The bacterial strains and plasmids used in this study are listed in Table 1. The strain A. caldus MTH-04 has been deposited in the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC 1.15711. Liquid Starkey-S⁰ and -K₂S₄O₆ inorganic media and solid Starkey-Na₂S₂O₃ plates for A. caldus MTH-04 culture were prepared as reported previously (Jin et al., 1992). Elemental sulfur (S⁰; boiling sterilized, 8g/L) or K₂S₄O₆ (membrane filtration, 3 g/L) were added prior to inoculation. Chloromycetin, kanamycin, and streptomycin were added to a final concentration of 34, 100, and 100 μg/mL in LB media, and at 60, 100, and 100 μg/mL in liquid and solid Starkey media. The culture conditions were 37°C, 200 r/min for Escherichia coli (E. coli), and 40°C, 150 r/min for A. caldus MTH-04 (Chen et al., 2012).

The wild type and mutant strains of A. caldus were initially grown on a solid Starkey-Na₂S₂O₃ plate. One colony from each plate was inoculated into 10 mL Starkey-S⁰ liquid medium and grown to stationary phase. The saturated 10 mL culture was then transferred to 150 mL Starkey-S⁰ liquid medium and allowed to grow to stationary phase. Finally, cells in the 150 mL culture were collected by centrifugation at 12000 × g for 5 min and diluted with Starkey liquid medium to a final concentration of OD₆₀₀ = 1.0. In order to measure growth or extract RNA, 1 mL of this culture was inoculated into 150 mL Starkey-S⁰ or -K₂S₄O₆ liquid medium. Cell growth in Starkey-S⁰ medium was measured at OD₆₀₀ after removal of elemental sulfur in the sample by low-speed centrifugation at 400 × g for 5 min (Yu et al., 2014). Only a small amount of cells (<1.5 %) were found attached to the sulfur particles, which were neglected in the cell growth measurement. All experiments were performed in triplicate.

To construct the basic suicide plasmid vector pSDUDI, the oriT region was initially amplified from the plasmid RP4 using primers oriT EcoR sen and oriT Sal ant and digested with EcoR I/Sal I. A ColE1-AmpR fragment was amplified by PCR from pMD19-T vector using primers pMD19 Sal sen and pMD19 EcoR ant and digested with EcoR I/Sal I. The two PCR products were ligated together to generate plasmid pMD-oriT. The plasmid pMD-oriT was then amplified by PCR using primers pMD19 Nde sen and oriT Apa ant to generate a linearized plasmid. This was digested with Nde I/Apa I, and ligated to Nde I/Apa I digested Km resistant gene amplified from plasmid pJRD215 using primers Km Apa sen and Km Nde ant. The resulting plasmid was the basic suicide plasmid pSDUDI. An I-Sce I recognition site (5′-TAGGGATAACAGGGTAAT-3′) and multiple cloning sites (MCS) were introduced into pSDUDI by PCR using primers Km Apa sen and pMD19 Nde sen/Km Nde ant, respectively. All primers used are listed in Table 2.

The I-Sce I-expressing plasmid pSDU1-I-Sce I was constructed by generating a linearized form of the vector pSUD1-tac by PCR amplification with pSDU1 Xba sen and tac Bam ant. The I-Sce I gene was then amplified from plasmid pACBSR using primers I-Sce Bam sen and I-Sce Xba ant, digested with BamH I and Xba I, and ligated into the BamH I/Xba I treated linearized vector pSUD1-tac. The resulting plasmid was designated as pSDU1-I-Sce I.

To generate the suicide plasmid for rsrR, the upstream and downstream homologous arms (UHA and DHA) of this gene were amplified using the primer pairs R1F/R1R and R2F/R2R, respectively. The two homologous arms were then linked using fusion PCR (Yon and Fried, 1989). Finally, the fused fragments were digested with Spe I and Not I, and ligated to Spe I/Not I digested plasmid pSDUDI, thus generating the suicide plasmid pSDUDI::rsrR (UHA + DHA).

