa and 63.7 kDa subunits, respectively). ATPS from hyperthermophile chemolithotrophic Aquifex aeolicus is an ortholog of the filamentous fungi enzyme, with a functional ASK domain (APS kinase domain) [9, 10] and presents high similarity to the true APS kinases in the "P-loop" region.
By contrast, ATPS from chemolithotrophic Riftia pachyptila symbiont bacteria presents only the ATS domain. It is postulated that ATPS present in chemolithotrophic organisms is more similar to ancestral ATPS that gave rise to all homooligomeric ATP sulfurylases (ATPSs) [1,10]. Other homooligomeric ATPSs are present in higher eukaryotic organisms with domains in inverted orientation (ASK domain at the N-terminus and ATS domain at the C-terminus) different in relation to the ATPS from A. aeolicus and fungi [10]. Their presence has been reported in several animals, such as the marine invertebrate Urechis carpo [11] ATPSs of Desulfuvibrio species are metalloproteins that bind cobalt and zinc, and present a C-X2-C-X8-CXH characteristic sequence (metal binding site) [7]. Homooligomeric ATPSs are not similar to heterooligomeric ATPSs found in E. coli and responsible for sulfate assimilation [3,7].
Homooligomeric ATPSs have V blocks in the ATS domain, II and IV blocks are rich in basic amino acids suggesting that they participate in the binding of MgATP 2-and SO 4 2- (Sperling et al. 1998 Briefly, 4.5 ml of an overnight culture grown in Luria broth plus ampicillin was centrifuged, and the resulting pellet was resuspended in 200 µl of lysozyme (50 mg/ml) and 4 μl of STET and incubated for 5 min at room temperature. After that, the suspension was placed in water at 100 o C for 45 s, centrifuged at 13000 x g for 10 min and 8µl of RNAse (10 mg/ml) was added, following incubation for 15 min at room temperature. 8 μl of 5% CTAB was added and incubated for 3 min at room temperature. The pellet obtained by centrifugation at 13000 x g for 10 min was resuspended in en 300 μl of 1.2 M NaCl. The plasmid DNA was then precipitated with 750 μl of absolute ethanol, incubated for 5 min at room temperature and centrifuged at 13000 x g for 10 min. The obtained pellet was resuspended in 70% ethanol and centrifuged at 13000 x g for 5 min. Supernatant was discarded and the pellet was dried for 30 min. Then, it was resuspended in 10 µl of ultrapure water.
Genomic DNA, plasmid and PCR products were visualized by 1% agarose gel electrophoresis (Gibco BRL ® ) in TAE 0,5X. The molecular weight marker used was the 1kb Plus DNA Ladder (Invitrogen). Ethidium bromide (0.5 µg/ml) was used for a 5 min gel staining. Gels were visualized with a UV transilluminator at 320 nm. The atpS gene obtained from plasmid pET101-atpS was sequenced to verify the correct insertion of the gene in the plasmid and to obtain the nucleotide sequence of the gene. Sequencing was done using the following primers: T7-TopoF-vector

ATPS enzymatic activity of A. ferrooxidans by bioluminescence
The protein concentrations of E. coli BL21 strain Star TM (DE3) cell extracts was analyzed under induction and no induction conditions with IPTG and were determined using the Bradford reagent [21] to an absorbance at 595 nm. A bioluminescent test ATP Determination Kit (Molecular Probes) was used to determine the ATP sulfurylase activity according to manufacturer's instructions. It produced the standard reaction solution (10 ml) containing: H 2 O, 8.9 ml reaction buffer (20X), 0.5 ml; DTT (0.1 M), 0.1 ml; D-luciferin (10 mM), 0.5 ml; and luciferase (5 mg/ml), 2.5 μl. A 100 µl reaction was started with the addition of luciferase. Testing with extracts de E. coli to the reaction was made by addition of 0.125 μl of APS (8 nmol/μl) and 0.5 μl of PPi (40 ρmol/μl) for ATP production (according to the reaction: PPi + APS ATP + SO 4 2-). Subsequently, ATP produced was detected by bioluminescence (according to the reaction: Luciferin + ATP + O 2 oxyluciferin + AMP + pyrophosphate + CO 2 + light). The following controls were used: a) Control without luciferase, APS and PPi, b) Control with APS and PPi without luciferase and c) Control of the positive reaction with 5 µl of ATP (5 µM). Relative light units (RLU) readings were carried out in a microplate reader luminometer Synergy ™ HT Multi-Detection Microplate Reader (Bio-Tek, USA). Readings were controlled by KC4 v3.0 software with PowerReports (Bio-Tek). All RLU measurements were performed in duplicate at a wavelength of 530 nm for 15 min of reaction at 25°C. The quantity of ATP produced was calculated from the URL of standard ATP concentrations (0.25 to 10 µM) in duplicate at a wavelength of 530 nm at 25ºC. An enzymatic unit produces 1.0 µmol of ATP from APS and PPi per minute at 30ºC.

