Acidithiobacillus caldus is a chemolithoautotrophic sulfur-oxidizing bacterium that is widely used for bioleaching processes. Acidithiobacillus spp. are suggested to contain sulfur dioxygenases (SDOs) that facilitate sulfur oxidation. In this study, two putative sdo genes (A5904_0421 and A5904_1112) were detected in the genome of A. caldus MTH-04 by BLASTP searching with the previously identified SDO (A5904_0790). We cloned and expressed these genes, and detected the SDO activity of recombinant protein A5904_0421 by a GSH-dependent in vitro assay. Phylogenetic analysis indicated that A5904_0421and its homologous SDOs, mainly found in autotrophic bacteria, were distantly related to known SDOs and were categorized as a new subgroup of SDOs. The potential functions of genes A5904_0421 (termed sdo1) and A5904_0790 (termed sdo2) were investigated by generating three knockout mutants (Δsdo1, Δsdo2 and Δsdo1&2), two sdo overexpression strains (OE-sdo1 and OE-sdo2) and two sdo complemented strains (Δsdo1/sdo1’ and Δsdo2/sdo2’) of A. caldus MTH-04. Deletion or overexpression of the sdo genes did not obviously affect growth of the bacteria on S0, indicating that the SDOs did not play an essential role in the oxidation of extracellular elemental sulfur in A. caldus. The deletion of sdo1 resulted in complete inhibition of growth on tetrathionate, slight inhibition of growth on thiosulfate and increased GSH-dependent sulfur oxidation activity on S0. Transcriptional analysis revealed a strong correlation between sdo1 and the tetrathionate intermediate pathway. The deletion of sdo2 promoted bacterial growth on tetrathionate and thiosulfate, and overexpression of sdo2 altered gene expression patterns of sulfide:quinone oxidoreductase and rhodanese. Taken together, the results suggest that sdo1 is essential for the survival of A. caldus when tetrathionate is used as the sole energy resource, and sdo2 may also play a role in sulfur metabolism.
Citation: Wu W, Pang X, Lin J, Liu X, Wang R, Lin J, et al. (2017) Discovery of a new subgroup of sulfur dioxygenases and characterization of sulfur dioxygenases in the sulfur metabolic network of Acidithiobacillus caldus. PLoS ONE 12(9): e0183668. https://doi.org/10.1371/journal.pone.0183668
Editor: Ligia O. Martins, Universidade Nova de Lisboa, PORTUGAL
Received: January 20, 2017; Accepted: August 8, 2017; Published: September 5, 2017
Copyright: © 2017 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: JqiangL received the National Natural Science Foundation of China (grant numbers 31370138, 31570036); LC received the National Natural Science Foundation of China (grant number 31400093); XP received the National Natural Science Foundation (grant number 30800011); JqunL received the National Basic Research Program of China (grant number 2010CB630902) and the State Key Laboratory of Microbial Technology Foundation (M2015-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Acidithiobacillus caldus is an acidophilic, chemolithoautotrophic, sulfur-oxidizing bacterium that is widely used in the bioleaching industry [1, 2, 3]. A. caldus effectively removes sulfur from the surfaces of minerals, while maintaining acidic conditions that promote the growth of other microorganisms important in the bioleaching process . A. caldus can oxidize various reduced inorganic sulfur compounds (RISCs), such as tetrathionate (S4O62-), thiosulfate (S2O32-), sulfite (SO32-), sulfide (S2-) and elemental sulfur (S0). The ATP and NAD(P)H generated during sulfur oxidation are then used by A. caldus to fix carbon dioxide and allow autotrophic growth [5, 6]. Given the commercial importance of A. caldus, the identification of sulfur-oxidizing enzymes can provide important insights into sulfur metabolism in Acidithiobacillus spp.
The ability of Acidithiobacillus spp. to oxidize sulfur was identified as early as 1959 , and identifying the sulfur-oxidizing enzymes has long been a focus of the field. In 1987, a periplasmic sulfur:ferric ion oxidoreductase was purified from Acidithiobacillus ferrooxidans and was shown to catalyze the oxidation of S0 to sulfite. The enzyme was named sulfur dioxygenase (SDO). The original oxidation experiments were catalyzed in the presence of reduced glutathione (GSH) by using Fe3+ or molecular oxygen as the electron acceptor . However, further studies showed that sulfur dioxygenase did not use S0 as its substrate, but rather used the sulfane sulfur atom of glutathione persulfide (GSSH) and its higher homologues (GSSnH, n>1), where sulfite was the first product [9, 10].
Although sulfur dioxygenase activity was first reported in Acidithiobacillus, the sdo genes have been identified only recently . ETHE1 proteins are mitochondrial sulfur dioxygenases in human and Arabidopsis that facilitate the catabolism of sulfide in mitochondria along with sulfide:quinone oxidoreductase (SQR) and rhodanese (Rhd) [12, 13, 14]. Comparing the amino acid sequences of these mitochondrial sulfur dioxygenases to heterotrophic bacteria has led to the discovery of three subgroups of SDOs called Blhs, ETHE1s and SdoAs . The 3D structures of an ETHE1 from Myxococcus xanthus and a SdoA from Pseudomonas putida revealed information of GSH binding and the catalytic mechanisms of these SDOs . An ETHE1 homologue ACAL_0790 (A5904_0790) was found recently in A. caldus MTH-04 by BLASTP search using the human ETHE1 sequence. Recombinant ACAL_0790 was shown to have sulfur dioxygenase activity in vitro . However, whether there are other sdo genes in the genome of A. caldus MTH-04 and whether these putative sdo genes have sulfur dioxygenase activity remains unknown.
Here, potential sdo genes were searched in the genome of A. caldus MTH-04. These putative genes were cloned, expressed in Escherichia coli and the recombinant proteins were assayed for SDO activity. In addition, the evolutionary relationships among all SDOs were analyzed. Furthermore, the putative sdo genes were deleted and complemented, as well as overexpressed, in A. caldus MTH-04 to test the function of these genes. The enzyme activities of the putative SDO proteins and their effects on cell growth, as well as the expression of sulfur oxidation related genes were characterized. This study provides important information for constructing improved sulfur oxidation models for Acidithiobacillus spp., which are important for optimizing bioleaching.