The suicide plasmid for rsrS was constructed in a similar manner by amplifying the two homologous arms (UHA and DHA) of rsrS using S1F/S1R and S2F/S2R. The UHA, DHA, and plasmid pSDUDI were then digested with Spe I/Hind III, Hind III/Kpn I, and Spe I/Kpn I, respectively. The three digested fragments were finally ligated to each other, and transformed into E. coli DH5α and screened for the suicide plasmid pSDUDI::rsrS (UHA + DHA).

The suicide plasmids for both genes were verified by restriction enzyme digestion and sequencing.

The suicide plasmids were transformed into E. coli SM10, and the transformed E. coli SM10 were conjugated with A. caldus as described earlier (Liu et al., 2007). After the first homologous recombination, the single crossover mutants were selected for kanamycin resistance on Starkey-Na₂S₂O₃ solid plates. Single recombination events were rapidly identified by PCR using R4F/R and S4F/R primers for rsrR and rsrS, respectively. The I-Sce I-expressing plasmid (pSDU1-I-Sce I) was then transferred into the single crossover mutants to induce a second homologous recombination, thereby generating the target mutants. The ΔrsrR and ΔrsrS strains were identified by PCR based screening using R4F/R and S4F/R primers. Finally, the PCR fragments amplified from ΔrsrR and ΔrsrS were sequenced using primers R5F/R and S5F/R, respectively to confirm their identity. All primers used in this section are listed in Table S1.

Elimination of the I-Sce I expression plasmid was achieved by spontaneous loss. The mutant containing I-Sce I expression plasmid was inoculated into the non-selective liquid Starkey-S⁰ medium and grown to stationary phase. An aliquot from the culture was diluted and plated on non-selective solid Starkey-Na₂S₂O₃ medium. Colony PCR was carried out to screen for loss of the plasmid using the primers RepA sen and RepC, which are also listed in Table S1. About 3–5 consecutive transfers were carried out to obtain the ΔrsrR and ΔrsrS mutants without the I-Sce I expression plasmid.

For RNA isolation, the culture was filtered through filter papers with a pore size of 15–20 μm to remove the sulfur powder, after which the cells were harvested by centrifugation at 12000 × g under refrigerated conditions. RNAprotect Bacteria Reagent (Qiagen Cor.) was added to resuspend the cells and inhibit changes to RNA transcripts. The suspension was mixed immediately by vortexing for 5 s, incubated for 5 min at room temperature, and centrifuged for 10 min at 5000 × g. The supernatant was discarded and the harvested cells were stored at −70°C. The RNAs were extracted by using RNAiso Plus kit (TaKaRa Cor.) according to the manufacturer's instructions. Reverse transcription was performed using the PrimeScript^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa Cor.). One microgram of total RNA was used for every 20 μL reverse transcription reaction system to obtain cDNA under the following reaction conditions: 42°C for 2 min, 37°C for 15 min, and 85°C for 5 s. The cDNAs from various cultures were used with SYBR® Premix Ex Taq^TM (TliRNaseH Plus; TaKaRa Cor.) for real-time quantitative PCR reactions, which were performed using LightCycler® 480 (Roche). Two-hundred nanograms of cDNA was used in a 20 μL RT-qPCR reaction. The conditions for qPCR were as follows: 95°C for 30 s followed by 40 cycles at 95°C for 5 s and 60°C for 30 s, and a final cycle at 95°C for 5 s, 60°C for 1 min and 95°C with continuous mode. The data and fold change were calculated using the LightCycler® 480 software. The primers used in this assay were designed using PRIMER PREMIER 5 software (PREMIER Biosoft Int., Palo Alto, CA, USA) and are listed in Table S2. The gapdH gene of A. caldus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as the reference gene for normalization (Livak and Schmittgen, 2001). Relative expression was calculated using the comparative ΔΔC_T method, and the values were expressed as 2^−ΔΔCT (Livak and Schmittgen, 2001). Three independent replicates were performed for each experiment. The values shown in this study are the means of three independent replicates showing fold changes (FC). FC ≥ 2, P ≤ 0.05 and FC ≤ 0.5, P ≤ 0.05 were regarded as significant changes, designated as up-regulation and down-regulation, respectively. No significant change was inferred when 0.5 ≤ FC ≤ 2, P ≥ 0.05. The SD-value was calculated using Origin software with “descriptive statistics,” and the P-value was calculated by using GraphPad Prism software with “unpaired t-test.”

