Acidithiobacillus caldus Sulfur Oxidation Model Based on Transcriptome Analysis between the Wild Type and Sulfur Oxygenase Reductase Defective Mutant

Background Acidithiobacillus caldus (A. caldus) is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs) for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox) system (omitting SoxCD), non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR). The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system. Results An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor) was created and its growth abilities were measured in media using elemental sulfur (S0) and tetrathionate (K2S4O6) as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR) of the wild type and the Δsor mutant in S0 and K2S4O6 media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO) and heterodisulfide reductase (HDR), the truncated Sox pathway, and the S4I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media. Conclusion An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.


Introduction
Acidithiobacillus caldus (A. caldus), a gram-negative, acidophilic, obligately chemolithotrophic, moderately thermophilic bacterium [1,2] and an important member of a consortium of microorganisms used for industrial bioleaching [3], plays key roles together with iron-oxidizing bacteria in bio-leaching processes [4,5]. A. caldus has the capability of oxidizing elemental sulfur and a wide range of reduced inorganic sulfur compounds (RISCs), but could not oxidize ferrous iron. It uses energy and electrons derived from sulfur oxidation for carbon dioxide fixation and other anabolic processes [1,2,6,7].
Oxidation of elemental sulfur and RISCs happens normally in some chemolithotrophic bacteria and archaea [8][9][10]. The sulfur oxidizing (Sox) enzyme system in lithoautotrophic Paracoccus pantotrophus (P. pantotrophus), responsible for the oxidation of sulfide, elemental sulfur, thiosulfate, and sulfite to sulfate, accompanied by electron transfer to cytochrome c, has been well studied [8][9][10][11][12]. It is located in the periplasm and constituted generally by four proteins: SoxYZ, SoxAX, SoxB and Sox(CD) 2 [13]. Initially, SoxAX initiates the oxidation of thiosulfate (S 2 O 3 22 ) producing SoxY-thiocysteine-S-sulfate (SoxYZ-S-S-SO 3 2 ) [14]; secondly, SoxB hydrolyzes sulfate (SO 4 22 ) from the thiocysteine-S-sulfate residue (SoxYZ-S-S-SO 3 2 ) producing S-thiocysteine (SoxYZ-S-S 2 ); thirdly, Sox(CD) 2 may oxidize the outer sulfur atom of Sthiocysteine producing SoxYZ-cysteine-S-sulfate (SoxYZ-S-SO 3 2 ); finally, sulfate is hydrolyzed and removed by SoxB from SoxYZ-S-SO 3 2 , and SoxYZ is regenerated. However, Sox(CD) 2 is absent in the so-called truncated Sox system of many prototypical a-Proteobacteria [8,10]. Another sulfur oxidation system based on the sulfur oxygenase reductase (SOR) has been elaborated in several acidophilic and thermophilic archaea [10,15]. SOR is able to catalyze the disproportionation of elemental sulfur, producing sulfite, thiosulfate, and sulfide [16,17]. The reaction has several characteristics: (1) it takes place in the cytoplasm; (2) it is dioxygen (O 2 )-dependent with no external cofactors or electron donors required [16]; (3) sulfur oxidation is not coupled with electron transfer or substrate-level phosphorylation in Acidianus ambivalens (A. ambivalens) [18]. The sulfur oxidation system in acidophilic Acidithiobacillus genus is related to three representative species widely used in bioleaching, which are A. ferrooxidans, A. thiooxidans and A. caldus. Although, the sulfur metabolic mechanism in A. ferrooxidans has been studied for many years, it is still not completely understood. The elemental sulfur in nature consists of a stable octasulfane ring (S 8 ), which forms orthorhombic crystals insoluble in water [19]. An elemental sulfur activation and oxidation model was proposed in Acidithiobacillus [20,21]. The S 8 is firstly activated to become thiol-bound sulfane sulfur atoms (R-S-SH) and then transported into the periplasm where it is oxidized by sulfur dioxygenase (SDO) [20,21]. SDO enzyme activity was detected from the crude cell extracts of A. ferrooxidans and A. thiooxidans, but SDO enzyme protein has not been purified and the gene(s) encoding for SDO activity not yet been identified [20,22]. Another elemental sulfur oxidation enzyme in A. ferrooxidans is the cytoplasmic heterodisulfide reductase complex (HdrABC), but it is a speculation from the genomic and transcriptomic analysis, not from biochemical experimental data [23]. The S 4 I pathway of thiosulfate oxidation via the form of tetrathionate intermediates exists widely in Acidithiobacillus: the periplasmic thiosulfate is oxidized to tetrathionate by thiosulfate quinine oxidoreduetase (TQO); then, tetrathionate is hydrolyzed by tetrathionate hydrolase (TetH) yielding thiosulfate and other products [10,24]. In addition, other RISC oxidation enzymes identified in Acidithiobacillus include: sulfide quinone reductase (SQR) being responsible for oxidation of hydrogen sulfide [25], and rhodanese or thiosulfate sulfurtransferase (TST) transferring a sulfur atom from thiosulfate to sulfur acceptors like cyanide and thiol compounds [26,27]. Recently, a model of sulfur oxidation in A. ferrooxidans was proposed, in which electrons from oxidation of RISCs are transferred via the quinol pool (QH 2 ) to terminal oxidases to produce ATP or to NADH complex I to generate NADPH, coupling the oxidation of RISCs with the generation of energy or reducing power [23].
