Complete Genome Sequence and Iron-Sulfur Oxidation Characteristics of The Newly Isolated Acidithiobacillus Ferrooxidans YQ-N3

Complete Genome Sequence and Iron-Sulfur Oxidation Characteristics of The Newly Isolated Acidithiobacillus Ferrooxidans YQ-N3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Complete Genome Sequence and Iron-Sulfur Oxidation Characteristics of The Newly Isolated Acidithiobacillus Ferrooxidans YQ-N3 Wenbo Li, Qiyan Feng, Ze Li, Di Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1430566/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Acidithiobacillus ferrooxidans ( A. ferrooxidans ) is a chemoautotroph that can simultaneously oxidize Fe 2+ , S, and reduced sulfur compounds. Therefore, this bacterium plays a key role in the natural cycles of Fe and S. In this study, a novel A. ferrooxidans strain was isolated from sediments of a river polluted by the acid mine drainage (AMD) of an abandoned mine in Shanxi, China, after which it was characterized via whole-genome sequencing. Furthermore, its functional genes related to iron and sulfur metabolism and response to environmental stress were analyzed, and its capacity to oxidize FeSO 4 ·7H 2 O, S 0 , and FeS 2 as an energy source was preliminarily discussed. The whole-genome sequencing results revealed that A. ferrooxidans YQ-N3 has a 3,217,720 bp genome, which is comprised of one circular chromosome and five circular plasmids (Plasmid A, Plasmid B, Plasmid C, Plasmid D, Plasmid E). Among these, Plasmid E had not been previously described in this species, and its genome contains various functional genes related to iron and sulfur, drug resistance, and heavy metal resistance. A. ferrooxidans YQ-N3 can increase the oxidation rate of Fe 2+ and S 0 and enhance the hydrophilicity of S 0 . Moreover, this strain can accelerate FeS 2 oxidation and the formation of secondary minerals. The present study demonstrated that the newly isolated A. ferrooxidans YQ-N3 could bio-oxidize iron and sulfur under acidic conditions, which was supported by our genome analysis results. Collectively, our findings provide important insights into the role and potential of A. ferrooxidans in biogeochemistry and industrial applications. Acidithiobacillusferrooxidans Complete genome iron-sulfur oxidation environmental adaptability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction A. ferrooxidans is an aerobic, acidophilic, gram-negative chemotrophic prokaryote (Kelly and Wood 2000). This bacterium is widely present in acidic mine soils, puddles, and environments containing iron or sulfur deposits (Barreto et al. 2003). Its major energy sources are Fe 2+ , S, and sulfide minerals, and its metabolites include sulfate and Fe 3+ (Adams et al. 1947). Therefore, it plays a key role in the natural biogeochemical cycles of Fe and S (Zhang et al. 2018). A. ferrooxidans participates in the oxidation of metal sulfide minerals in mining areas via the oxidation of Fe 2+ and reduced sulfur compounds, resulting in acid mine drainage (AMD) in mining areas. Due to its low pH and high sulfate and heavy metal contents, AMD can severely impact the surrounding soil and groundwater (Surber and Simonton 2017). In fact, the United States Environmental Protection Agency (EPA) has reported that the environmental risks posed by AMD are second only to global warming and ozone depletion (Iakovleva et al. 2015). However, despite considerable efforts to identify the generation mechanisms of AMD and potential removal strategies, little progress has been made toward the development of effective AMD mitigation methods. Due to its capacity to oxidize Fe 2+ and reduced sulfur compounds at an industrial scale, A. ferrooxidans has been widely applied in bioleaching, biological desulfurization, biosynthesis, and biochemical production (Valdés et al. 2008). For instance, Lorenzo-Tallafigo et al developed a new process for the recovery of lead, silver, and gold from polymetallic sulfide ores using A. ferrooxidans and demonstrated that the process was cleaner than traditional hydrometallurgical methods (e.g, hot brine leaching) (Lorenzo-Tallafigo et al. 2019). The process included a bio-oxidation stage, where sulfides were oxidized in the presence of extremophiles, followed by pickling and citric acid leaching, after which lead was successfully recovered. Nie et al isolated A. ferrooxidans Z1 from printed circuit board waste and confirmed that A. ferrooxidans Z1 was able to extract 96% copper from metal concentrates at an initial Fe 2+ concentration of 12 g L − 1 after 7 days (Nie et al. 2015). A single A. ferrooxidans bioreactor can reportedly remove 98.0% SO 2 and 99.0% H 2 S from gas (Yu et al. 2007). Biological desulfurization of coal is beneficial for its clean utilization. Rout et al reported that A. ferrooxidans cells catalyzed the removal of sulfur in both organic and inorganic forms in coal samples, and approximately 79% of the total sulfur was removed from coal samples during a microbiological desulfurization process in a 500 mL flask within 14 d (Rout et al. 2021). Additionally, A. ferrooxidans can also be used to produce a variety of biomaterials, including schwertmannite, jarosite, iron-sulfur clusters (ISC), and magnetosomes (Mengran et al. 2021). Therefore, an in-depth study of A. ferrooxidans would not only provide key insights into the mechanisms of AMD generation and potential AMD removal strategies but would also shed light on the metabolic processes that enable microbes to adapt to harsh environments. In turn, this would broaden our understanding of redox metabolism in bacteria and its value in industrial applications. Currently, most studies on A. ferrooxidans have focused on the model strain ATCC23270, whereas newly discovered A. ferrooxidans strains have remained largely uncharacterized. Therefore, examining novel A. ferrooxidans strains may provide new insights into the involvement of A. ferrooxidans in biogeochemical cycles and its industrial applicability. In this study, a