Materials and methods
SDO homologues were searched in the genome of A. caldus MTH-04 (CGMCC 1.15711) (GenBank accession number LXQG00000000) using NCBI BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The ExPASy Compute pI/Mw tool (http://web.expasy.org/compute_pi/) was used to predict the isoelectric points (pI) and the molecular weights of the homologous proteins. Multiple sequence alignment was performed using ClustalX version 1.81. A neighbor-joining phylogenetic tree of the homologous proteins was constructed with ClustalX version 1.81 and MEGA version 5 with a p-distance distribution, pairwise deletion and bootstrap analysis of 10,000 repeats. Finally, subcellular localizations of proteins were predicted using Softberry ProtCompB tool (http://linux1.softberry.com/berry.phtml?topic=protcompan&group=programs&subgroup=proloc), SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and PSORTB v3.0 (http://www.psort.org/psortb/).
Bacterial strains, plasmids, media and growth conditions
Bacterial strains and plasmids used in this study are listed in S1 Table.
A. caldus was grown at 40°C with shaking at 150 rpm in liquid Starkey-S0 medium (pH 2.5), liquid Starkey- K2S4O6 medium (pH 2.5), liquid DMSZ 71- Na2S2O3 medium (pH 4.7) or on solid Starkey-Na2S2O3 medium (pH 4.8) [17, 18]. Liquid Starkey medium contained S0 (8 g/L) or K2S4O6 (2.27 g/L) and liquid DMSZ 71 medium contained Na2S2O3·5H2O (5 g/L) as the energy source for A. caldus. K2S4O6 and Na2S2O3 were sterilized by filtration (0.22-μm pore size) and added to the media before inoculation. S0 was sterilized by boiling for at least 4 h and was added to the media after inoculation. Kanamycin (200 μg/ml) or chloromycetin (68 μg/ml) were used in liquid Starkey-S0 medium, liquid Starkey- K2S4O6 medium or liquid DMSZ 71- Na2S2O3 medium as required, and solid Starkey-Na2S2O3 medium was made with kanamycin (80 μg/ml) or chloromycetin (27.2 μg/ml) for selection. E. coli strains were grown at 37°C at 170 rpm in liquid Luria-Bertani (LB) broth or on LB agar plates with either ampicillin (100 μg/ml), kanamycin (100 μg/ml), or chloromycetin (34 μg/ml).
Restriction enzyme digestion, ligation, gel electrophoresis and other general molecular techniques were performed as previously described . A. caldus genomic DNA was isolated using the TIANamp Bacteria DNA Kit (TIANGEN). Plasmids were isolated using plasmid mini kit I (Omega Bio-Tek). DNA fragments were extracted from agarose gels using a gel extraction kit (Omega Bio-Tek). DNA polymerase, restriction enzymes and T4 DNA ligase were purchased from TaKaRa, and primers were generated by Invitrogen.
Cloning, expression and purification of SDO homologues
The coding sequences of A5904_0421, A5904_0790 and A5904_1112 were amplified from A. caldus MTH-04 genomic DNA using PrimeSTAR® HS DNA Polymerase (TaKaRa) with the primer pairs 0421orfF/0421orfR, 0790orfF/0790orfR and 1112orfF/1112orfR (S2 Table), respectively. The fragments were digested with NdeI-XhoI and ligated into NdeI-XhoI treated pET22b(+) to generate the recombinant plasmids pET22b-0421, pET22b-0790 and pET22b-1112. Correct clones were confirmed by sequencing, then the plasmids were transformed into E. coli BL21(DE3) cells. Expression of recombinant proteins was induced by the addition of 0.4 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at 25°C for 5 h. Cells were collected by centrifugation and washed twice with ice-cold 20 mM NaH2PO4 buffer (pH 7.4) containing 30 mM imidazole and 500 mM NaCl, and lysed by sonication at 4°C. Supernatants were collected by centrifugation at 13,400×g for 30 min at 4°C and the recombinant proteins were analyzed by 10% (wt/vol) SDS-PAGE gels. Proteins were purified using HisTrap™ HP Crude columns (GE Health) according to the manufacturer’s instruction. The buffer was exchanged to 50 mM Tris-HCl buffer (pH 7.4), and glycerol was added to a final concentration of 20% before storage at– 20°C. The enzymes were stable for several weeks under the storage conditions. Finally, protein concentrations were measured using the Bradford assay.
Sulfur dioxygenase activity
Sulfur dioxygenase activity was measured using purified proteins from E. coli according to a previously published method with minor modifications . Dispersed elemental sulfur suspension was prepared by mixing 32 mg elemental sulfur powder and 5 μl of Tween-20 to 5 ml of 50 mM Tris-HCl buffer (pH 8.0), and sonicating (90 kHz, 15 s on, 5 s off) the mixture for 5 min on ice. The reactional mixture (0.8 ml) contained 0.5 ml dispersed elemental sulfur suspension, 127 μg purified A5904_0421 recombinant protein or 31 μg purified A5904_0790 recombinant protein and GSH was added into the reaction mixture to a final concentration of 0.8 mM before measurement. The assays were performed at 40°C with shaking at 150 rpm for 30 min. All samples were then centrifuged for 5 min at 13,400 × g and 4°C to remove remaining sulfur, and the supernatant was used to measure the amount of sulfite produced during the reaction. The amount of sulfite was measured according to a previously published method . Heat inactivated purified proteins were used as controls for each experiment. When the activity was calculated, the sulfite produced by heat inactivated proteins was deducted. One unit of SDO activity was defined as the production of 1 μmol of sulfite per minute. Specific SDO activity was measured as units per milligram of protein.
pH and temperature optima
The influence of pH and temperature on the SDO activity of the purified recombinant proteins was measured using the method described above. SDO activity over a range of pH values was measured at 40°C in 50 mM citric acid/NaH2PO4 buffer (pH 3.0–6.0), 50 mM Tris/HCl buffer (pH 7.0–8.0) or 50 mM glycine/NaOH buffer (pH 9.0–10.0). The influence of temperature was measured at pH 8.0 over the range 30–70°C.