An unique sulfur oxidation system exists in A. caldus, which is quite different from that of A. ferrooxidans according to comparative genome analysis [28]. The sulfur oxidation system of A. caldus can be classified into three subsystems: the truncated Sox subsystem, non-Sox sulfur subsystem, and SOR subsystem. The truncated Sox subsystem in A. caldus contains two copies of essential soxABXYZ genes but no soxCD genes. The non-Sox sulfur subsystem, similar to the sulfur oxidation system in A. ferrooxidans, contains the sulfur oxidation enzyme genes (tetH, sqr, rhd, and hdrABC) and terminal oxidase genes. The SOR subsystem is characterized by the sulfur oxygenase reductase gene (sor) in A. caldus, which was only found in several acidophilic and thermophilic archaea but not in the two species A. ferrooxidans and A. thiooxidans [10,29,30]. Therefore, A. caldus has a complex and integrated sulfur oxidation system. The currently known sulfur oxidation pathways in A. caldus are mainly acquired from the genome sequence analysis, which are not clear and remain unanswered questions: (1) How is the elemental sulfur oxidized and how many elemental sulfur oxidizing pathways exist? (2) How does the truncated Sox subsystem work? (3) How do the various pathways interconnect to complete the sulfur oxidation? Although, a rough model for ISCs metabolism in A. caldus was proposed [31], the above questions are still awaiting clear answers.
Recently, a method for A. caldus mutant construction was developed in our laboratory, which is different from the previous reported gene knock-out method based on the homologous recombination in A. caldus [32]. In our experiments, construction of gene mutant via the transposition insertional mutagenesis of the insertion sequences (IS elements) in A. caldus was discovered for the first time. The first report about IS element transposition in Acidithiobacillus was the ISAfe1 insertional inactivation of resB (a putative cytochrome c-type biogenesis protein) producing the A. ferrooxidans mutant, which lost Fe (II) oxidation ability [33,34]. IS elements are also widely distributed in A. caldus [35]. The loss of the sor gene resulting from the ISAtc1 transposition in A. caldus SM-1 was proposed according to the comparison of A. caldus SM-1 genome sequence and the sor gene cloned from SM-1 [30,35].
In this research, A. caldus MTH-04 Dsor mutant is constructed by electroporation of a suicide plasmid. The cell growth of the mutant is measured using S 0 and K 2 S 4 O 6 as the substrates. Then, comparative transcriptome analysis is carried out by wholegenome microarray and quantitative RT-PCR. Finally, an integrated sulfur oxidation model in A. caldus is proposed.

Construction of Dsor mutant
The suicide plasmid pMD19sor::V-Cm ( Figure 1A) derived from pUC19 is unable to replicate in A. caldus. It carries the homologous sequence of the disrupted sor gene by insertion of chloramphenicol resistance gene (cat, 816 bp) generating two homologous sequences, the L-arm (1,321 bp) and the R-arm (1,332 bp) ( Figure 1A). A. caldus MTH-04 was electroporated using the suicide plasmid and the mutants were screened using colony PCR, then the chromosomes of the screened mutants were extracted and analyzed by PCR ( Figure 1B) and southern blot analyses ( Figure 1D). As shown in Figure 1B, two sets of primers (SorA fwd and SorA rev, SorB fwd and SorB rev) specific to the sor gene and a set of primers (Big fwd and Big rev) specific to the homologous sequence were used to verify the mutant. Fragments of 684 bp (lane 2, Figure 1B) and 971 bp (lane 3, Figure 1B) specific to the sor gene were amplified from the chromosome of the wild type, but not from the mutant. An 8 kbp fragment amplified from the mutant using the primers Big fwd and Big rev (lane 7, Figure 1B) was larger than the fragment of about 6 kbp amplified from the wild type (lane 6, Figure 1B). The fragments of lane 6 and lane 7 in Figure 1B were sequenced for validation. The results are shown in Figure 1C: the sor gene region (from 2,403 bp to 4,343 bp) in A. caldus MTH-04 wild type was different from the corresponding region (from 2,403 bp to 6,537 bp) which is replaced by the IS element (ISAtc1) and the transposase gene in the A. caldus MTH-04 mutant. In order to further confirm the mutant, southern blot analysis was carried out using the sor gene as the probe. As shown in Figure 1D, an EcoR I-fragment (6.7 kbp) was isolated from the wild type chromosome, but not from the mutant. In addition, the analysis of the sor regions in different A. caldus strains is depicted in Figure 1C, ISAtc1 is positioned upstream of the sor gene in the wild type A. caldus MTH-04 genome, similar to A. caldus SM-1 but different from A. caldus ATCC 51765, while the sor gene region in the Dsor mutant was replaced by the IS elements.