novel A. ferrooxidans strain was isolated from sediments of a river polluted by the AMD of an abandoned mine in Shanxi, China. The oxidation features of Fe 2+ , S 0 , and FeS 2 were studied and the whole genome of the newly isolated strain was sequenced. Additionally, the capacity of the newly discovered strain to oxidize iron and sulfur and its adaptability to the environment were analyzed from a functional gene perspective. Therefore, our study provides a theoretical basis for studying the formation and processing of AMD, as well as for the development and optimization of industrial applications using A. ferrooxidans YQ-N3. Materials And Methods Isolation, purification, and identification Previous studies have demonstrated that A. ferrooxidans is ubiquitous in acid mining sites. This bacterium is strictly aerobic, acidophilic, chemoautotrophic, and can oxidize metallic sulfides. Specifically, A. ferrooxidans can use Fe 2+ and reduced sulfur as energy sources. This bacterium grows at 20–40°C with a pH of 1.5–3.5 and can use CO 2 as a carbon source and NH 4+ as a nitrogen source (Rawlings 2005 ). According to the growth characteristics of A. ferrooxidans , Leathen or 9K medium are often used during the isolation and purification of this bacterium (Lavalle et al. 2005 ). In this study, 9K medium was selected for isolation and purification of A. ferrooxidans . 9K liquid medium is composed of solutions A and B. In turn, solution A is composed of 3.00 g (NH 4 ) 2 ∙SO 4 , 0.10 g/L KCl, 0.655 g/L K 2 HPO 4 ·3H 2 O, 0.50 g/L MgSO 3 ·7H 2 O, and 0.01 g/L Ca(NO 3 ) 2 ·4H 2 O. After the preparation of solution A, the pH of the solution was adjusted to 1.8 with 1:1 concentrated sulfuric acid and sterilized with high temperature and humidity at 121°C. Solution B contains FeSO 4 ·7H 2 O and was sterilized using a microporous membrane (d = 0.22 µm). Upon cooling down to room temperature, solution A was evenly mixed with solution B before use. Furthermore, 9K solid medium contains solution A, solution B, and solution C. Solutions A and B were prepared as described above, whereas solution C was a 7.5 g/L agarose solution. After solution C was heated and dissolved, it was sterilized with high temperature and humidity at 121°C. Once solution A was cooled to approximately 60°C, it was mixed with solution C. When the pH was adjusted to 2.0, solution B was quickly added to the mixture to make a solid medium (Kai et al. 1989 ). The strains used in this study were isolated from sediments of a river polluted by AMD from an abandoned mine in Shanxi, China. Following aseptic procedures, the sediment samples were collected and stored at 4°C and then transported to the laboratory. Next, 10 g of sediment sample was added to 100 mL of sterile water and the mixture was incubated at 30°C and 180 r/min for three days. The samples were then filtered to obtain a bacterial solution, which was stored at 4°C. The bacteria-containing solution was inoculated into an Erlenmeyer flask containing 9K liquid medium at a 10% inoculum volume. Next, the flask was covered with a perforated sealing film and transferred to a constant temperature incubator at 30°C and 180 r/min for continuous enrichment culture. Once the bacteria had multiplied until the medium turned reddish-brown, the culture was inoculated into a new 9K liquid medium at a 10% inoculum volume rate, and isolation and purification were performed after 5–6 consecutive enrichments. The enrichment experiments were conducted in triplicate, including blank controls. An alternate solid-liquid culture method was used for the isolation and purification of A. ferrooxidans (Feng et al. 2017 ). The gradient dilution method was adopted during the isolation. Sterile sulfuric acid solution (pH = 1.8) was used to dilute the enriched bacterial liquid samples, and each sample was sequentially diluted from the initial concentration 10, 10 2 , 10 3 , 10 4 , 10 5 , and 10 6 times. Next, 0.2 mL of bacterial solutions at different concentrations were inoculated into the solidified 9K solid medium and evenly coated using a sterile coating rod. After coating, the Petri dish was placed upright in a constant temperature incubator at 30°C for 10 h, after which the Petri dish was incubated upside down to prevent contamination caused by the backflow of water to the 9K solid medium. Isolation and purification were performed when the liquid medium turned reddish-brown. This process was repeated until pure strains were obtained. A small amount of bacterial solution was taken after isolation and purification, after which a 1-1.5 cm diameter bacterial smear was evenly spread on a glass slide with a sterile inoculation loop, and ammonium oxalate crystal violet staining solution was added in a dropwise fashion to the smeared area, which was then stained for 1 min, and washed with water. After blotting with absorbent paper, anhydrous ethanol was also added in a dropwise fashion to cover the entire smear area and then washed with water for 30 s. After blotting with absorbent paper, safranin dye solution was added in a dropwise fashion and stained for 1 min. The samples were then washed with water, blot dried with absorbent paper, and placed under a light microscope to observe bacterial morphology and Gram staining. Afterward, 100 mL of bacterial liquid cultured to the log phase was frozen and centrifuged at high speed, fixed with 2.5% glutaraldehyde overnight, and centrifuged to store the bacteria. The bacteria were subjected to gradient dehydration with 30%, 50%, 75%, 90%, and 100% Ethanol (10 min per step). The samples were then freeze-dried at -20, -40, -60, and − 80°C and kept at each temperature for 12 h. After sample loading and gold spraying, the samples were examined via scanning electron microscopy (SEM). Bacterial cultures at the logarithmic phase were frozen and centrifuged to obtain the bacterial cells for bacterial species identification. The amplicons were sequenced using a 3730 first-generation sequencer (paired-end