The influence of metal ions (Mg2+, Mn2+, Fe3+, Ni2+, Zn2+, Cu2+, Co2+ and Hg2+), EDTA, DTT and N-ethylmaleimide (NEM) on SDO activity was evaluated by performing the SDO activity assay as described above in the presence of each reagent in 50 mM Tris/HCl buffer (pH 8.0) at 40°C.
Kinetic analysis was carried out in 50 mM Tris/HCl buffer (pH 8.0) at 45°C with 127 μg purified A5904_0421 recombinant protein or 31 μg purified A5904_0790 recombinant protein and various concentrations of GSSH (range over 95.3–953 μM). GSSH was prepared by mixing equal volumes of 17 mM glutathione in 50 mM Tris/HCl buffer (pH 8.0) with a saturated sulfur solution, containing 17 mM elemental sulfur. The GSSH concentration was determined according to a previously published method . The initial reaction rate was determined for each GSSH concentration. Km values were determined by nonlinear regression analysis of the initial reaction rates against GSSH concentrations fitted to the Michaelis–Menten equation using the software program Origin 8.0 (Originlab Software).
Total GSH-dependent sulfur oxidation activity assays of A. caldus
The total GSH-dependent sulfur oxidation activity of A. caldus was measured using A. caldus cell extracts according to the method described for sulfur dioxygenase activity. A. caldus strains were sonicated on ice for 10 min, centrifuged for 5 min at 13,400 × g and the supernatant was removed and used as the cell extract for the GSH-dependent sulfur oxidation activity assays. The assay was performed at 40°C in 50 mM Tris/HCl buffer (pH 8.0). Heat inactivated A. caldus cell extracts were used as controls for each experiment. One unit of the total GSH-dependent sulfur oxidation activity was defined as the production of 1 μmol of sulfite per minute. The total GSH-dependent sulfur oxidation activity was measured as units per milligram of protein.
Generation of the markerless sdo knockout mutant of A. caldus
Markerless sdo knockout mutants were generated as described previously with minor modifications . All primers used in this section are listed in S2 Table. The suicide plasmids pSDUDI-sdo1 (specific to A5904_0421) and pSDUDI-sdo2 (specific to A5904_0790) were made using pSDUDI as the plasmid backbone. The upstream and downstream homologous arms of sdo1 were amplified using the primer pair sdo1UF/sdo1UR to amplify the 1,211 bp upstream homologous arm, and primers sdo1DF/sdo1DR to amplify the 722 bp downstream homologous arm. The homologous arms of sdo2 were amplified using primer pair sdo2UF/sdo2UR to amplify the 1,109 bp upstream homologous arm and primers sdo2DF/sdo2DR to amplify the 1,275 bp downstream homologous arm. The sequences were ligated with pSDUDI after digestion with appropriate restriction enzymes. Successful insertions were confirmed by sequencing, and the resulting recombinant plasmids were named pSDUDI-sdo1 and pSDUDI-sdo2.
pSDUDI-sdo1 or pSDUDI-sdo2 was transformed into E. coli SM10 by selecting for ampicillin resistant clones and these transformants were used as donor strains for conjugation. A. caldus MTH-04 was used as the recipient to generate the A. caldus Δsdo1 or Δsdo2 strains. Δsdo1 was used as the recipient to construct the Δsdo1&2 strain. pSDUDI-sdo1 or pSDUDI-sdo2 was transferred from E. coli SM10 to A. caldus by conjugation. Both donor cells and recipient cells were cultivated until exponential phase, collected by centrifugation, and mixed at a ratio of approximately 1:3, then spotted onto a 25 mm diameter, 0.22-μm pore size sterilized filter membrane that was placed on the mating medium. After incubation at 37°C for 3 days, cells on the filter membrane were suspended in ddH2O and plated on selective Starkey-Na2S2O3 plates containing kanamycin. Colonies were selected and analyzed by colony PCR using primer pairs located in the homologous arms to amplify a partial sequence of the homologous arms. Primer pair sdo1inF/sdo1inR was used to detect single crossovers of Δsdo1, where 1,906 bp and 1,168 bp fragments were detected when a single crossover had occurred. Primer pair sdo2inF/sdo2inR was used to detect the single crossover of Δsdo2 or Δsdo1&2, where 1,721 bp and 974 bp fragments were detected in the case of a single crossover. Colonies with the correct PCR fragments were inoculated into liquid Starkey-S0 medium and the genomic DNA of each selected colony was isolated for PCR analysis to further confirm the single crossover-recombination event.
Plasmid pSDU1-I-Sce I was introduced into the single-crossover cells of A. caldus to express I-SceI endonuclease to digest its recognition site (5’-TAGGGATAACAGGGTAAT-3’) that was introduced by the suicide plasmid. This creates double-stranded DNA breaks (DSBs) on the chromosome to induce the second homologous recombination event, generating knockout mutants or wild type individuals. The colonies grown on selective Starkey-Na2S2O3 plates containing chloromycetin were tested for genetic changes by colony PCR as described above. sdo1inF/sdo1inR primers were used to detect Δsdo1 or Δsdo1&2. sdo2inF/sdo2inR primers were used to detect Δsdo2 or Δsdo1&2 (predicted sizes of the fragments amplified by PCR were listed in S3 Table). Sdo gene knockout mutants were cultured in liquid Starkey-S0 medium at 40°C. The genomic DNA of each colony was isolated for PCR analysis to confirm sdo gene knockout mutants. Primer pairs 0421orfF/0421orfR, sdo1inF/sdo1inR and sdo1outF/sdo1outR were used to confirm the sdo1 deletion, and primer pairs 0790orfF/0790orfR, sdo2inF/sdo2inR and sdo2outF/sdo2outR were used to confirm the sdo2 deletion (S3 Table, S1 Fig). Primer pairs sdo1outF/sdo1outR and sdo2outF/sdo2outR were located outside the homologous arms. The amplified PCR products from the mutants using primer pairs of sdo1outF/sdo1outR and sdo2outF/sdo2outR were sequenced to confirm the mutants.