The hypothesis that IS element transposition results in the loss of sor gene in A. caldus SM-1 has been proposed according to sequence analysis [35]. Fortunately, it was confirmed for the first time in our experiments. Firstly, the same result was obtained in the repeated experiments and no mutant was obtained when plasmid pMD19 or pSDU1 was used, which showed that deletion of sor gene was not a natural occurrence. Secondly, no artificial or exogenous IS elements was introduced into the suicide plasmid. Finally, the IS element (ISAtc1) causing the loss of sor gene could be found on A. caldus MTH-04 wild type chromosome. For above reasons, it is clear that the transposition of IS elements in A. caldus MTH-04 chromosome leads to the loss of sor gene. Moreover, it also implies the role of IS elements in gene evolution and metabolic diversity in A. caldus.

Growth curve analysis of the wild type and the Dsor mutant
The growth curves of the wild type and the mutant in Starkey-S 0 or Starkey-K 2 S 4 O 6 liquid media are shown in Figure 2. When S 0 was utilized as the sole substrate, the cell concentration of the mutant was a little higher than the wild type in the first six days, but was slightly lower for the next six days (Figure 2A). When K 2 S 4 O 6 was used as the sole substrate, the mutant had an obvious growth advantage compared to the wild and its maximum cell concentration was 70% higher than the wild ( Figure 2B). The mutant still had S 0 oxidation ability, indicating that SOR is not the sole and determinative elemental sulfur oxidation pathway and there are other additional elemental sulfur oxidation pathways in A. caldus. In addition, the maximum value of OD 600 of A. caldus in S 0 medium in Figure 2A could reach about 0.35 much higher than that in K 2 S 4 O 6 medium (OD 600 <0.065), so it is suggested that A. caldus has a high efficient elemental sulfur oxidizing ability to enable it grow well.

Comparative transcriptome analysis
Hybridization scheme, microarray experiments, and data analysis are described in materials and methods. Unsupervised hierarchical cluster analysis of sulfur oxidation genes are depicted in Figure 3. It shows the normalized transcription levels of sulfur oxidation genes for each sample compared to the common reference. Several remarkable differences are revealed in this figure: firstly, there was a bright green region related to the genes in the sox operons of the wild but a red color region of the mutant in K 2 S 4 O 6 medium, suggesting that genes in the sox operons had a low expression level in the wild and a high expression level in the mutant when K 2 S 4 O 6 was applied; secondly, the bright green color appeared in the row of sor gene of the mutant was resulted from the deletion of sor, moreover the sor gene of the wild displayed red color in K 2 S 4 O 6 medium and green color in S 0 medium, indicating the higher expression level of sor in K 2 S 4 O 6 medium; thirdly, the green region at the lower right corner of the figure indicated that genes involving rhd, dsrE, and tusA had lower expression levels in the mutant when K 2 S 4 O 6 was applied.