sequencing) to obtain ABI sequencing peak map files. The bacteria obtained in the experiment were identified as pure A. ferrooxidans , after which their growth conditions were studied, including the optimum growth temperature and inoculum size. A. ferrooxidans was inoculated in 9K liquid medium with 10% of the inoculum, and the pH of the medium was adjusted to 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2. The samples were then placed and cultured in a 30 ℃ incubator at 180 r/min. The bacterial concentration of the culture medium was calculated using a hemocytometer to explore the optimal initial pH for growth, and 10% of the inoculum was inoculated into 9K liquid medium. The culture temperature was set to 15, 20, 25, 30, 35, and 40°C, and the initial pH of the medium was 1.8. A hemocytometer was used to calculate the bacterial concentration to study the optimal growth temperature. To determine the optimal inoculum volume, A. ferrooxidans was inoculated into 9K liquid medium at a 5%, 10%, 15%, and 20% inoculum volume rate, after which the samples were incubated at 30°C and 180 r/min. The initial pH of the medium was also 1.8, and the bacterial concentration of the culture medium was calculated using a hemocytometer to study the optimal inoculum amount. Whole-genome sequencing The bacteria collected above were sent to Beijing BMK Biological Co, Ltd, and whole-genome sequencing was carried out using PacBio sequencing technology. The experimental process was performed according to the standard protocol provided by PacBio, including sample quality detection, library construction, library quality detection, and library sequencing. PacBio sequencing technology consists of using a SMRT chip as the sequencing carrier. In the nanopore inside the SMRT chip, DNA polymerase is combined with the template, and four kinds of bases (dNTPs) are labeled with 4 different fluorescent dyes. In the base-pairing stage, the addition of different bases will emit different wavelengths, and the type of bases entering can be determined according to the wavelength and peak value of the emitted light (Rhoads and Au 2015 ). Low-quality and short fragments with a length of less than 2,000 bp were removed from the reads obtained after sequencing, and clean reads were obtained for genome assembly and functional annotation. To fully display the features of the genome, a genome circle diagram of a single sample was generated using the Circos software and a variety of information was displayed in the diagram to provide a comprehensive and intuitive understanding of the characteristics of the strain genome. Upon comparing the 16S rRNA sequences in the NCBI database, the 19 strains that were closest to the species level were selected, and the NJ (Neighbor-Joining) method was used to construct a phylogenetic tree using the MEGA 6.0 software to visualize the evolutionary relationship of the sample and the near-source species. Chromosome genes were predicted using Glimmer (Delcher et al. 2007 ) and plasma genome was predicted using GeneMarkS (Besemer and Borodovsky 2005 ). The predicted genome was compared with the COG and KEGG databases based on protein sequence, and functional annotation was performed to obtain the corresponding gene function information. Oxidation characteristics of Fe2+, S0, and FeS2 by A. ferrooxidans YQ-N3 As the dominant microbe in metallic sulfide mining areas, A. ferrooxidans plays a vital role in Fe and S biogeochemical cycling (Akcil et al. 2007 ). Therefore, studies on the Fe 2+ , S 0 , and FeS 2 oxidation capacities of novel A. ferrooxidans strains are essential, specifically due to their theoretical and practical value. This study characterized A. ferrooxidans strain YQ-N3 isolated in the previous stage, and its ability to oxidize FeSO 4 ·7H 2 O, S 0 , and FeS 2 as an energy source was discussed. The preserved YQ-N3 strain was transferred to 9K medium containing FeSO 4 ·7H 2 O, S 0 , and FeS 2 with 10% of the inoculum, then placed in a constant temperature culture shaker at 30°C and 180 r/min for multiple activations. The bacterial density was determined using a hemocytometer, and acclimated log-phase bacteria were collected. The domesticated strains were inoculated into 9K medium containing FeSO 4 ·7H 2 O, S 0 , and FeS 2 at a 10% inoculum volume proportion, and the bacterial concentration and Fe 2+ , Fe 3+ , SO 4 2− , pH, and Eh in the culture system were monitored thereafter. The specific experimental design is shown in Table 1 . Three parallel groups and one blank control were set for each group of experiments. Table 1 Study design for the assessment of Fe 2+ , S 0 , and FeS 2 oxidation by A. ferrooxidans YQ-N3 Group Energy source Bacterial inoculum A.f Oxidation Fe 2+ FeSO 4 ·7H 2 O (8.95 g) 10% Control FeSO 4 ·7H 2 O (8.95 g) 0% A.f Oxidation S 0 S 0 (0.5 g) 10% Control S 0 (0.5 g) 0% A.f Oxidation FeS 2 FeS 2 (5 g) 10% Control FeS 2 (5 g) 0% Note : All experiments were conducted with 200 mL of 9K medium without Fe. At the end of the experiment, mineral samples generated by oxidation of Fe 2+ and FeS 2 before and after the reaction were collected. After dust removal, the samples were ground and screened for mineral composition analysis. The phases and components of the collected samples were determined by X-ray diffraction (XRD) using an X-ray diffractometer (Bruker D8 ADVANCE, Germany) with Cu Kα radiation operated at 40 kV and 30 mA. The samples were scanned at a 0.02 °/s rate to record the patterns within a 2θ range of 3-105°, the mineral composition of the sample was then analyzed. Results Growth characteristics of A. ferrooxidans YQ-N3 In this study, sediment was collected from a river polluted by the AMD of an abandoned mine in Shanxi, China. The sediment samples were treated with sterile water and inoculated in 9K-Fe 2+ liquid medium for enrichment culture. After cultivation, all three replicate cultures exhibited the same characteristics. The color of the medium gradually changed from light blue-green