The expression and purification of RsrR was performed as described. The rsrR gene of A. caldus MTH-04 was amplified by PCR using primers rsrR-F and rsrR-R listed in Table S3. The PCR product was cloned into plasmid pET-22b, generating the expression plasmid pET-22b-rsrR. A positive clone was verified by sequencing, and transformed into E. coli BL21 (DE3). RsrR was purified using HisTrap HP column (GE Healthcare), and the concentration of the purified protein was determined using the Bradford assay.

DNA fragments were obtained by PCR amplification using different sets of primers listed in Table S3. The G360, T360, T148, and T90 fragments were obtained using primer-pairs G360-F and G360-R, T360-F and T360/148/90-R, T148-F and T360/148/90-R, and T90-F and T360/148/90-R, respectively. The G360+58IRS fragment was obtained after two rounds of PCR. The first round of PCR was performed with G360 fragment as the template using the primer-pair G360-F and G360+58IRS–R1. In the second round of PCR, the PCR product from the first round was used as the template with the primer-pair G360-F and G360+58IRS–R2, to generate the G360+58IRS fragment. The T360Δ19 fragment was obtained by fusion PCR. Two fragments were generated by PCR from the T360 fragment using the primer-pairs T360-F and T360Δ19-R, and T360Δ19-F1 and T360/148/90-R. Fusion PCR was then performed using the two generated fragments as templates with the primer pair T360-F and T360/148/90-R to obtain the T360Δ19 fragment without the 19 bp IRS. After PCR amplification, the amplified fragments were purified using QIAquick Gel Extraction Kit (Qiagen Corp.), desalted, and concentrated using ultrafiltration. Ultrafiltration was carried out using Amicon Ultra-15 mL, 3 kDa Centrifugal Filter Unit (Millipore Corp.) at 5000 × g in a refrigerated centrifuge.

EMSA assays were performed as described in Pardo and Orejas (2014). Initially, a 15 μL reaction mixture containing 1.5 μL 10 × Binding Buffer, 1 μg of salmon sperm DNA, and 1 μg RsrR was mixed and used as the reaction solution. Reaction solution containing PBS (phosphate buffer saline) instead of RsrR was used as a negative control. Both solutions were prepared and incubated at room temperature for 20 min. Two-hundred nanogram of DNA fragments were then added to both mixtures and incubated at room temperature for another 20 min. The DNA-protein complexes in both reactions were separated on 6.5% nondenaturing polyacrylamide gels in 0.25 × TBE (22.25 mM Tris-Boric acid, 0.5 mM EDTA) on ice (150 volts for 1.5 h). Visualization of the bands was done using ethidium bromide staining as described earlier (Bidart et al., 2014).

The primers used for constructing the three IRS-probe vectors and the primers that were used to verify the function of the 19bp-IRS in vivo are listed in Table S4.

The nucleotide sequences of rsrR and rsrS have been deposited with GenBank accession numbers KX161704 and KX161705, respectively. This Whole Genome Shotgun project for A.caldus MTH-04 has been deposited at DDBJ/ENA/GenBank under the accession LXQG00000000.