The results of significance analysis of microarrays (SAM) for sulfur oxidation genes under four cases are shown in Table 1 (the numbers without brackets). The expression patterns observed with microarrays were validated by using real-time quantitative RT-PCR (qRT-PCR) for some sulfur oxidation genes ( Table 1, the numbers in the brackets). The results from microarrays and qRT-PCR were highly consistent. Genes that met the criterions of fold change $1.5 with q-value#0.05 and fold change #0.667 with q-value#0.05 were up-regulated and down-regulated, respectively. The expression of sulfur oxidation genes showed distinct differences under the four experimental cases (Table 1). In the first case, the comparison was between the mutant and the wild using S 0 as sulfur source shown in the column of MT/WT (S 0 ). No obvious difference was found in the expression levels of sulfur oxidation genes except that the expression of sor was not detected in the mutant. The results implied that deletion of sor had no obvious influence on the other sulfur oxidation pathways in S 0 medium at the early phase. In the second case, K 2 S 4 O 6 was used as sulfur source to analyze the differential gene expressions between the mutant and the wild. The loss of sor led to significant gene expression changes in K 2 S 4 O 6 medium, in particular: (1) the remarkable up-regulation of genes in the two sox operons; (2) the significant up-regulation of genes in tetrathionate hydrolase operon (tetH and doxD); (3) the obvious down-regulation of sulfur transferase genes, such as dsrE (ACAL_2473), tusA (ACAL_2474), and rhd (ACAL_2475, ACAL_2860). The results showed that deletion of sor stimulated the up-regulation of the sox pathway and the S 4 I pathway in K 2 S 4 O 6 medium. In the third case, the effect of different sulfur sources on the gene expression of the wild type was showed in the column of S 0 /K 2 S 4 O 6 (WT). Sulfur oxidation genes of the two sox operons, tetrathionate hydrolase operon, and heterodisulfide reductase complex operon had higher expression levels of varied degrees when S 0 was added, in contrast, sor had a lower expression level. Therefore, the lower expression level of sor gene at the early fast growth phase in S 0 medium and the better growth of the mutant at the early days in S 0 medium implied that SOR does not play an important role at the early stage of the elemental sulfur oxidation process. The fourth case showed the differential gene expressions of the mutant in the two kinds of media. An obvious change compared to the third case was that the majority of sulfur oxidation genes of sox operons and tetrathionate hydrolase operon had higher expression levels in K 2 S 4 O 6 medium than that in S 0 medium. From above results together with the following facts that the lower expression levels of these genes of the wild type in the K 2 S 4 O 6 medium (Table 1, column of S 0 /K 2 S 4 O 6 (WT)) and the no differential expression of these genes between the mutant and the wild in S 0 medium (Table 1, column of MT/WT (S 0 )), it can be concluded that there was a significant increase of the expression levels of above genes in the mutant compared with that in the wild type in K 2 S 4 O 6 medium. Above conclusion showed that deletion of SOR pathway stimulated the sox and S 4 I pathways in K 2 S 4 O 6 medium.

Model of the sulfur oxidation system in A. caldus
The role of SOR in elemental sulfur oxidation. Our experimental data can be explained when SOR is considered a cytoplasmic enzyme oxidizing elemental sulfur in the cytoplasm without coupling with the electron transfer chain or substrate-level phosphorylation in A. caldus. A hypothetical model is shown in Figure 4, sulfur atoms (S) produced from other sulfur oxidation pathways in the periplasm can be accumulated in form of polymeric sulfur (S n ), then S or S n are transported via an unknown mechanism into the cytoplasm where they are immediately oxidized by SOR. The higher expression level of sor gene in K 2 S 4 O 6 medium can be explained by cytoplasmic located SOR. K 2 S 4 O 6 is much easily and quickly entering into the periplasm, where it is hydrolyzed by TetH to produce sulfur atoms and transported into the cytoplasm inducing the expression of sor. While S 0 needs to be activated before being transported into the periplasm, which is slower than that of K 2 S 4 O 6 , resulting in the lower expression level of sor in S 0 medium. The role of SOR is supported by the facts that the sor (ACAL_1435) gene that encodes a sulfur oxygenase reductase with the ability to oxidize S 0 , has been cloned and expressed in E. coli and the enzymatic activity of S 0 oxidation has been detected without addition of glutathione in our laboratory (unpublished data). The location of SOR in cytoplasm is in agreement with the reports that there are no intracellular sulfur globules in A. caldus, whereas sulfur globules are accumulated in A. ferrooxidans which acks sor gene [36], and SOR is reported existed in the cytoplasm in archaea [10].