to turbid, after which it became clear red-brown after continued cultivation. After culturing on 9K-Fe 2+ solid medium for 30 days via the dilution coating method, colonies as shown in Fig. 1 -A appeared on the plate. The colonies were round, with a prominent center, neat edges, and yellow surrounding. After repeated solid-liquid alternating culture, the bacteria were collected by high-speed freezing and centrifugation, and DNA was extracted for gene sequencing. After gene amplification, there was a single clear target band, which was tentatively named A. ferrooxidans YQ-N3 according to the comparison between the sequencing results and BLAST. The collected bacteria were diluted several times with normal saline and then observed by Gram staining, thus confirming that the bacteria were gram-negative. Optical microscope observations showed that the bacteria were rod-shaped (both as single cells and aggregates) and were able to swim rapidly. SEM analyses indicated that the bacteria had a short rod shape with blunt rounded ends, as shown in Fig. 1 -B, with a length of approximately 0.8–1.2 µm and a width of 0.2–0.5 µm. Upon comparing the 16S rRNA sequences in the GenBank database, the 19 strains that were closest at the species level were selected, and the NJ (Neighbor-Joining) method was used to construct the phylogenetic tree of A. ferrooxidans YQ-N3 using the MEGA 6.0 software, as shown in Fig. 2 . Phylogenetic tree analyses demonstrated that A. ferrooxidans YQ-N3 was distinct from Pseudomonas , Oceanicoccus , Luteimonas , and Xanthomonas , but appeared to be related to A. thiooxidans , A. ferridurans , and A. ferrivorans . Particularly, the 16S rDNA sequence of A. ferrooxidans YQ-N3 was more than 99.93% similar to that of A. ferrooxidans ATCC23270, and it was therefore concluded that the isolated strains were A. ferrooxidans . After screening and obtaining pure A. ferrooxidans YQ-N3 cultures, the optimum growth conditions were explored. The results showed that A. ferrooxidans YQ-N3 could grow at a pH range of 1.6–2.4 and a temperature range of 20–35 ℃, which was consistent with previous studies. The strain achieved optimal growth at an initial pH of 1.8, a 30 ℃ temperature, and an inoculum volume of 10%. Under these conditions, A. ferrooxidans YQ-N3 could reach a density of up to 2.3 × 10 8 cells/mL after 16 h. Genome overview of A. ferrooxidans YQ-N3 Upon sequencing the whole genome of A. ferrooxidans YQ-N3, short-length circular consensus sequencing (ccs) reads were filtered out from the raw data as a quality control measure. The filtered ccs reads were denovo assembled, and the assembled genome was corrected for errors. Once the assembly was completed, genome analysis and functional annotation were performed. Whole-genome analysis indicated that the genome size of A. ferrooxidans YQ-N3 was 3,217,720 bp, including one circular chromosome and five circular plasmids (plasmid A, plasmid B, plasmid C, plasmid D, and plasmid E). The sequence length of the circular chromosome was 3,043,496 bp and the GC content was 58.64%. To fully demonstrate the genome features of A. ferrooxidans YQ-N3, the genome circle was drawn using the Circos software (Fig. 3 ). The coding sequences (CDs) in the genome were predicted using the glimmer, GeneMarkS, and prodigal software. A total of 3,200 predicted CD genes, 6 rRNAs, 46 tRNAs, and 18 sRNAs were predicted. The sequence length of the circular Plasmid A was 79,659 bp and the GC content was 61.64%. The length of Plasmid B was 34,460 bp and the GC content was 60.54%. The sequence length of Plasmid C was 29,178 bp and the GC content was 62.67%. The sequence length of plasmid D was 23,017 bp and the GC content was 60.69%. The sequence length of plasmid E was 7,910 bp and the GC content was 52.40%. Among them, the Plasmid E sequence had not been annotated in the reference database. The complete genome sequences for the main chromosome and plasmids A-E of A. ferrooxidans YQ-N3 were submitted to the GenBank database under accession numbers CP084172.1, CP084173.1, CP084174.1, CP084175.1, CP084176.1, and CP084177.1, respectively. Genes associated with iron and sulfur metabolism In-depth characterization of its iron and sulfur metabolism system from a functional gene perspective would not only provide key insights into the physiology of these microorganisms but could establish a theoretical basis for the efficient application of these bacteria in industry. According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation results of A. ferrooxidans YQ-N3, the genes related to iron and sulfur metabolism are summarized in Table 2 . Our findings demonstrated that this strain possesses multiple genes related to Fe 2+ oxidation metabolism, including cyc2, rus, petA, petB, coxA, coxB, and coxC. A. ferrooxidans can oxidize various reductive inorganic sulfides, including sulfur, sulfides, thiosulfates, tetrathionates, and sulfites. A. ferrooxidans YQ-N3 possesses multiple genes related to sulfur metabolism, including sqr, doxDA, moaD, cysD, hdrA2, hdrB2, hdrC2, and thiS. Table 2 General features and genomic comparison between A. ferrooxidans YQ-N3 and selected representatives. Strain Geographic origin BioProject Genome size (Mb) GC% CDS Plasmids Scaffolds A. ferrooxidans YQ-N3 Shanxi, China PRJNA543567 3.21772 58.7316% 3132 5 6 A. ferrooxidans YNTRS-40 - PRJNA543563 3.25704 58.4696% 3168 1 2 A. ferrooxidans ATCC23270 Bituminous coal mine effluent PRJNA543564 2.9824 58.8% 2927 - 1 A. ferrooxidans BYM Baiyin, China PRJNA543565 3.2555 58.4696% 3134 1 2 A. ferrooxidans NFP31 Volcanic ash deposits on Miyake-jima, Japan PRJNA543566 3.24985 58.4724% 3193 1 2 A. ferrooxidans ATCC53993 - PRJNA543568 2.88504 58.9% 2811 - 1 A. ferrooxidans CCM4253 Mine waters, Czech Republic PRJNA5435690 3.19656 58.6% 3073 - 15 A. ferrooxidans DSM16786 Wudalianchi Heilongjiang, China PRJNA543570 3.67579 58.4% 3636 - 49 A. ferrooxidans YQH-1 Wudalianchi volcano water, China PRJNA543571 3.11122 58.60% 3012 - 96 A. ferrooxidans