Bioinformatics and phylogenetic analysis of the amino acid sequence of the ATPS of A. ferrooxidans
From the amino acid sequence of the ATPS of A. ferrooxidans obtained in this work (CAQ76453) was determined the molecular weight and pI using the ProtParam program

Result: Identification of ATPS gene in the A. ferrooxidans ATCC 23270 genome
An open reading frame (ORF) for the atpS gene (1674 bp) encoding ATP sulfurylase (ATPS) was located at nucleotide positions 2327613 to 2329286 in the genome of A. ferrooxidans by bioinformatic analysis. Comparing this sequence with other ATPS of GenBank indicates the presence of an ATP sulfurylase domain (ATS) conserved typical of this type of enzyme, which is associated with an APS kinase domain (ASK) as occurs in some ATPSs [15]. The expected size of amplified atpS gene was obtained (1674 bp) and cloned in the pET101-TOPO vector resulting in the plasmid pET101-atpS, which was used to transform E. coli cells. Secuencing of atpS gene was performed from the plasmid extracted from recombinant E. coli TOP10.

Expression of recombinant A. ferrooxidans ATCC 23270 ATPS protein in E. coli
The cells of recombinant E. coli BL21 Star TM (DE3) on induction conditions with IPTG expressed a recombinant ATPS protein of approximately 63.5 kDa (Figure 1). E. coli BL21 Star TM (DE3) without plasmid was subjected to the same experiment and did not express the protein in any condition. The weight of the monomer of A. ferrooxidans ATPS is 60.49 kDa based on their amino acid sequence.

Enzymatic activity assay of the ATPS protein of A. ferrooxidans
Bioluminescence carried out at a wavelength of 530 nm at 25ºC for 15 min of reaction showed enzyme activity in cell extracts of recombinant E. coli strain BL21 (+IPTG) expressing ATPS, which is higher compared with those obtained from extracts of recombinant E. coli BL21 (-IPTG), and from negative controls. Specific activity of recombinant ATPS from A. ferrooxidans was calculated from cell extracts of E. coli, obtaining 106.9 μM ATP/mg/min.

Bioinformatics analysis of the amino acid sequence of ATPS from A. ferrooxidans
The biochemical characteristics of the ATP sulfurylase from A. ferrooxidans was determined from its amino acid sequence (CAQ76453) using bioinformatics programs. According to these results the ATPS of A. ferrooxidans would be a soluble protein of cytoplasmic localization similar to other homooligomeric ATPSs that have been described. It has a molecular weight de 60.49 kDa and has not signal peptide. The comparison of the amino acid sequence of ATP sulfurylase from A. ferrooxidans (CAQ76453) with other ATP sulfurylase, shows 93% identity and 95% similarity with ATP sulfurylase from A. ferrivorans (YP_004784724), 70% identity and 80% similarity with ATP sulfurylase from A. caldus ATCC 51756 (EET28427) and 44% identity and 60% similarity with the ATP sulfurylase from A. aeolicus (O67164), but also has similarity to ATPS found in Coxiella burnetii RSA493, fungi and yeasts. The high similarity of ATPS from A. ferrooxidans with homooligomeric ATP sulfurylases is demonstrated by multiple alignments using the Clustal W program (Figure 2).   3 HXhpXGXXKXXDhpXXXXR 215 , which is conserved among various families of homoligomeric ATP sulfurylases (hp = hydrophobic residues). The ASK domain of the ATPS from A. ferrooxidans has a high similarity with the ASK domain of the bifunctional ATPS from A. aeolicus and is identical to the active site of the true kinases ( 376 GLSASGKST 384 ) (Figure 3).