SOR is not coupled with the electron transfer chain or substrate-level phosphorylation. The obvious growth advantage of the Dsor mutant in the first six days in S 0 medium or during the whole growth period in K 2 S 4 O 6 medium ( Figure 2) should be caused by the sulfur oxidation pathway shifting from SOR to other pathways coupled with the electron transfer chain. Moreover, it is not an efficient way to produce electrons for sulfur atoms to be oxidized by SOR when sulfur atoms in cytoplasm are insufficient caused by the delay of sulfur activation and transportation at the early growth stage in S 0 medium. In contrast, when sulfur atoms are sufficient at the late growth stage in S 0 medium, SOR oxidizes the sulfur atoms to produce other sulfur compounds, which enter other sulfur oxidation pathways in the cytoplasm to produce electrons. These could be the reason for the lowered cell growth of the Dsor mutant in S 0 medium than the wild type after six days of cultivation ( Figure 2A). SOR plays a central role in the cytoplasmic sulfur oxidation pathways. The possible products from the SOR catalyzed reactions are thiosulfate, sulfite and sulfide [17,37]. Thiosulfate is considered to be catalyzed by rhodanese (TST) in the cytoplasm of A. caldus according to the reasons: (1) TST widely exists in the cytoplasm of both prokaryotes and eukaryotes, and it transfers a sulfur atom from thiosulfate to thiol compounds producing persulfide and sulfite [27,38]; (2) there is an obvious relevance between the expressions of sor and rhodanese (rhd) genes. As shown in Figure 3, when K 2 S 4 O 6 was applied, the expressions of sor and rhd (ACAL_2475, ACAL_2860) showed red and dark colors, respectively, for the wild type, while the expressions of the two rhd genes showed bright green for the Dsor mutant; when S 0 was applied, the expressions of the two rhd genes showed a little brighter for the Dsor mutant than that for the wild. Above results showed that the expressions of the two rhd genes down-regulated when sor was deleted. Consequently, a hypothetical thiosulfate oxidation pathway was put forward and shown in Figure 4: the thiol proteins (RSH) in the cytoplasm of A. caldus can be used as sulfur atom acceptors for the catalysis of thiosulfate by TST, producing sulfane sulfate (RSSH) which is used as the substrate of the heterodisulfide reductase complex (HDR), thus a cycle is formed, in which RSH obtains a sulfur atom to form RSSH catalyzed by TST and then RSSH is oxidized by HDR to regenerated RSH. Sulfite is toxic to the cell and needs to be oxidized rapidly. An APS (adenosine-59-phosphosulfate) pathway for sulfite oxidation maybe exists in A. caldus which is similar to the sulfite oxidation pathway in A. ferrooxidans [23]. A putative sulfate adenylyltransferase dissimilatory-type/denylylsulfate kinase (sat) gene was discovered in A. caldus MTH-04 but the APS reductase gene that catalyzed sulfite to adenosine-59-phosphosulfate (APS) was not determined (see Figure 4). Sulfide could be converted to hydrogen sulfide and then oxidized by sulfide:quinine reductase (SQR) located in the cytoplasmic membrane. Therefore, it is presumed that SOR plays a central role in the cytoplasmic elemental sulfur oxidation in A. caldus.
Other elemental sulfur oxidation pathways. An important elemental sulfur oxidation pathway in the periplasm near the outer membrane of A. caldus is hypothesized as shown in Figure 4: the extracellular elemental sulfur (S 8 ) is activated by thiol groups of special outer-membrane proteins and transported into the periplasmic space as persulfide sulfur (R-SH), and then oxidized by the sulfur diooxygenase (SDO), meanwhile hydrogen sulfide is produced during the activation of S 8 . Sulfur diooxygenase gene (sdo) in the A. caldus MTH-04 genome was discovered, cloned and expressed in E. coli in our laboratory, and the enzymatic activity of SDO was detected with the addition of glutathione (data to be published). Although several outer-membrane proteins related to sulfur oxidation in A. ferrooxidans have been reported, the thiolbearing membrane proteins are not yet identified [39,40]. Another proposed elemental sulfur oxidation pathway locating in the cytoplasm near the inner membrane is shown in Figure 4: heterodisulfide reductase (HDR) catalyzes sulfane sulfate (RSSH) to produce sulfite and regenerate RSH. Two subunits genes (hdrB and hdrC) of HDR were found in the draft genome sequence of A. caldus MTH-04 but all genes (hdrA, hdrB and hdrC) of HDR were found in A. caldus ATCC 51756 [31].
In summary,there are three elemental sulfur oxidation pathways in A. caldus: (1) the pathway based on SDO in the periplasm; (2) the pathway based on the HDR in cytoplasm near the inner membrane; and (3) the pathway based on the SOR in the cytoplasm.