Hel18 Flue dust PRJNA543572 3.10916 58.6% 3065 - 123 A. ferrooxidans PQ506 Santiago, Chile PRJNA543573 3.37146 58.3% 3342 - 277 A. ferrooxidans PQ50 5 Santiago, Chile PRJNA543574 3.51657 58.4% 3534 - 305 A. ferrooxidans CF3 Santiago, Chile PRJNA543575 3.01139 58.7% 3057 - 310 A. ferrooxidans F221 - PRJNA543576 3.00995 58.7% 3006 - 360 A. ferrooxidans BY0502 - PRJNA543577 2.97667 56.8% 3026 - 295 A. ferrooxidans BY-3 Gansu, China PRJNA543578 3.83234 57.8% 3777 - 194 A. ferrooxidans RVS1 Andacollo gold mining area, Argentina PRJNA543579 2.82631 58.8% 2705 - 49 A. ferrooxidans DLC-5 - PRJNA543580 4.18422 57.6% - - 2090 A. ferrooxidans COP1 - PRJNA543581 3.008 58.8% 3855 - 1561 A. ferrooxidans S10 Santiago, Chile PRJNA543582 2.953 58.8% 3998 - 1827 Note: “-” indicates no data. Table 3 List of selected genes identified in strain YQ-N3 strain via KEGG annotation, including genes for iron and sulfur metabolism. Gene name Gene length KEGG gene ID KEGG orthology description fdxA 327bp afr: AFE_0014 ferredoxin feoA 288bp afr:AFE_2523 ferrous iron transport protein A feoB 2349bp afr:AFE_2524 ferrous iron transport protein B hemH 1014bp afr:AFE_0179 protoporphyrin/coproporphyrin ferrochelatase cyc2 1458bp afr:AFE_3153 iron: rusticyanin reductase [EC:1.16.9.1] resB 1845bp afr:AFE_3112 cytochrome c biogenesis protein hyaC 756bp afr:AFE_2429 Ni/Fe-hydrogenase 1 B-type cytochrome subunit coxA 1884bp afe:Lferr_2747 cytochrome c oxidase subunit I coxB 765bp afe:Lferr_2748 cytochrome c oxidase subunit II coxC 555bp afr:AFE_3148 cytochrome c oxidase subunit III porA 984bp tti:THITH_06955 pyruvate ferredoxin oxidoreductase alpha subunit porB 1164bp tig:THII_3692 pyruvate ferredoxin oxidoreductase beta subunit porC 603bp tti:THITH_06960 pyruvate ferredoxin oxidoreductase gamma subunit - 2340bp afe:Lferr_1935 iron complex outermembrane receptor protein fdxA 621bp afr:AFE_1844 ferredoxin fdx 306bp afr:AFE_1541 ferredoxin, 2Fe-2S rus 564bp afr:AFE_3146 rustycanin - 1035bp afe:Lferr_1212 iron complex transport system substrate-binding protein petA 621bp afe:Lferr_2707 ubiquinol-cytochrome c reductase iron-sulfur subunit petB 1209bp afe:Lferr_2708 ubiquinol-cytochrome c reductase cytochrome b subunit petC 729bp afr:AFE_3111 ubiquinol-cytochrome c reductase cytochrome c1 subunit erpA 372bp afj:AFERRID_10140 iron-sulfur cluster insertion protein moaD 243bp afr:AFE_0975 sulfur-carrier protein iscA 324bp afr:AFE_0675 iron-sulfur cluster assembly protein doxDA 1083bp afe: Lferr_0045 thiosulfate dehydrogenase (quinone) doxA 1083bp afj:AFERRID_13680 thiosulfate dehydrogenase (quinone) small subunit cysN 1353bp afr:AFE_3125 sulfate adenylyltransferase subunit 1 cysD 939bp afe:Lferr_2723 sulfate adenylyltransferase subunit 2 cysJ 1767bp afr:AFE_3121 sulfite reductase (NADPH) flavoprotein alpha-component cysI 1692bp afr:AFE_3122 sulfite reductase (NADPH) hemoprotein beta-component cysH 738bp afr:AFE_3123 phosphoadenosine phosphosulfate reductase sqr 1140bp afr:AFE_2601 sulfide:quinone oxidoreductase hdrA2 1056bp afr:AFE_2553 heterodisulfide reductase subunit A2 hdrB2 912bp afr:AFE_2550 heterodisulfide reductase subunit B2 hdrC2 720bp afr:AFE_2551 heterodisulfide reductase subunit C2 thiS 201bp afr:AFE_0642 sulfur carrier protein Gene traits related to environmental adaptability Based on protein sequence alignments, the coding genes predicted by A. ferrooxidans YQ-N3 were compared with the COG (Clusters of Orthologous Groups of proteins) and KEGG databases for functional annotation, and the corresponding functional annotations were obtained Fig. 4 shows the COG annotation classification statistics of A. ferrooxidans YQ-N3. Our findings indicated that A. ferrooxidans YQ-N3 has a total of 2,571 genes annotated in COG, accounting for approximately 80.34% of the total number of genes, and these genes are classified into 22 COG types. Among these genes, a total of 728 with unknown function were identified. The proportion of genes related to replication, recombination and repair (L), cell wall/membrane/envelope biogenesis (M), energy production and conversion (C), and inorganic ion transport and metabolism (P) was slightly higher than that of genes with other functions, accounting for 9.45%, 6.76%, 7.0%, and 6.2% of all annotated genes, respectively. Additionally, defense mechanisms accounted for 5.98% of all annotated genes, suggesting that this strain has a strong ability to self-repair and resist harsh environments (Zhang et al. 2019 ). The results of A. ferrooxidans YQ-N3 genome annotation using the KEGG pathway database indicated that the gene functions of this bacterium could be mainly divided into six categories, including Metabolism, Cellular Processes, Human Diseases, Genetic Information Processing, Organismal Systems, and Environmental Information Processing, as shown in Fig. 5 . Among these classifications, 1,353 functional genes related to Metabolism could be divided into 12 types, 81 functional genes related to Cellular Processes could be divided into 3 types, 124 genes related to Human Diseases could be divided into 10 types, 183 functional genes related to Genetic Information Processing could be divided into 6 categories, 56 genes related to Organic Systems could be divided into 8 categories, and 177 genes related to Environmental Information Processing could be divided into two categories. Gene island prediction of the A. ferrooxidans YQ-N3 genome was performed using IslandViewer (Bertelli et al. 2017 ). The prophage prediction was performed using Phage_Finder (Fouts 2006 ). CRISPR-Cas (Clustered Regularly Interspersed Short Palindromic Repeats) prediction was performed using Minced (Bland et al. 2007 ). The predicted results indicated that the chromosome of A. ferrooxidans YQ-N3 contained 10 gene islands, whereas Plasmid A contained one gene island, and these gene islands contained a total of 181 CDs. Interestingly, our analyses indicated that a prophage genome had integrated into the chromosome of A. ferrooxidans YQ-N3. The sequence length