The pSDU1-I-Sce I plasmid in the mutant cells was eliminated by propagating the Δsdo mutants 5 to 10 times in liquid S0 medium without chloromycetin, and plating the cells on non-selective solid Starkey-Na2S2O3 medium. Colony PCR was carried out to verify the loss of the plasmid using the primer pairs pSDU1cxs-pSDU1cxa. No fragment was expected if pSDU1-I-Sce I had been lost.
Construction of A. caldus sdo overexpression strains and sdo complemented strains
The coding sequence of sdo1 and sdo2 were amplified from A. caldus MTH-04 genomic DNA using sdo1F/sdo1R and sdo2F/sdo2R primer pairs, respectively (S2 Table). Sequences were digested with restriction enzymes and inserted downstream of the tac promoter in the plasmid pSDU1-tac to produce pSDU1-sdo1 and pSDU1-sdo2, and the insertions were confirmed by sequencing. The original plasmid pSDU1 was used as a control. The transfer of plasmids from E. coli SM10 into A. caldus MTH-04 or Δsdo strains was performed by conjugation as described above.
Growth measurements of A. caldus strains
The A. caldus MTH-04 wild type, sdo knockout mutants, control strain (wild type carrying plasmid pSDU1), sdo overexpression strains, and sdo complemented strains were grown in Starkey-S0 medium for 7 days and cells were collected by centrifugation and adjusted to the same cell density (OD600 = 20.0). A 50 μl aliquot of the treated cells was inoculated into 150 ml fresh Starkey-S0 medium, Starkey- K2S4O6 medium or DMSZ 71- Na2S2O3 medium. A. caldus growth in Starkey-S0 medium or Starkey- K2S4O6 medium was monitored by measuring the optical density at 600 nm after low-speed centrifugation at 400 × g for 5 min, according to the previous report of Wang et al. . Growth of A. caldus in DMSZ 71- Na2S2O3 medium was monitored by cell counting. In order to determine the dry weight cell yield, wild type A. caldus MTH-04 cells grown to stationary growth phase on the energy source of tetrathionate were collected by centrifugation (5,000 × g, 5 min) and dried to a constant weight. The consumption of K2S4O6 was determined by high performance liquid chromatography (HPLC, UL-3000 system; Dionex) equipped with a UV detector, according to a previously published method with minor modifications . The HPLC employed a C18 column (250×4.6 mm, inner diameter, 5 μm; Sepax) with a flow-rate of 0.6 ml per min and the detector was set at 230 nm. The mobile phase consisted of 20% (vol/vol) acetonitrile and 80% (vol/vol) water (containing 6 mM tetrapropylammoniumhydroxide, pH 5.0). The injection volume was 10 μl. The production of sulfate and the consumption of Na2S2O3 were determined by ion chromatography (ICS-1100 system; Dionex) with a mobile phase that contained 25 mM KOH at a flow rate of 1 ml per min. All measurements were performed in triplicate from at least three independent biological replicates. The P-value was calculated using a t-test.
RNA extraction and RT-qPCR
To measure relative RNA transcript levels of the genes involved in sulfur metabolism, A. caldus strains were grown in Starkey-S0 medium or Starkey- K2S4O6 medium, collected by centrifugation at 12,000 × g for 5 min, resuspended in RNAlater® Solution (Ambion), stored overnight at 4°C and harvested from the RNAlater® suspension by centrifugation. Cells were resuspended in100 μl of lysis buffer (1 mg/ml lysozyme, 10 mM Tris and 1 mM EDTA, pH 8.0) and incubated at 26°C for 6 min. Total RNA was extracted using Trizol® reagent (Ambion) according to the manufacturer’s instruction. RNA quality was determined by denaturing formaldehyde agarose gel electrophoresis and RNA concentration was determined using a NanoDrop-1000 spectrophotometer (NanoDrop Technologies). Genomic DNA was removed using the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time)(TaKaRa) and confirmed by PCR using primer pair alaSF/alaSR (S4 Table). cDNA was synthesized using the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time)(TaKaRa).
RT-qPCR reactions were performed on a LightCycler® 480 system (Roche) with SYBR®Premix Ex Taq (TaKaRa), according to the manufacturer’s instructions. All RT-qPCR reactions in this study were performed in technical triplicates from at least three independent biological replicates. Primers used for gene expression analysis are listed in S4 Table. Changes in the relative RNA transcript levels of the sulfur-oxidizing genes in the wild type, knockout mutants and overexpression strains were normalized against the internal control alaS, and calculated using the comparative ΔΔCT method and shown as 2-ΔΔCT . Genes showing a fold change ≧2 or ≦0.5 and P < 0.05 were considered to be differentially expressed. The P-value was calculated using a t-test.
Sequence accession numbers
The genome of A. caldus MTH-04 (CGMCC 1.15711) has been deposited in the NCBI GenBank database under the accession number LXQG00000000. A5904_0421, A5904_0790 and A5904_1112 sequences have been deposited in the NCBI GenBank database under accession numbers OAN04050.1, OAN03714.1 and OAN03376.1, respectively.
Discovery and identification of SDOs in A. caldus MTH-04
A BLASTP search was conducted using the previously identified SDO (A5904_0790)  against the A. caldus MTH-04 draft genome, which resulted in the identification of two more putative sdo genes, A5904_0421 and A5904_1112. The ORF of A5904_0421 shares 33% amino acid identity with A5904_0790, and encodes a 245 amino acid metallo-β-lactamase with a theoretical isoelectric point of 5.02 and molecular mass of 26.2 kDa. The ORF of A5904_1112 is 34% identical to A5904_0790, and encodes a 243 amino acid metallo-β-lactamase with a theoretical isoelectric point of 7.09 and molecular mass of 27.0 kDa (S2 Fig).