The Sox pathway. The truncated Sox pathway in A. caldus MTH-04 contains two sox gene clusters, sox operon I (soxX-soxY-soxZ-soxA-hyp-soxB) and sox operon II (coxB-cox-cyoC-hyp-hyp-soxY-soxZ-soxB-hyp-resB-soxX-soxA-resC). There is also a strong connection between the expressions of the sox genes and the terminal oxidase genes. As shown in the columns MT/WT (K 2 S 4 O 6 ) and S 0 /K 2 S 4 O 6 (WT) in Table 1, the expression of terminal oxidase genes, especially the cytochrome bo 3 ubiquinol oxidase genes, upregulated significantly when sox genes had high expression levels. The well-studied Sox pathway in P. pantotrophus demonstrated that it couples the sulfur oxidation with the electron transfer [8]. Besides, the sox operon II together with a bo 3 ubiquinol oxidase operon make up a big gene cluster on the chromosome, which implies the feasible regulation and control on their transcriptional level. The hypothetical pathway is shown in Figure 4: electrons produced from the Sox system via QH 2 were transferred to the terminal oxidases (bd and bo 3 ) and the NADH   complex to produce ATP and NADPH, respectively. However, an important question is raised as to how the truncated Sox system works in A. caldus in the absence of SoxCD. Two possible ways to regenerate SoxYZ are put forward. One way is that the sulfur atom of the sulfane intermediate (SoxYZ-S-S 2 ) is dropped from SoxYZ, which has been reported in other bacteria [10,41,42]. Another way is that the sulfur atom of the sulfane intermediate is oxidized by sulfur dioxygenase (SDO), because SoxY is actually a kind of thiol-bearing protein that may be used as an oxidation substrate of SDO. In addition, SoxY is presumed to play a role in the activation of S 8 for the reasons that elemental sulfur (S 8 ) assembled on the thiol of SoxY via nonenzymatic conjugation has been reported [10,42], and the expression of soxY gene of the wild type is much higher in S 0 medium than that in K 2 S 4 O 6 medium (Table 1). Therefore, the Sox system in A. caldus has capabilities to oxidize a wide range of sulfur compounds including sulfite, thiosulfate, sulfide and elemental sulfur and couple the process  with electron transfer, which make the Sox pathway play a central role in the periplasmic sulfur oxidation system. The S 4 I pathway. The reaction cycle linking the hydrolysis of tetrathionate and the oxidation of thiosulfate is an important sulfur oxidation pathway in the periplasm of A. caldus. Tetrathionate hydrolase (TetH), a soluble periplasmic enzyme, is responsible for the hydrolysis of tetrathionate in A. caldus [43]. One of the hydrolysates of tetrathionate catalyzed by TetH is thiosulfate, but the other products are uncertain [43,44]. The higher expression level of the sor gene in K 2 S 4 O 6 medium than that in S 0 medium (Table 1) implied that sulfur atoms (S) may be one of the products [44]. Thiosulfate quinine oxidoreduetase (TQO), which is constituted of subunits DoxA and DoxD, catalyzes thiosulfate producing tetrathionate. The expression levels of tetH and doxD of the wild type in S 0 medium were much higher than that in K 2 S 4 O 6 medium ( Electron transfer from sulfur oxidation to respiratory system. Little is known about the electron transfer chain in A. caldus, but it has been well studied in A. ferrooxidans. One of the electron transfer chains in A. ferrooxidans is that electrons from Fe (II) oxidation flow through Cyc2 to rusticyanin, and then reduce oxygen via a cytochrome aa 3 complex (downhill pathway) or reduce NAD + via a bc1/quinone pool/NADH complex (uphill pathway) [23,46,47]. The other one is that electrons from RISCs oxidation are transferred via the quinol pool (QH 2 ) either to terminal oxidases bd, bo 3 and aa 3 to produce a proton gradient, or to NADH complex I to generate reducing power [23,46]. A. caldus only has the sulfur oxidation system and its genome information shows that four copies of bd ubiquinol oxidase genes (cydAB), two gene clusters encoding bo 3 ubiquinol oxidase (coxB and cyoC) and a cytochrome c oxidase gene (cox, ACAL_2516) exist in the A. caldus MTH-04 draft genome. SoxAX, a c-type cytochrome complex that consists of SoxA as a diheme subunit and SoxX as a monoheme [48], can carry two electrons during the catalyzing process in the Sox pathway. The comparative transcriptome analysis showed that once the expression of genes in Sox pathway upregulated, the expression of bd and bo 3 oxidases genes were also upregulated (Table 1), which implied that electrons could be transported from the Sox pathway to bd and bo 3 oxidases in A. caldus. Therefore, a hypothesized electron transfer chain based on the quinol pool in A. caldus is depicted in Figure 4: electrons from SQR, TQO, HDR and SoxAX are transferred via the quinol pool either directly to terminal oxidases bd or bo 3 to produce the proton gradient to generate ATP, or directly to NADH complex I to generate reducing power. Furthermore, there are several features of the electron transfer in A. caldus including: (1) SoxAX connects the Sox pathway with the electron transfer chain, so that makes the Sox pathway become an important sulfur oxidation pathway for producing mass electrons; (2) Quinol terminal oxidases (bd and bo 3 ) are the dominant terminal oxidases as more electrons are produced from S 0 oxidation than that from Fe (II) oxidation; (3) The quinol pool (QH 2 ) located in the cytoplasmic membrane may play a regulatory role in the electron transfer chain. Overall, a powerful electron transfer and respiratory system including the quinol pool (QH 2 ), terminal oxidases and NADH complex exist in A. caldus.