of the prophage was 17,089 bp, and had 19 CDs, and a 58.64% GC content. The chromosome of A. ferrooxidans YQ-N3 was predicted to contain four CRISPRs with average repeat lengths of 27, 28, 23, and 27 bp, respectively. The average lengths of the spacer sequences were 27, 32, 43, and 45 bp, respectively. Oxidation of Fe 2+ , S 0 , and pyrite by A. ferrooxidans YQ-N3 During Fe 2+ oxidation by A. ferrooxidans YQ-N3, the color of the medium gradually shifted from clear light green to yellow-green at first, and after 16 h, the medium completely changed to reddish-brown. Then, a yellow precipitate gradually appeared in the reaction system. The blank control group did not exhibit any obvious color change. Figure 6 shows the trends of pH, ORP, Fe 2+ content, and total iron content of the medium during Fe 2+ oxidation. According to Fig. 1 a, the pH in the experiment involving A. ferrooxidans YQ-N3 increased from 1.8 to 2.16 after 48 h, and then rapidly decreased to 1.7. ORP also increased throughout the experiment but this trend decelerated slightly after 48 h. In the blank control group, pH and ORP showed a slight upward trend throughout the experiment. This can be attributed to H + consumption by oxidation of Fe 2+ into Fe 3+ (Offeddu et al. 2015 ). Fe 3+ , SO4 2− , and other cations (e.g, K + , Na + , NH 4 + ) begin to react as the Fe 3+ content increases, which results in the production of H + and the minerals jarosite (KFe 3 (SO 4 ) 2 (OH) 6 ) and ammoniojarosite ((NH 4 ) 2 Fe 6 (SO 4 ) 4 (OH) 12 ) (see Fig. 7 ), which is consistent with previous studies (Nazari et al. 2014 ). Therefore, the pH rapidly decreased and the ORP increase rate slowed down in the later stage of the experimental group (Li et al. 2021 ). Figure 1 b shows that the Fe 2+ content in the experiment involving A. ferrooxidans YQ-N3 continued to decrease rapidly, and the Fe 2+ content reached 0 mg/L when the reaction reached 60 h. Total iron concentration decreased by approximately 2700 mg/L during the whole reaction period, whereas only 14.5% Fe 2+ was oxidized and the total iron concentration in the blank control group remained unchanged for 96 h. This can be attributed to the fact that A. ferrooxidans YQ-N3 accelerates the oxidation of Fe 2+ , and the Fe 3+ generated by the reaction leads to a decrease in the total iron content in the system. Fe 2+ is naturally oxidized and generates free Fe 3+ in the blank control group during the reaction, and therefore the total iron content remained unchanged in the system. During oxidation of S 0 by A. ferrooxidans YQ-N3, the medium gradually changed from clear and colorless to light yellow, the particle size of sulfur powder gradually became smaller, and its hydrophobicity gradually weakened. However, no obvious change was observed during the reaction of the blank control group. This phenomenon was mainly because A. ferrooxidans adsorbed on the surface of S when oxidizing it and secreted hydrophilic organic substances that covered the surface of S to enhance its hydrophilicity (Konishi et al. 1995 ; Knickerbocker 2000). During the oxidation of S 0 by A. ferrooxidans YQ-N3, the pH value of the medium decreased from 2.2 to 1.74, whereas the SO 4 2− concentration of the medium increased to 3896.66 mg/L after 45 d (Fig. 8 ). This can be attributed to the release of H + through the oxidation of S 0 to SO 4 2− . Figure 9 illustrates the trends of pH, ORP, SO 4 2− , Fe 2+ , and Fe t during pyrite oxidation by A. ferrooxidans YQ-N3. In the experimental group, the pH value decreased from 1.9 to 1.2 after 45 d, the ORP increased from 353 to 632 mV after 45 d, the SO 4 2− concentration increased to 5433.8 mg/L after 45 d, the Fe 2+ content decreased drastically from 43.6 mg/L to a negligible level after 23 d, and the Fe t content increased to 3,853 mg/L after 45 d. Characterization by XRD revealed that pyrite oxidized by A. ferrooxidans YQ-N3 contains jarosite and FeOOH, in addition to quartz impurities (Fig. 10 ). In the blank control group, the pH value decreased slightly, the ORP decreased and then slightly increased, the SO 4 2− concentration increased to 563 mg/L after 45 d, the Fe 2+ content increased to 178.2 mg/L after 45 d, and the Fe t concentration increased to 197.7 mg/L after 45 d. This could be because the H + released by the oxidation of S 0 into SO 4 2− exceeded the levels consumed by Fe 2+ oxidation. A. ferrooxidans YQ-N3 can accelerate the oxidation of Fe 2+ to Fe 3+ and pyrite oxidation can be carried out continuously and rapidly. The dissolved Fe mainly exists in the form of Fe 3+ in this system, and therefore ORP continues to increase. Discussion A. ferrooxidans can grow in a variety of extreme environments and have great potential applications in industries such as bioleaching and biological desulfurization (Gonzáleztoril et al. 2003 ; Valdés and Holmes 2009 ; Umanskii and Klyushnikov 2013 ). However, there are few published genomes of A. ferrooxidans strains (Zhang et al. 2019 ), and therefore the whole genome sequencing and analysis of A. ferrooxidans YQ-N3 could provide key insights into its genetic properties and potential applicability. To understand the differences between the genomes of A. ferrooxidans YQ-N3 and other A. ferrooxidans strains, the genome information of A. ferrooxidans YQ-N3 and A. ferrooxidans that have been published in GenBank were selected for tabulation analysis, as shown in Table 1 . The genome size distribution of A. ferrooxidans was 2.82631–4.18422 Mb, the GC content was 56.8%-58.9%, and the CD number distribution was 2,705-3,998. The genome size, GC%, and number of CDs of A. ferrooxidans YQ-N3 are similar to those of other A. ferrooxidans strains. However, the assembled A. ferrooxidans YQ-N3 genome in the published database included no more than five plasmids. A. ferrooxidans is a chemoautotrophic bacterium with a complex energy metabolism pathway, which obtains energy through the oxidation of Fe 2+ and reducing sulfur compounds, and generates energy to sustain its