Discussion:
The enzymes ATP sulfurylases (ATPSs) belong to a superfamily of proteins and are widely distributed among microorganisms, protista, plants, animals and humans [2, 4, 5, 35] involved in desassimilative sulfate reduction, activation of sulfate assimilation and sulfur oxidation. The direct oxidation pathway of sulfite is the most widely distributed, but photolithotrophic and chemolithotrophic bacteria belonging to the β-and γ-

Proteobacteria also have an indirect oxidation pathway [7],
where ATPS participate in the final oxidation reaction, producing ATP and SO 4 2-from APS and PPi [1]. We have cloned and overexpressed the ATP sulfurylase from A. ferrooxidans in E. coli as a recombinant protein of approximately 63.5 kDa bound to six histidines (Figure 1), being the monomer molecular weight of 60.49 kDa protein (based on its 557 amino acids) similar to the monomer molecular weight of ATP sulfurylase (62.8 kDa) of the thermophilic chemolithotroph A. aeolicus [9]. Probably, the ATPS from A. ferrooxidans has a dimeric structure similar to chemolithotroph A. aeolicus [9] because it has conserved amino acids involved in the dimerization in the ATPS from A. aeolicus (results not shown), whereas in the ATPS from P. chrysogenum the subunits dimerize and then form a triad of dimers [36]. These results are consistent with the proposal by Sperling (1998) et al. [3], which each subunit of the monomeric or homooligomeric ATPSs would range from 41 to 69 kDa. The specific activity of ATPS from A. ferrooxidans for the synthesis of ATP determined from crude E. coli extracts is 106.9 units/mg/min. The specific activity for the ATPS purified from A. aeolicus was 13.1 units/mg proteins [9]. In contrast, The ATPS from T. denitrificans synthesizes around 400 units/g cell weight of ATP and the levels in the R. pachyptila symbiont is similar assuming that the bacterium represents 50% of trophosomal tissue [9]. The ATPS of A. ferrooxidans is functional in the direction of ATP synthesis involved in the oxidative metabolism of sulfite, and possibly with a low activity in the direction of APS synthesis (experiments not conducted) similar to the ATPS from A. aeolicus and the R. pachyptila symbiont [1,9]. In organisms such as sulfate assimilators P. chrysogenum and S. cerevisiae, ATPS activity by the molybdolisis method was 60 units/mg of protein and 39 units/mg of protein, respectively [37]. However, by the luminometer method the reported activity was 140 units/mg proteins for ATPS from S. cerevisiae due to the addition of APS in the filtration step of purification [38]. Moreover, in higher eukaryotes, the ATPS that integrates human PAPS synthetase has an activity of 18.7 units/mg [5]. In plants, ATPS activity in leaves of spinach (Spinacia oleracea L.) is 23.1 nmol ATP/mg/min [39] and Arabidopsis thaliana is 0.012 units/mg [40]. Some chemolithotrophic bacteria such as Acidithiobacillus could have three enzymes: sulfite-dependent cytochrome oxidase (APS), adenylyl phosphate (APAT) and ATP sulfurylase (ATPS), which could catalyze the reaction of terminal sulfate production in the complete oxidation of reduced sulfur compounds while others seem to possess one or two enzymes (9). The gene for the APS reductase (cysH gene) involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle [41] has been reported in A. ferrooxidans. The organisms that possess this APS reductase pathway are widely distributed in natural environments with high concentrations of sulfide or other reduced sulfur compounds and use this pathway to generate ATP or by attaching electrons to reduce CO 2 [42]. Although the conserved hypothetical gene (orf2) embedded in the hdr locus of sulfur oxidizers could also be involved, that strongly suggests that these microorganisms have a novel sulfur oxidation pathway in which sulfite is hypothesized to be produced in the cytoplasm by heterodisulfide reductase that in turn would catalyze the oxidation of sulfite to APS. Furthermore, the