The tetH clusters in various bacteria and archaea were compared for analysis. As shown in Figure 1, the two functional genes (tetH and doxDA) of the S₄I pathway were distributed in chemoautotrophic sulfur oxidizers including bacteria and archaea of the genera Acidithiobacillus and Acidianus, respectively. However, only A. caldus and Acidithiobacillus ferrooxidans have a specific two-component system (TCS) upstream the tetH gene. The cluster of A. caldus MTH-04 was identical to that of A. caldus SM-1 and shared 99% identity with that of A. caldus ATCC 51756. Furthermore, the tetH cluster in Acidithiobacillus sp. GGI-221 has certain components including tetH and doxDA with 61 and 59% identity, respectively. The 16S rRNA gene sequence of Acidithiobacillus sp. GGI-221 shows 99.7% identity to Acidithiobacillus ferrooxidans indicating that this is a strain of Acidithiobacillus ferrooxidans (Williams and Kelly, 2013). However, the TCS of A. ferrooxidans is σ⁵⁴-dependent and has a different order from tcsS to tcsR encoding the sensor histidine kinase and the cognate response regulator when compared to A. caldus. The tetH and doxDA genes are arranged in a cluster in A. caldus and Acidithiobacillus thiooxidans, while they were separately distributed in the genomes of Acidianus and other Acidithiobacillus spp. Two copies of doxDA genes are located separately in the genomes of A. ferrooxidans and Acidianus hospitalis W1, while in Acidianus ambivalens DSM 3772 there are two copies of tetH. Moreover, doxDA in Acidithiobacillus spp. encodes a protein with two domains DoxD and DoxA, but DoxD and DoxA in Acidianus hospitalis W1 and Acidianus ambivalens DSM 3772 are two individual subunits encoded by two separate genes, doxD and doxA (Müller et al., 2004; Valenzuela et al., 2008; Valdés et al., 2009; Mangold et al., 2011). BLAST and multiple alignment (Figure S1) results demonstrated that the two subunits DoxD and DoxA are fused as one protein in the three A. caldus strains, MTH-04, SM-1, and ATCC 51756, indicating that the doxD gene in this tetH cluster encodes a protein that has two domains corresponding to DoxD and DoxA (Müller et al., 2004). Thus, we renamed the doxD gene in these strains as doxDA or tqo.

A mobile suicide vector (pSDUDI, Figure 2A) was employed to introduce homologous sequences of the target genes into A. caldus cells, and to integrate these homologous sequences into the genome by homologous recombination, thus generating cointegrates (Figure 2C). The backbone of the suicide plasmid was derived from pUC19 and therefore cannot replicate in A. caldus. Second, the origin of transfer (oriT) of plasmid RP4 was cloned into this plasmid, which allows it to be mobilized into A. caldus with high efficiency. Third, an 18 bp I-Sce I endonuclease recognition site (5′-TAGGGATAACAGGGTAAT-3′) was also introduced into this plasmid to facilitate cleavage of the cointegrate by I-Sce I endonuclease.

The I-Sce I-expressing plasmid (pSDU1-I-Sce I) shown in Figure 2B was derived from pJRD215, which can replicate and remain stable in A. caldus. The Ptac promoter was introduced into this plasmid to express I-Sce I in A. caldus. Mobilization of pSDU1-I-Sce I into the cointegrate of A. caldus would lead to the generation of double-stranded breaks (DSBs) at the I-Sce I site. The subsequent second homologous recombination event, would ultimately lead to generation of the mutant or wild type strains (Figure 2C).

Verification of the ΔrsrR and ΔrsrS strains is shown in Figure 3. Smaller fragments were amplified from ΔrsrR and ΔrsrS strains compared to the wild type using primers R4F/R and S4F/R (lane 1, 5.1 kb; lane 2, 4.6 kb; lane 3, 1.0 kb, and lane 4, 551 bp), and R5F/R and S5F/R (lane 1, 5.0 kb; lane 2, 3.9 kb; lane 3, 2.1 kb, and lane 4, 910 bp; Figures 3C,D lanes 1–4). No band could be amplified from rsrR and rsrS knockout strains using primers R3F/R and S3F/R (Figures 3C,D lanes 6), while there were 413 and 850 bp bands for rsrR and rsrS genes, respectively in the wild type as shown in Figures 3C,D lanes 5. The sizes of the observed PCR bands were as expected in different strains. The precise sequences of the two mutants were confirmed by sequencing the PCR fragments (Figures 3C,D lane 2) derived from ΔrsrR and ΔrsrS.