A GSH-dependent in vitro SDO activity assay was conducted using recombinant proteins A5904_0421, A5904_0790 and A5904_1112 that had been expressed and purified from E. coli. SDO activity was not detected between pH 3 and pH 10 for A5904_1112. However, SDO activity was detected between pH 5.0 and 9.0 for A5904_0421, with an optimum at pH 8.0 (Fig 1A). The temperature profile of the purified A5904_0421 was determined at pH 8.0 and showed that this protein exhibited optimal activity between 45°C and 50°C (Fig 1B). Based on the in vitro SDO activity, A5904_0421 was designated as SDO1 and A5904_0790 was designated as SDO2.
The values corresponding to the 100% activity are 57 mU/mg in (A) and 66 mU/mg in (B), respectively.
The kinetic parameters of the SDOs were determined in 50 mM Tris/HCl buffer (pH 8.0) at 45°C. The Km of SDO1 for GSSH was 267±31 μM and kcat was 5.4 s-1. The Km of SDO2 for GSSH was 298±13 μM and kcat was 48.1 s-1.There was no significant difference between the Km values of SDO1 and SDO2 (P>0.05). The kcat/Km of SDO1 and SDO2 were 20.2 and 161.4 mM -1s-1, respectively. SDO2 had the highest catalytic efficiency of the two enzymes according to the kcat/Km ratio (Table 1). The Km values of SDO1 and SDO2 were larger than that of Urechis unicinctus ETHE1 (82.5 μM ), smaller than that of human ETHE1 (340 μM ) and within the range of those of the bacterial SDOs (108 μM– 342 μM ).
The influence of metal ions on the SDO activity of SDO1 was also determined (S5 Table). Ni2+, Cu2+, Co2+ and Hg2+ were found to strongly inhibit the activity of SDO1, while SDO activity was inhibited to some extent by Mg2+, Zn2+, Mn2+ and Fe3+. The divalent metal chelator EDTA completely inhibited the SDO activity of SDO1, indicating that a divalent metal ion might be essential for the activity of this enzyme. 0.05 mM DTT did not significantly affect SDO activity while the thiol modifying reagent NEM strongly inhibited SDO activity, suggesting that free SH group(s) are needed for the catalytic activity of SDO1, as previously reported for other SDOs .
Distribution of SDOs in Acidithiobacillus spp and other sulfur oxidizers
BLASTP analysis of bacterial and archaeal genomes using the SDO1 or SDO2 amino acid sequences as a query revealed that SDO homologues are widespread in Acidithiobacillus spp., and there were two to three copies of SDO in different strains of Acidithiobacillus spp. bacteria (Table 2). The sdo genes in A. caldus MTH-04 are co-localized with other genes related to sulfur-metabolism. A dsrE-like gene (A5904_0418), which potentially encodes a protein involved in sulfur transfer, is downstream of sdo1. A sqr-like gene (A5904_0792) is downstream of sdo2. A5904_1112 (with no in vitro SDO activity) is not co-localized with genes related to sulfur-metabolism (Fig 2).
The predicted functions of genes are shown. The locus-tags are given inside of the gene arrows.
A phylogenetic analysis was conducted to better understand the relationship between previously described SDOs (ETHE1s, Blhs, SdoAs)  and the putative SDOs detected in Acidithiobacillus spp. and other sulfur oxidizing bacteria (Fig 3). A. caldus SDO2 showed a closer evolutionary relationship to known ETHE1s from the mitochondria of eukaryotes, while A. caldus SDO1 and its homologues, mainly found in autotrophic bacteria, were distantly related to other known SDOs. A new group, containing SDO1 from A. caldus MTH-04, and SDOs from autotrophic sulfur-oxidizing bacteria, such as Acidithiobacillus spp., Thiobacillus spp., Acidithiobacillales sp., Sulfuricella sp., Thioalkalivibrio sp., was designated as subgroup SdoS.
GenBank accession numbers are presented in the parentheses. The proteins with SDO activities demonstrated in this study or previous studies are marked by asterisk (*). The SDOs with known 3D structures are highlighted in bold. The proteins from A. caldus MTH-04 are underlined.
Construction and characterization of sdo knockout mutants, sdo overexpression strains and sdo complemented strains of A. caldus MTH-04
To further study the function of the SDOs in A. caldus, a markerless gene knockout system was used to generate the sdo single knockout strains Δsdo1 and Δsdo2, as well as the sdo double knockout strain Δsdo1&2. The complete sequences of sdo1 and sdo2 were deleted in the knockout strains and the mutants were identified by PCR using different primer sets (S1 Fig). All observed PCR fragments were in accordance with the predicted sizes as listed in S3 Table. The correct sequences of the mutants were confirmed by sequencing the mutated regions. The sdo overexpression strains (OE-sdo1 and OE-sdo2), sdo complemented strains (Δsdo1/sdo1’ and Δsdo2/sdo2’) and a control strain (wild type carrying plasmid pSDU1) were generated as described in the Materials and Methods section.
To understand the effect of sdo genes on the growth of A. caldus, wild type and recombinant A. caldus strains (including knockout, complemented and overexpression strains) were grown in Starkey-S0 medium, Starkey- K2S4O6 medium and DMSZ71- Na2S2O3 medium. In general, the Δsdo1, Δsdo2 and Δsdo1&2 strains only showed slight differences in growth when compared to wild type on S0. Δsdo1 had a slightly reduced growth rate compared to the wild type (P<0.05), and Δsdo2 had a similar growth rate to Δsdo1 (P>0.05), while Δsdo1&2 had a slight growth advantage over Δsdo1and Δsdo2 during the first 5 days (P<0.05) (Fig 4A). Δsdo1 and Δsdo1&2 did not survive on tetrathionate. Interestingly, Δsdo2 entered the logarithmic growth phase one day earlier than wild type when grown on tetrathionate (Fig 4B). The complemented sdo1 strain rescued the wild-type phenotype when grown on tetrathionate (Fig 4E). These results suggested that A. caldus requires sdo1, but not sdo2, to grow on tetrathionate. When thiosulfate was used as the energy source, Δsdo2 still entered the logarithmic growth phase one day earlier than wild type. Δsdo1 and Δsdo1&2 showed a slightly reduced growth capacity compared to wild type (P<0.05) (Fig 4C). Overexpression of sdo genes did not increase the growth rate compared to the control strain (wild type carrying plasmid pSDU1) on S0 (Fig 4D), tetrathionate (Fig 4E) or thiosulfate (Fig 4F).