growth by fixing carbon and nitrogen in the air (Hallberg et al. 2010 ; Bonnefoy and Holmes 2012 ). The oxidative metabolism of ferrous and sulfur compounds by A. ferrooxidans makes this bacterium especially well-suited for industrial applications (Ponce et al. 2012 ). Previous studies have demonstrated that Fe 2+ oxidation by A. ferrooxidans in the electron transport chain has two patterns, including the downhill potential gradient and the uphill potential gradient (Johnson 2008 ). Most electrons generated by Fe 2+ oxidation pass along the downhill potential gradient. Specifically, electrons generated by Fe 2+ oxidation enter the cytoplasm from the outer cell membrane, then pass to the cytochrome protein cyc2, after which they are received by rus, a ceruloplasmin, and passed to cyc2, cytochrome protein. Afterward, they are received by cytochrome oxidase aa3 and then passed to O 2 , where they finally combine with protons to generate H 2 O (Bird et al. 2011 ). In this process, the oxidation process of the rus operon plays a dominant role, and the rus operon is composed of multiple genes including cyc2, coxA, coxB, coxC, and rus (Andrés et al. 2004 ). Furthermore, studies on the regulation of the expression of key rus operon genes in model microorganisms show that the expression of the rus operon is induced by Fe 2+ (Amouric et al. 2009 ; Kucera et al. 2013 ). The sqr gene, which encodes the sulfide quinone reductase enzyme, has been previously identified in various A. ferrooxidans strains (Wakai et al. 2017). doxDA encoding thiosulfate quinone reductase was also identified in ATCC23270 (Fabian et al. 2004 ) and CCM4253 (Janiczek et al. 2007 ). When elemental sulfur was used as an energy source, the transcription level of the heterodisulfide reductase (HdrABC) complex encoded by ten genes including hdrB, hdrA, hdrC2, and hdrB2 was significantly up-regulated, indicating that this complex was related to sulfur metabolism in A. ferrooxidans (Liu t al. 2012). Therefore, YQ-N3 can oxidize Fe 2+ and sulfur and could thus have industrial applications. However, its metabolic pathways remain to be further explored. A. ferrooxidans is widely found in metal mines, high-sulfate coal mines, and other extreme environments due to its high environmental resistance, as well as its tolerance to metal ions and high acidity (Acosta et al. 2005 ). The characterization of genes associated with environmental resistance in A. ferrooxidans YQ-N3 would provide insights into the mechanisms that mediate its adaptability, which also lays the foundation for future research on its survival state and industrial application value in extreme environments. In this study, the environmental resistance-related gene signatures in A. ferrooxidans YQ-N3 were investigated from the perspective of gene annotation and mobile genetic elements. The results of COG annotation show that the proportion of genes with uncharacterized functions was much higher than that of the other categories, suggesting that this strain likely possesses several novel genes. In addition, YQ-N3-gene-1491 and YQ-N3-gene-2286 located in the chromosome were annotated as ENOG410ZI6F and their COG description was “heavy metal transport detoxification protein,” which further suggested that this strain was resistant to heavy metals. The results of KEGG annotation show that the majority of the gene types in the A. ferrooxidans YQ-N3 genome summary were associated with Metabolism, followed by Environmental Information Processing. The genes related to Environmental Information Processing were divided into signal transduction and membrane transport, among which multiple genes related to signal transduction were annotated, including cusA, cusB, cusR, and cusS. Previous studies have demonstrated that the cus proton pump system is widely distributed in gram-negative bacteria and is associated with copper and silver resistance in A. ferrooxidans (Mealman et al. 2012 ). In fact, cusA, cusB, and cusF possess metal-binding sites (Chacón et al. 2018 ), and Cu 2+ is directly transferred from protein to protein in the cus system during ion transport, thereby reducing the potential toxicity of free Cu 2+ to cells (Almárcegui et al. 2014 ; Chacón et al. 2018 ). Nan et al linked the adaptability of A. ferrooxidans to chemotactic movement and quorum sensing (QS), both of which allow this bacterium to grow, develop, and reproduce in extreme environments (Nan et al. 2011 ). Mobile genetic elements refer to some exogenous gene fragments that can be incorporated into bacterial genomes to adapt to environmental changes or improve the likelihood of survival. These fragments generally contain genes that encode enzymes of other proteins with specific functions to help bacteria overcome adverse conditions or take advantage of resources that otherwise could not be exploited. The gene islands often carry functional genes or contain integrase, plasmid conjugation-related factors, etc. (Coutinho et al. 2015 ). The prophage sequence often contained some functional genes, such as antibiotic resistance genes and virulence genes, among others, all of which enhanced the adaptability of this bacterium to its environment (Dominguez-Mirazo et al. 2019 ). The CRISPR/Cas system is a prokaryotic immune system designed to resist the invasion of exogenous genetic material (Cady et al. 2012 ). In summary, our findings suggested that A. ferrooxidans YQ-N3 may have strong adaptability to a variety of extreme environments, making it a promising candidate for various industrial applications such as biological desulfurization, bioleaching, metal processing, and others. Studying the oxidation features of Fe and reduced sulfur compounds by A. ferrooxidans YQ-N3 is helpful to further exploit its industrial application potential. In this study, the oxidation of different energy substances (Fe 2+ , S 0 , and pyrite) by A. ferrooxidans YQ-N3 was investigated. The results indicate A. ferrooxidans YQ-N3 can accelerate the oxidation of Fe 2+ and