Conclusions
The growth measurements of the wild type and the mutant, and the comparative transcriptome analysis extend our understanding of the complicated sulfur oxidation system in A. caldus. Several distinctive features of the sulfur oxidation system of A. caldus are summarized as follows: (1) The sulfur oxidation system of A. caldus involves the Sox subsystem, a non-Sox sulfur subsystem similar to that in A. ferrooxidans, and SOR which work together to complete the oxidation of elemental sulfur and various sulfur compounds, so that A. caldus has a growth advantage over any other bacteria in the bio-mining reactor [35]. (2) Regional division of the sulfur oxidation system in A. caldus is an obvious characteristic. The first region is the outer membrane of the periplasm where the extracellular elemental sulfur (S 8 ) is activated and transported into the periplasmic space as persulfide sulfur(R-SH). The second region is the periplasm where the Sox pathway, SDO and TetH perform their functions, and this is an important place for sulfur oxidation. Moreover, nonenzymatic reactions producing thiosulfate from sulfite and sulfur atoms and forming polysulfur (S n ) are complementary to the periplasmic sulfur oxidation system. The third region is the cytoplasmic membrane involving SQR, TQO and HDR with a common feature of coupling sulfur oxidation with electron transport. The fourth region is the cytoplasm with an SOR-based sulfur oxidation system. (3) There are complex controls on the sulfur oxidation process. On one hand, the sulfur oxidation related genes belong to different pathways regulated at the transcriptional level to adapt to the production and consumption of various sulfur compounds during the process of elemental sulfur oxidation. On the other hand, metabolic control on the substrate level may be an important regulatory method, as the poly-sulfur (S n ) and poly-thionates (S 4 O 6 22 ) accumulated in the periplasm could be the main forms for energy storage.
In summary, an integrated sulfur oxidation model of A. caldus is proposed based on comparative transcriptomic analysis, which provides new insights and guides for the future study of the sulfur oxidation metabolism. In view of the diversity of the RISCs and the complexity of the sulfur oxidation system of A. caldus, many fundamental questions such as identification of sulfur oxidation genes and determination of enzyme activities remain to be resolved. Fortunately, the establishment of genetic manipulation of A. caldus provides effective and powerful tools for elucidation of the sulfur oxidation mechanism of A. caldus [32,49,50].

Bacteria and culture conditions
The bacterial strains and plasmids used are listed in Table 2. The media of Luria broth (LB) or agar plate for E. coli were described in reference [51]. Liquid Starkey-S 0 and Starkey-K 2 S 4 O 6 inorganic medium and solid Starkey-Na 2 S 2 O 3 medium for cultivation of A. caldus MTH-04 were prepared as described in reference [52]. Elemental sulfur (S 0 ) (boiling sterilized, 20 g/L) and K 2 S 4 O 6 (membrane filtration, 5 g/L) was added before inoculation. Chloromycetin (Cm) was added to the final concentrations of 34 mg/ml in LB medium, 60 mg/ml in liquid Starkey-S0 medium and solid Starkey-Na 2 S 2 O 3 medium, respectively. The cultivation temperatures were 37uC for E. coli and 40uC for A. caldus MTH-04. The shaking speed for liquid cultivation of A. caldus MTH-04 was 125 r/min if not specifically stated.

Generation of mutant
The suicide plasmid was constructed as follows. The essential part of the suicide plasmid was amplified from pSIM-PLE19hsdM::V-Cm using the primers of PMD fwd and PMD rev, then digested with Sal I and Not I. A 2.6 kbp homologous sequence arm holding the sor gene was amplified from A. caldus MTH-04 chromosome using the primers of Whol fwd and Whol rev, digested with Sal I and Not I, and ligated to the essential part of the suicide plasmid. The resulting plasmid was named pMD19sor, then linearized in the middle of the sor gene, using the primers of Mid fwd and Mid rev. The linear plasmid pMD19sor was digested with Kpn I and Bgl II, and ligated to the chloramphenicol resistance gene (cat) amplified from plasmid pSDU1 using primers of Cat fwd and Cat rev digested with Kpn I and Bgl II. The generated plasmid carrying the mutant allele of sor disrupted by the cat was the suicide plasmid pMD19sor::V-Cm. The suicide plasmid was sequenced by Invitrogen Corp. for sequence confirmation. The sequences of the primers are listed in Table 3. The restriction enzymes and Prime STAR HS Taq were purchased from TaKaRa Corp. The suicide plasmid was electroporated into A. caldus MTH-04 using the methods described in the reference [49].
Colony PCR was used to screen the mutants using the primers of Clnfwd and Cln rev. The mutants were incubated in liquid Starkey-S 0 medium, and then collected to extract the chromosome. The chromosomes of the mutants were analyzed using PCR. Firstly, two sets of primers (SorA fwd and SorA rev, SorB fwd and SorB rev) specific to the sor gene were used to verify the mutants. Then, PCR amplification was done using primers of Big fwd and Big rev specific to the mutant allele using LA taq (TaKaRa Corp.) and the amplified fragments were sequenced by Invitrogen Corp. for sequence confirmation. The sequences of the primers used are listed in Table 3.
Southern hybridization was done to further confirm the mutants. Genomic DNA of the wild type and the mutants were extracted using a QIAmpH DNA Mini kit (Qiagen Corp.), digested with EcoRI(NEB Corp.), electrophoresed on agarose gel, and transferred by capillary blotting to positively charged Hybond-N membranes (Roche Corp.). The sor probe was obtained by incorporation of alkali-labile DIG-dUTP (Roche Corp.) during PCR elongation with primers SorAfwd and SorA rev. Hybridization was carried out under stringent conditions as recommended by the manufacturer.