(A-C) Growth of sdo knockout mutants compared with wild type on S0 (A), tetrathionate (B) and thiosulfate (C). (D-F) Growth of sdo overexpression strains and sdo complemented strains compared with the control strain (wild type carrying pSDU1) on S0 (D), tetrathionate (E) and thiosulfate (F). WT represent the wild type, Δsdo1, Δsdo2, Δsdo1&2 represent the sdo knockout mutants, WT (pSDU1) represent the control strain, OE-sdo1 and OE-sdo2 represent the sdo overexpression strains, Δsdo1/sdo1’ and Δsdo2/sdo2’ represent the sdo complemented strains of A. caldus MTH-04, respectively. OD600 indicates the optical density at 600 nm, all measurements were performed in triplicate and error bars correspond to the standard deviations.
When S0 was used as the energy source, sulfate was the only detected product, and the knockout mutants produced more sulfate (Fig 5A) but grew less than the wild type (Fig 4A). This result indicated that the wild type grew more efficiently as it had a complete metabolic network. When grown on tetrathionate, the wild type and Δsdo2 strains used all the 7.5 mM tetrathionate (Fig 5B) to produce an equal amount of sulfate (approximately 31 mM) (Fig 5C). No tetrathionate was consumed by the Δsdo1 and Δsdo1&2 strains (Fig 5B), which was in agreement with lack of observed growth (Fig 4B). When thiosulfate was used as the energy source, no thiosulfate was detected in the end and sulfate was the only detected product. Na2S2O3 easily degrades into S0 and SO32- at pH lower than 4.4, and the produced S0 could be used by A. caldus strains as well. As the pH decreased soon after the growth began, degradation might contribute to the exhaustion of thiosulfate as well. There was no significant difference in sulfate production among wild type and sdo knockout mutants (P>0.05)(Fig 5D).
(A) Sulfate production in S0 medium. (B) Tetrathionate consumption in tetrathionate medium. (C) Sulfate production in tetrathionate medium. (D) Sulfate production in thiosulfate medium. WT represent the wild type, sdo1 mutant, sdo2 mutant, sdo1&2 mutant represent the sdo knockout mutants. At least three independent biological replicates were tested and error bars correspond to the standard deviations. Asterisks denote statistically significant changes (*P < 0.05, *** P < 0.001).
Analysis of the total GSH-dependent sulfur oxidation activity of A. caldus
We tested the sulfur oxidation activity of the Δsdo strains to study the effect of sdo gene deletion on the sulfur metabolic network of A. caldus. The total GSH-dependent sulfur oxidation activities of the wild type and mutant A. caldus strains grown in Starkey-S0 medium were measured using the cell extracts of these strains. As shown in Fig 6, the GSH-dependent sulfur oxidation activities of Δsdo1 and Δsdo1Δsdo2 increased 72% and 48%, respectively, compared to the wild type, while the activity of the Δsdo2 strain was approximately the same as that of the wild type (Fig 6). The high growth rates of sdo single- and double-knockout mutants in Starkey-S0 medium and the high level of GSH-dependent sulfur oxidation activity in these mutants, indicated that there may be undetected S0-oxidizing enzymes in A. caldus.
Transcriptional analysis of A.caldus strains
Cells of the wild type A. caldus grown on S0 were collected on the 4th (early logarithmic phase), 7th (late logarithmic phase) and 10th (early stationary phase) days of growth (Fig 4A) to purify total RNA for transcriptional analysis. Wild type A. caldus cells grown on tetrathionate were collected on the 2nd day (late logarithmic phase) (Fig 4B) to purify total RNA for transcriptional analysis. When S0 was used as the sole energy source, the RNA transcript level of sdo1 on the 7th (late logarithmic phase) and 10th (early stationary phase) days were 1.8-fold and 1.4-fold, respectively, compared with that of the 4th day (early logarithmic phase) (P<0.05) (Fig 7A), while the RNA transcript level of sdo2 on the 7th (late logarithmic phase) and 10th (early stationary phase) days were 5.4-fold and 0.8-fold, respectively, compared with that of the 4th day (early logarithmic phase) (P<0.01) (Fig 7B). These results showed that sdo genes were highly transcribed in the late logarithmic growth phase on S0. In the late logarithmic phase, the RNA transcript level of sdo1 on S0 medium increased to 3.5-fold compared with that on tetrathionate medium, and the RNA transcript level of sdo2 on S0 medium decreased to 0.6-fold compared with that on tetrathionate medium (P<0.001) (Fig 7C). This suggests that the metabolic substrates available to A. caldus significantly influenced sdo1 expression.
(A-B) RNA transcript level of sdo1 (A) and sdo2 (B) on the 7th (late logarithmic phase) and 10th (early stationary phase) days compared with that of the 4th day (early logarithmic phase) in S0 medium. (C) RNA transcript level of sdo genes in S0 medium compared to that in tetrathionate medium at the late logarithmic phase. Asterisks denote statistically significant changes (*P < 0.05, **P < 0.01, *** P < 0.001).