enzyme responsible for the second step in this pathways, has been reported by quantitative RT-PCR analysis of the atpS gene expression of A. ferrooxidans in the presence reduced inorganic sulfur compounds oxidation [43] and the presence of the an ORF to ATPS in the genome of the new specie described A. ferrivorans [44], which along with our results in the functionality of ATPS, leads us to suspect the involvement of ATPS from A. ferrooxidans in the oxidation of sulfite through the reverse path of APS reductase or a novel sulfur oxidation pathway. The ATS domain of ATPS from A. ferrooxidans has five highly conserved regions or blocks similar to all the homooligomeric ATPSs from archaea to higher eukaryotes (Figure 2), with blocks II and IV rich in basic amino acids that participate in the binding of MgATP 2-and SO 4   The ASK domain activity of ATPS from A. ferrooxidans remains to be established. However, it is possible an APS kinase activity because it has conserved amino acids compared to the ATPS from A. aeolicus that presents APS kinase activity (Figure 3 & Figure 6) [9], showing "P-loop" 376 GLSASGKST 384 motifs, whose sequence is identical to that found in true APS kinases ( 32 GLSASGKST 40 ) and is similar to the ATPS from A. aeolicus that presents the 379 GLPCAGKST 387 motif. By contrast, in ATPS from filamentous fungi this motif in the ASK-like domain has changed and only conserves four amino acids: 403 GYMNSGKDA 411 [9]. Other key residues of the ASK domain presents in the ATPS de A. ferrooxidans that have been described in the ATPS from A. aeolicus are present: a) Asp405 (Asp407 in A. aeolicus) that interacts with Mg +2 + ATP; b) Phe419 (Phe421 in A. aeolicus) and Phe503 (the same position in A. aeolicus), which would bind to the adenine ring of APS; c) the amino acid Arg410 (Arg412 in A. aeolicus) and Arg424 (Arg426 in A. aeolicus), which would associate the phosphosulfate group of APS, and d) Tyr453 (Tyr455 in A. aeolicus), which would help to align the Arg424 (similar to Arg426 in A. aeolicus). Additionally, e) the residue Lys489 (similar to A. aeolicus), as in true APS kinase, while in P. chrysogenum is Arg515 [46]. Furthermore, the arrangement of some helices, beta sheets and loops of this domain with true ASK kinase is very similar.
The evolutionary origin of homooligomeric ATPSs has not been determined; they have no similarity to heterooligomeric ATPSs. These two classes of ATPS with the same function are probably originated by convergent evolution [3]. The phylogenetic tree of homooligomeric ATPSs rooted with archaeal ATPSs leads us to suppose that probably the evolution of this enzyme started from the existence of ancestral atpS gene similar to subgroup 1 of the homooligomeric ATPSs with only ATS domain, presents in archaea and in Gram-positive bacteria mainly. Later, from ATPS ancestral gene would have originated in two subgroups: a) The subgroup 2 of the homooligomeric ATPSs which has two domains (ATS in the N-terminal and ASK or ASK-like in the Cterminal) and b) subgroup 3 of the homooligomeric ATPSs who have both domains in the reverse order to subgroup 2. Possibly the subgroup 2 is originated from a fusion of ancestral atpS gene with a gene encoding the APS kinase protein giving rise to a primitive bifunctional ATPS, whose homologous representative could be the bifunctional ATPS found recently in chemolithotrophic bacteria A. aeolicus from which the ATP sulfurylase of fungi would have evolved [10]. The ATPS from A. ferrooxidans has a high level of homology to the C-terminal domain (kinase domain) of the ATPS from A. aeolicus and presents an identical P-loop region to that of true APS kinases (Figures 3 & 6), which probably suggests that A. ferrooxidans possesses a functional enzyme of the subgrupo 2 more ancestral than that in A. aeolicus. The most basal location of the enzyme from A. ferrooxidans with A. ferrivorans and A. caldus in the phylogenetic tree would corroborate our hypothesis (Figure 