To verify the influence of the lack of RsrR or RsrS on the S₄I pathway, relative RNA transcript levels of genes in the tetH cluster of ΔrsrR, ΔrsrS, and the wild type strains were tested by RT-qPCR. Strains were grown to mid-log phase in S⁰-medium (at the 4th day), followed by addition of equal volumes of either K₂S₄O₆ or H₂O to the stimulation and control groups, respectively. Cells at the 4th and 4.5th days were collected to purify total RNA for transcriptional analysis. The ratio of relative RNA transcripts levels of the genes at the two time points were calculated and are shown in Figure 4. After stimulation with exogenous K₂S₄O₆ at a final concentration of 3 g/L, the relative RNA transcript levels of rsrS, rsrR, tetH, and doxDA in the wild type increased by about 5, 20, 60, and 80 fold, respectively (Figure 4A). However, the relative transcript levels of these genes in the two mutants did not show any obvious increase (0.5 ≤ ratio ≤ 2.0; Figure 4A). In the control group, addition of water did not result in a significant change in expression of any of the four genes in the three strains (0.5 ≤ ratio ≤ 2.0; Figure 4B). This result supported the notion that there is a K₂S₄O₆-dependent positive regulation of RsrS-RsrR on tetH-doxDA.

Positive control of the cotranscription of tetH and doxDA (Rzhepishevska et al., 2007) is likely achieved by binding of RsrR to a cis-regulatory element at the promoter region of tetH. To confirm the above hypothesis, three fragments about 360, 148, and 90 bp upstream of the “ATG” of tetH were amplified to test for their interaction with RsrR by electrophoretic mobility shift assays (EMSAs) (Figure 5A). A 360 bp fragment amplified from the gapdH gene of A. caldus was used as a negative control. The results showed that the binding region is located in a 58 bp region between 90 and 148 bp upstream of “ATG” (Figure 5B). When the 58 bp region was fused with the 360 bp gapdH fragment, the fusion could bind to RsrR (Figure 5C), indicating that this cis-regulatory element was located in the 58 bp region. To further narrow down the binding region of RsrR, the software package Repeat Around-2.1 was used to analyze this region and a 19 bp (AACACCTGTTACACCTGTT) inverted repeat sequence (IRS) within the 58 bp region was predicted to be the binding sequence (Figure 5A). Upon removal of the 19 bp-IRS from the 360 bp-fragment upstream of tetH, binding of RsrR could not be observed (Figure 5D), which suggested that the 19 bp-IRS was the key binding site of RsrR.

To verify the function of the 19 bp-IRS on the transcription of tetH and doxDA in vivo, the 360, 148, and 90 bp fragments upstream of the “ATG” of tetH were fused to the reporter gene gusA to generate three IRS-probe vectors, as shown in Figure 5E. The resulting plasmids were designated as pJRD-P360IRS, pJRD-P148IRS, and pJRD-P90, respectively and were mobilized into wild type and ΔrsrR strains. All strains were grown in S⁰-medium, and the relative RNA transcript levels of gusA in each strain were measured after stimulation with K₂S₄O₆. As shown in Figure 5F, a significantly low level of gusA transcript was observed in the wild type strain harvesting plasmid pJRD-P90 when compared with that with pJRD-P360IRS and pJRD-P148IRS, indicating that the 19 bp-IRS had a positive effect on the transcription of tetH promoter. The relative gusA RNA transcript levels from plasmids pJRD-P360IRS and pJRD-P148IRS in ΔrsrR were much lower than those in the wild type strain, indicating that IRS is needed for the positive effect of RsrR.

Structural simulation and protein sequence alignment between RsrS-RsrR and EnvZ-OmpR were also carried out in order to help understand the mechanism of regulation of the tetH cluster by RsrS-RsrR, which showed that the two TCSs are highly identical at the protein level (Figure S2).