To further understand the influence of deletion or overexpression of sdo genes on other sulfur-oxidizing genes in A. caldus, the relative mRNA levels in wild type, mutants, overexpression strains and control strain (wild type carrying plasmid pSDU1) were measured using RT-qPCR. Cells were grown to late logarithmic phase either with S0 or tetrathionate as the sole energy source. Relative RNA transcript levels of these genes were determined against the wild type for sdo knockout strains, or the control strain (wild type carrying pSDU1) for sdo overexpression strains. When S0 was used as the sole energy, the RNA transcript levels of tetH and tqo were increased in sdo knockout strains and decreased in sdo overexpression strains, indicating a strong correlation between sdo genes and the tetrathionate intermediate pathway. Although the deletion or overexpression of sdo genes did not result in significant growth rate changes on Starkey-S0 medium, both strains showed altered transcriptions of genes in sox operon and heterodisulfide reductase complex operon (increased in sdo knockout strains and decreased in sdo overexpression strains)(Table 3). When tetrathionate was used as the sole energy, the overexpression of sdo1 resulted in decreased transcriptions of most assayed genes (e.g. hdrC2 and dsrE2), which was in agreement with the changes observed on S0. Δsdo2 increased the transcriptions of almost all assayed genes except sdo1, dsrE1, sqr1 and rhd3, which was in agreement with the observed increased growth of this mutant grown on tetrathionate compared with the wild type (Fig 4B). Lastly, the OE-sdo2 strain showed increased transcriptions of sqr and decreased transcription of rhd, indicating that sdo2 may have a functional connection with SQR and Rhd (Table 3).
Discovery and identification of SDOs in A. caldus MTH-04
Two new putative sdo genes (A5904_0421 and A5904_1112) were identified in the genome of A. caldus MTH-04 by conducting a BLASTP search with A5904_0790, however, only A5904_0421 had SDO activity. The proteins with SDO activity contain two conserved metal binding sites: site I consists of H113, H115, H169 and D188 and site II consists of D117, H118, H169 and H229 (in U. unicinctus ETHE1) . Both of these metal binding sites were found in A5904_0421 and A5904_0790, respectively. However, A5904_1112 only had H113 of metal binding site I and H118 from metal binding site II (Fig 8). The metal binding sites play important roles in SDO activity in eukaryotic ETHE1s [14, 21, 25]. The absence of conserved metal binding sites in A5904_1112 may explain the lack of SDO activity of this gene. A conserved Y residue is present in the GSH binding site of U. unicinctus ETHE1 (Y231) and P. putida SdoA (Y214), however, a corresponding tyrosine residue was not found in A5904_0421 (Fig 8) [16, 21]. GSH was reported to activate the specific SDO activity of human ETHE1 . Study of U. unicinctus ETHE1 showed that the lack of conserved residues in GSH binding site does not always affect the Km for GSSH, but results in reduced GSH binding capacity and lower SDO activity . Thus the lack of conserved residues in GSH binding site may underlie the low SDO activity in A5904_0421 due to the low GSH binding capacity. In addition, we analyzed the sequences using ProtCompB, SignalP 4.1 and PSORTB v3.0, which predicted subcellular localization for the A. caldus SDOs. The SDO homologues do not contain signal peptides or transmembrane regions, suggesting that they might be cytoplasmic proteins, similar to other bacterial SDOs [11, 15]. However, whether the SDO proteins are localized to the cytoplasm or the periplasmic space requires further investigation.
The proteins encoded by A5904_0421, A5904_0790 and A5904_1112 from A. caldus MTH-04 are abbreviated to Ac-0421 (OAN04050.1), Ac-0790 (OAN03714.1) and Ac-1112 (OAN03376.1), respectively. The SDOs from Acidithiobacillus thiooxidans, Agrobacterium tumefaciens, Homo sapiens, Pseudomonas putida and Urechis unicinctus are abbreviated to At SdoS (WP_010641840.1), At Blh (Q8UAA9), Homo ETHE1 (NP_055112), Pp SdoA (ABQ76243) and Uu ETHE1 (AEV92813.1), respectively. Identical amino acids residues are highlighted in grey. The conserved residues in the metal binding site are marked under the row with triangle (▲). The conserved residues interacting with GSH are marked above the row with star (★).
Distribution of SDOs in Acidithiobacillus spp and other sulfur oxidizing bacteria
Sequence analysis revealed that SDO homologues were widespread in Acidithiobacillus spp., and some sdo genes in Acidithiobacillus spp. are co-localized with other genes related to sulfur-metabolism. Both sdo1 and sdo2 are co-localized with other genes related to sulfur-metabolism in A. caldus MTH-04 (Fig 2) while the sqr gene (AFE_0267) is downstream of sdo (AFE_0269) in A. ferrooxidans ATCC 23270 as well [11, 26]. The co-localization of these genes on the chromosome suggests that they act cooperatively in sulfur metabolism. In addition, genes encoding transcriptional regulators are widely distributed upstream or downstream of sdo genes in Acidithiobacillus spp., indicating that sdo genes may be regulated at the transcriptional level. Transcriptional analysis revealed that the type of metabolic substrates that were available to A. caldus significantly influenced the expression of sdo1, which might be in accord with this hypothesis.
Our phylogenetic analysis of SDOs from other organisms identified a new SDO subgroup named SdoS, which predominantly contains SDOs from autotrophic sulfur-oxidizing bacteria. Previous studies on the structure of P. putida and M. xanthus SDOs supports the notion that SDOs may be classified into different subgroups by differences in the GS-moiety binding sites . Blhs and SdoAs (also known as type II persulfide dioxygenase due to their use of glutathione persulfide as a substrate) have the following conserved residues in the GSH binding pocket: D78, H149, D170, R181, Y214, R250, R253 and L262. ETHE1 enzymes, also known as type I persulfide dioxygenase, do not contain R250, R253 and L262. Members of the SdoS group contain the conserved residues D78, H149, D170 and R181 (Fig 8). Differences in GS-moiety binding sites and different SDO activities suggest that there may be different mechanisms for substrate binding among different SDO subgroups. The presence of sdo gene duplications in different subgroups suggests that there may also be different mechanisms for sulfur oxidation. The identification of a new SDO subgroup may aid in the discovery of further sdo genes in other organisms. Further studies of sdo genes in these strains may shed light on the different types of SDO subgroups, and how they may be involved in sulfur oxidation through similar or complementary mechanisms.