4). Moreover, the subgroup 3 would have originated during the course of evolution after the divergence of an ancestral ATPS similar to subgroup 1 gave rise first to the ATPS of plants and then by fusion with the gene for APS kinase generated a bifunctional enzyme called PAPS synthetase in metazoans (ASK domain and ATS domain) as postulated earlier [47]. Alternatively, the domains of the ATPSs subgroup 2 or During the evolution of the homoligomeric ATPSs occurred horizontal atpS gene transfer events between some organisms whose expression allowed them to adapt their metabolism and lifestyle [1]. Similar results were obtained by Patron et al. (2008) [35] whose proposed that the inheritance of the enzymes of the ATPS, APR and PAPR have multiple origins in lineages that comprise opisthokonts (fungi and animals), gene fusions with other enzymes of sulphate assimilation pathway and evidence an eukaryote-to-prokaryote lateral gene transfer. Some organisms (Thiobacillus denitrificans, Phaeodactylum tricornutum, Thalassiosira pseudonana) present yet ATPS with only ATS domain and ATPS with ATS; ASK domains or fusion with pirophosphatase domain. The crystallography of the putative bifunctional ATPS (with ATS and ASK domains) from Thiobacillus denitrificans has shown that it only presents APS kinase activity, and exhibits numerous structural and sequence differences in the ATS domain to other ATPSs that render it inactive with respect to ATP sulfurylase activity, probably has unknown function [49]. The fusion of ATPS has happened not only with the gene for APS kinase, but with the inorganic pyrophosphatase enzyme in stramenopiles (in the diatom Thalassiosira pseudonana and Phaeodactylum tricornutum, and the oomycete Phytophtohora sojae) and on haptofites (algae such as Pavlova lutheria and Emiliania huxleri). Furthermore, in Heterocapsa triquetra is found the fusion of ATPS to APR, which probably would ensure a rapid transition from APS to the site of its reduction by increasing the production rate of sulfite [35]. Genomic analysis of the atpS gene region of A. ferrooxidans ATCC 23270 (16 kb) shows that the atpS gene is not associated with other genes in the metabolism of sulfur compounds especially with the APS reductase involved in the indirect route of oxidation of sulphite, but is associated with other transferases and chaperonins. An ORF that encoded the APS reductase of A. ferrooxidans was found in the genome the A. ferrooxidans ATCC 23270 strain but not adjacent to the atpS gene. By contrast, in the phototrophic sulfur oxidizer Chromatium vinosum, genes encoding for ATP sulfurylase and APS reductase (aprMBA, aprM encodes a membrane anchor protein) form an operon [50]. In the sulfate reducing archaeon A. fulgidus, the aprC gene that encodes a soluble protein with no known function, is inserted between the atpS gene (called sat) and the aprBA gene [3]. In the genome of C. tepidum, the genes for ATP sulfurylase and APS reductase are located adjacent to each other [7].

Conclusion:
We have demonstrated the expression of the gene encoding the enzyme ATP sulfurylase in the chemolithotrophic bacterium A. ferrooxidans 23270 that it would participate in the indirect pathway of sulfide oxidation to obtain energy. It presents an homooligomeric ATPS with: a) similarity at the sequence level and structure to homooligomeric ATPS with conserved motifs and mobile loop at the active site, b) Five blocks present in all homooligomeric ATPS, c) Enzyme activity producing ATP from APS and PPi, d) Presence of amino acids similar to ATPS from A. aeolicus involved in dimer formation, e) similarity to subgroup 2 of the homooligomeric ATPS, and f) size protein and cytoplasmic location. Subsequent studies of the ATPS present in A. ferrooxidans as purification and crystallization involved in the indirect oxidation of sulfite will be vital in understanding the mechanism of acid drainage generation used in bioleaching processes to improve the recovery rate of metals.