As shown in Figure 6, the growth rates of the rsrR or rsrS knockout mutants were not similar to the wild type. When grown in S⁰-medium, both ΔrsrR and ΔrsrS had a slight growth advantage over the wild type strain from the 5th to the 11th day, which corresponds to the logarithmic growth phase prior to entering into the stationary phase. In contrast, the wild type strain grew slightly better than the mutants after the 15th day (Figure 6A). When K₂S₄O₆ was used as the sole sulfur substrate, the three strains showed very slow overall growth (up to 0.06) compared to S⁰-medium due to differences in RISCs metabolism for different substrates (Zyl et al., 2008; Chen et al., 2012; Zhang et al., 2014). In K₂S₄O₆-medium, ΔrsrR and ΔrsrS had a 2 and 4 day growth delay in lag phase, respectively when compared to the wild type. However, the three tested strains reached approximately the same growth when they reached stationary phase (Figure 6B). In addition, complementation of the mutations with wild type alleles resulted in a growth pattern similar to the wild type in both media (Figures 6C,D). Therefore, the recovery of growth of the mutants in K₂S₄O₆-medium after a delay of several days suggested that RsrS-RsrR might be the primary, but not the sole signal transduction pathway that regulates the metabolism of tetrathionate.

In A. caldus, the sulfur-oxidizing mechanisms include the periplasmic Sox system, the S₄I pathway, and the RISCs oxidation enzymes such as SOR, SDO, SQR, HDR, RHD, DsrE, and TusA (mentioned in the Introduction Section). To investigate the effect of the absence of RsrS and RsrR on the S₄I pathway, we analyzed the relative RNA transcript levels of genes attributed to play a role in the A. caldus RISCs metabolism. Cross-talk often occurs between a sensor kinase and a non-cognate response regulator in the TCSs (Procaccini et al., 2011), so genes encoding single- and two-component systems (SCSs and TCSs) were also analyzed for their relative RNA transcript levels. The transcriptional analysis of these sulfur-oxidizing and regulatory genes during cultivation on S⁰ was carried out by RT-qPCR. All data were calculated and the statistically valid data were showed as the mean value of three independent replicates in Figure 7 (original data with values for standard deviation and P-value are listed in Supplementary Text S1). As shown, several genes in the mutants had significant changes in expression (FC ≥ 2, P ≤ 0.05, up-regulation; FC ≤ 0.5, P ≤ 0.05, down-regulation) when compared with the wild type. The deletion of rsrR resulted in a sharp up-regulation (FC ≥ 6, P ≤ 0.05) of tetH-doxDA, sox operon I, sdo-1, and sdo-2, clear down-regulation (FC ≤ 0.01, P ≤ 0.05) of tusA, and weak transcriptional changes (2 ≤ FC ≤ 6, P ≤ 0.05) in sox operon II, sqr, and rhd. The absence of rsrR also resulted in significant up-regulation (FC ≥ 2, P ≤ 0.05) of a majority of regulator genes in TCSs including ompR (A5904_2590), phoB (A5904_0374), cheY (A5904_1450), tspR (A5904_2485), A5904_0219, A5904_0936, A5904_1207, A5904_1342 and A5904_1480, and in SCSs including A5904_0420, A5904_0789 and A5904_1113. The sensor histidine kinase (HK) genes rsrS and envZ in TCSs are up-regulated significantly in the rsrR knockout strain. However, the HK genes phoR (A5904_0373), cheA (A5904_1448), tspS (A5904_2484), kdpD (A5904_1340), fleS (A5904_1479), A5904_0218 and A5904_0934 had no obvious transcriptional changes in this mutant. In the ΔrsrS strain, the genes of the Sox operon (except soxYZ in operon II), tetH, and doxDA showed significant changes in expression when compared to the wild type. The expression of soxYZ in operon II was reduced to almost undetectable levels. Almost all the other genes involved in the sulfur oxidation system, TCSs, and SCSs did not show significant changes. However, tspS showed a significant change (2 ≤ FC ≤ 7, P ≤ 0.05) in the ΔrsrS strain.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.