Growth measurements of the wild type and the Dsor mutant
A. caldus MTH-04 and the Dsor mutant were inoculated into 150 ml of fresh Starkey-S 0 medium with bacterial loading at 10% (vol/vol). Cells were harvested at the stationary growth phase. The solid sulfur in the cultivation broth was removed by low-speed centrifugation (1006g) before cell harvesting. Then, the cells were harvested by high-speed centrifugation (10,0006 g), washed twice using deionized distilled H 2 O (ddH 2 O), and diluted to the final  Design of the hybridization scheme on the gene chips A complex hybridization scheme was designed using two-color platforms and common reference samples to determine the levels of the differential expressions between the group pairs [53]. As shown in Table 4, there were four experimental groups: WT (S 0 ): the wild type in S 0 medium, WT (K 2 S 4 O 6 ): the wild type in K 2 S 4 O 6 medium, MT (S 0 ): the mutant inS 0 medium, and MT (K 2 S 4 O 6 ): the mutant in K 2 S 4 O 6 medium. Each group contained triplicate biological repeats, so there were totally 12 samples. An aliquot of each sample was taken, mixed and used as common reference. Samples labeled with Cy5-dCTP and common reference labeled with Cy3-dCTP were hybridized on a chip. The gene expression level in the samples was determined based on the common reference. Therefore, there were four cases based on group pairs, which were the gene differential expression in S 0 medium between the mutant and the wild type (MT/WT (S 0 )), the gene differential expression in K 2 S 4 O 6 medium between the   mutant and the wild type (MT/WT (K 2 S 4 O 6 )), the gene differential expression of the wild type between S 0 medium and K 2 S 4 O 6 medium (S 0 /K 2 S 4 O 6 (WT)), and the gene differential expression of the mutant between S 0 medium and K 2 S 4 O 6 medium (S 0 /K 2 S 4 O 6 (MT)).

RNA isolation and microarray experiments
The wild type and the Dsor mutant were cultured as described in the section of growth measurements of the wild type and the Dsor mutant. Cells were collected at exponential growth phase (4 th day), washed twice using sterile RNase-free ddH 2 O and treated with RNA protect Bacteria Reagent (Qiagen Corp.). Total RNA was extracted using RNeasy Mini Kit (Qiagen Corp.) in accordance with the manufacturer's instructions.
Total RNA was purified using NucleospinH RNA Clean-up (MN Corp.) and tailed with polyA using Poly (A) Polymerase (Ambion Corp.). The tailed RNA was used to produce cy5/cy3labeled cDNA employing an RNA amplification strategy (Capi-talBio eukaryotic RNA Amplification and Labeling Kit, Capital-Bio Corp.). Cy5/cy3-labeled cDNA were hybridized with the microarray at 42uC for 16 h. Following hybridization, the arrays were washed using two consecutive solutions (0.2% SDS, 26 SSC at 42uC for 5 min, and 0.26SSC for 5 min at room temperature). Arrays were scanned using a confocal LuxScan TM scanner and the images obtained were then analyzed using LuxScan TM 3.0 software (both from CapitalBio Corp.). For individual channel data extraction, faint spots with intensities below 400 units after background subtraction in both channels (Cy3 and Cy5) were removed. A space-and intensity-dependent normalization based on a LOWESS program was employed [54]. The raw data were Log 2 transformed and median centered by arrays and genes using the Adjust Data function of CLUSTER 3.0 software and then further analyzed using hierarchical clustering with average linkage [55]. To determine the significant differentially expressed genes whose ratio changes $1.5 fold with p#0.05, Significance Analysis of Microarrays (SAM, version 3.02) was performed using two-class unpaired comparison in the SAM procedure [56].

Real-time quantitative RT-PCR (qRT-PCR)
Total RNA was extracted as described above and was treated with RNase-Free DNase Set (Qiagen Corp.) to eliminate the traces of genomic DNA. The total RNA of 2 mg was reversely transcribed using M-MLV Reverse Transcriptase (Invitrogen Corp.) under the following conditions: 25uC for 10 min, 37uC for 60 min, and 70uC for 10 min. RT reaction products of 1 ml were used for PCR amplification using Power SYBR Green PCR Master Mix (Applied Biosystems). The conditions for the PCR reaction were as follows: 95uC for 10 min followed by 40 cycles at 95uC for 15 s and 60uC for 1 min using a 7900 HT Fast RealTime PCR system (Applied Biosystems). The glyceraldehyde-3-phosphate dehydrogenase gene (gapdh) was used as the reference gene for normalization. The relative expression was calculated using the comparative DDC T method, and the values were expressed as 2 2DDCT [57]. Primers used in this study are listed in Table 5.