Sulfur dioxygenases in the sulfur metabolic network of A.caldus
SDOs from Acidithiobacillus thiooxidans and A. ferrooxidans are presumed to play a role in oxidizing extracellular elemental sulfur [9, 27]. However, the Δsdo1&2 strain grew well when elemental sulfur was used as the sole energy source (Fig 4A), and the sdo overexpression strains did not show increased growth rates. The results indicated that the SDO tested in this study does not play an essential role in the oxidation of extracellular elemental sulfur in A. caldus. Sulfur oxygenase reductase (SOR) is another elemental sulfur oxidation enzyme in A. caldus, which catalyzes the disproportionation of elemental sulfur to generate thiosulfate, sulfite, and sulfide. However, the sor gene in A. caldus MTH-04 has been lost during subcultivation in S0 media under laboratory conditions . Deletion of sdo1 significantly increased S0-oxidizing activity, indicating that the lack of sdo1 may potentially stimulate other sulfur oxidation pathways. Deletion of sdo increased S0-oxidizing activity in A. ferrooxidans as well . The high growth rates of the single and double sdo knockout strains in S0-media, and the high levels of elemental sulfur oxidation activity in these mutants, indicates that there are potentially undetected S0-oxidizing enzyme(s) in A. caldus.
When tetrathionate was used as the sole energy source, the growth was much lower than that on sulfur (Fig 4). Similar low cell growth phenomena on tetrathionate measured by OD600 were also observed before [22, 28, 29]. Wild type A. caldus MTH-04 cells grown on tetrathionate until stationary phase yielded 3.56±0.44 g (dry wt.) cell material per mol tetrathionate, which was in agreement with the result reported by Shiers et al. . The tetrathionate intermediate (S4I) pathway plays an important role in the metabolism of tetrathionate and thiosulfate in Acidithiobacillus spp. [26, 28, 31, 32, 33, 34]. The S4I pathway consists of a thiosulfate-quinone oxidoreductase (TQO), which oxidizes thiosulfate to tetrathionate, and a tetrathionate hydrolase (TetH) that hydrolyzes tetrathionate to thiosulfate, elemental sulfur and sulfate . Deletion of sdo1 caused complete inhibition of growth on tetrathionate, indicating that sdo1 is necessary for A. caldus growth on tetrathionate. Deletion or overexpression of sdo genes resulted in the differential expression of tetH, tqo, and dsrE2. Previous studies have suggested that DsrE plays a role in thiosulfate transfer [36, 37]. This result was in agreement with the correlation between sdo1 and the tetrathionate intermediate pathway. Based on the results of this study, we propose that sdo1 plays a key role in the metabolism of TetH metabolites, for example, elemental sulfur (Fig 9). Due to the difficulties in analyzing intermediate metabolites in A. caldus, the specific mechanisms of the pathway will require further investigation.
Abbreviations: TetH, tetrathionate hydrolase; TQO, thiosulfate-quinone oxidoreductase; Sox Complex, sulfur oxidation complex; SQR, sulfide:quinone oxidoreductase; Rhd (TST), rhodanese (also known as thiosulfate sulfurtransferase, TST); SDO, sulfur dioxygenase; HDR, heterodisulfide reductase. SDO oxidizes the elemental sulfur generated from the S4I and SQR pathways, sulfite is the product.
Thiosulfate is a key intermediate in RISC metabolism in Acidithiobacillus spp, and is metabolized through the S4I pathway and the truncated sulfur oxidation (Sox) pathway [28, 31, 32]. The truncated Sox pathway consists of SoxYZ, SoxAX and SoxB. Deletion or overexpression of the sdo genes also resulted in the differential expression of genes involved in the Sox pathway (Table 3). Previous studies have suggested that SDO may substitute for the role of Sox(CD)2 in A. caldus [28, 31]. However, there was no corroborating evidence for this hypothesis from this study. The transcriptional changes that were observed in the Δsdo1 mutant may be due to a compensatory mechanism for the loss of sdo1.
Previous studies have established that SDO (ETHE1) catalyzes the oxidation of persulfides by interacting with SQR and Rhd [12, 13, 14, 38]. The transcriptional changes of sqr and rhd genes in the sdo2 deletion or overexpression strains grown on different sulfur-substrates showed that sdo2 may have a functional connection with SQR and Rhd (Table 3). Sulfide (H2S) is an important signaling molecule in human, animals and plants, and its accumulation can lead to cell damage [12, 13, 14]. The sqr-sdo-rhd pathway plays an important role in sulfur detoxification in mitochondria [12, 13, 14]. However, Hildebrandt et al. and Goubern et al. showed the possibility of getting energy by metabolizing sulfide (H2S) in mammalian, invertebrate and human cells, respectively [38, 39]. In A. caldus, sulfide (H2S) is an important intermediate metabolite of the sulfur metabolism and it can also be used as the energy source [5, 6]. In the reported sulfur metabolism models of Acidithiobacillus spp., sulfide (H2S) is suggested to be metabolized by SQR and produced electrons go to the quinol pool [26, 28, 31, 32, 33, 34, 40]. So, the sqr-sdo-rhd pathway may play a role in sulfur metabolism of A. caldus. Taken together, sdo1 and sdo2 may play different roles in A. caldus.
S1 Fig. Identification of the sdo knockout mutants of A. caldus MTH-04 by PCR.
WT represent the wild type, Δsdo1, Δsdo2, Δsdo1&2 represent the sdo knockout mutants, respectively. 1, 2, 3, 4, 5, 6 represent the primer pairs 0421orfF-0421orfR, sdo1inF-sdo1inR, sdo1outF-sdo1outR, 0790orfF-0790orfR, sdo2inF-sdo2inR and sdo2outF-sdo2outR, respectively. The numbers on the right indicate the sizes of the fragments based on the molecular size maker (lane M).
S2 Fig. Purification of recombinant SDOs from E. coli BL21(DE3).
The proteins are loaded on 10% (wt/vol) SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. 1, 2, 3 represent the purified recombinant proteins of A5904_0421, A5904_0790 and A5904_1112, respectively. M: Blue Plus™ II Protein Marker (TransGen Biotech).
S1 Table. Bacterial strains and plasmids used in this study.
S3 Table. Predicted sizes of the fragments amplified by PCR with the primer pairs used in confirming the Δsdo mutants.
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