contributes to the formation of minerals such as jarosite and ammoniojarosite while also accelerating the oxidation of S 0 and enhancing its hydrophilicity. A. ferrooxidans YQ-N3 can also achieve rapid and continuous oxidation of pyrite by accelerating Fe 2+ oxidation, thereby increasing the ORP and the SO 4 2− concentration, decreasing the pH value, and generating minerals (e.g, jarosite and FeOOH), all of which are hallmarks of the AMD generation process (Kuang et al. 2013 ). Thus, A. ferrooxidans YQ-N3 can accelerate the production of AMD. Conclusions In this study, a novel A. ferrooxidans strain was isolated from sediment from a river polluted by the AMD of an abandoned mine in Shanxi, China. Our whole-genome sequencing analyses revealed that the genome size of A. ferrooxidans YQ-N3 is 3,217,720 bp. This genome includes one circular chromosome and five circular plasmids (Plasmid A, Plasmid B, Plasmid C, Plasmid D, and Plasmid E), among which Plasmid E had not been previously annotated in public databases. The sequence length and GC content of the circular chromosome were 3,043,496 bp and 58.64%, respectively. Additionally, 3,200 CD genes, six rRNAs, 46 tRNAs, and 18 sRNAs were predicted. This newly identified strain possesses multiple genes related to iron and sulfur metabolism and environmental resistance. However, additional studies are required to characterize its energy metabolism pathways. A. ferrooxidans YQ-N3 can accelerate the oxidation of Fe 2+ , S 0 , and FeS 2 , as well as the generation of secondary minerals during the oxidation process. This study not only establishes a theoretical foundation for future research on the generation and treatment of AMD but also provides key insights into the role and potential of A. ferrooxidans in biogeochemistry and industrial applications. Declarations Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No. 41977159); Open fund project of Key Laboratory of coal measures mineral resources of China Coal Geology administration (KFKT-2020-3). 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Microorganisms 8(1): 2. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 13 Feb, 2023 Reviewers invited by journal 21 Mar, 2022 Editor assigned by journal 11 Mar, 2022 Submission checks completed at journal 08 Mar, 2022 First submitted to journal 08 Mar, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1430566","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":89335466,"identity":"b917dcd8-d845-4061-8d8f-9e5d6b992ca7","order_by":0,"name":"Wenbo Li","email":"","orcid":"","institution":"China University of Mining and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenbo","middleName":"","lastName":"Li","suffix":""},{"id":89335467,"identity":"62e97652-13b4-4b85-bc54-7b34aa295959","order_by":1,"name":"Qiyan Feng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYBAC9gYILcfG3nyAOC08EHUGxnw8xxJI05I4TyJHgUgt7DmGn3lz/qS3MeQwMPyo2EaEFp43xtK82wxy2xjOHmDsOXObsBZ7iRwzZrAWxr4EZsY2IrTwQLWkszHzGJCmJYGNjWgtPM+KJeduMzZs42FLOEiUX3jYkzd+eLtNTl5+/uODD35UEKGFgSHDAM48QIx6IEh/QKTCUTAKRsEoGLEAADJYNLDshUslAAAAAElFTkSuQmCC","orcid":"","institution":"China University of Mining and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Qiyan","middleName":"","lastName":"Feng","suffix":""},{"id":89335468,"identity":"83744b8f-948e-4352-9b12-a24f5f4429b4","order_by":2,"name":"Ze Li","email":"","orcid":"","institution":"China University of Mining and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ze","middleName":"","lastName":"Li","suffix":""},{"id":89335469,"identity":"1e36a0c7-d7f5-4921-a23a-8bbaa1830044","order_by":3,"name":"Di Chen","email":"","orcid":"","institution":"China University of Mining and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2022-03-08 10:14:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1430566/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1430566/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19130095,"identity":"94d09bb3-6a39-4302-9d25-4207428e69c5","added_by":"auto","created_at":"2022-03-11 15:49:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":439020,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic features of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/841b8659180d9f2e68cf06ce.png"},{"id":19130424,"identity":"a284b598-2c47-4b6d-93da-e2a54684ad09","added_by":"auto","created_at":"2022-03-11 15:52:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44075,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 sequences and other sequences obtained from the GenBank database\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/96ec6a82897b5f2c9a218dbd.png"},{"id":19130099,"identity":"c722602b-57c5-4e08-a171-494114305a12","added_by":"auto","created_at":"2022-03-11 15:49:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103525,"visible":true,"origin":"","legend":"\u003cp\u003eCircular genome map of\u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3. From outside to center: genes on the direct strand, genes on the complementary strand, tRNAs (orange), rRNA(purple), CRISPR (blue), and genomic island (green), GC-skew, sequencing depths are also displayed; a: Chromosome, b: Plasmid A, c: Plasmid B, d: Plasmid C, e: Plasmid D and f: Plasmid E).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/63b2b1432bdebd167d8ad12c.png"},{"id":19130093,"identity":"ea45867e-d64e-42cd-957c-b77c27e2ae6f","added_by":"auto","created_at":"2022-03-11 15:49:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73653,"visible":true,"origin":"","legend":"\u003cp\u003eCOG annotation classification statistics map of \u003cem\u003eA. ferrooxidans\u003c/em\u003eYQ-N3\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/f86d4bf38b061d3d833022ff.png"},{"id":19130101,"identity":"061c87ef-0b80-45b2-a1a1-7c5ee1963ad1","added_by":"auto","created_at":"2022-03-11 15:49:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":124663,"visible":true,"origin":"","legend":"\u003cp\u003eKEGGmetabolism pathway annotation of \u003cem\u003eA. ferrooxidans\u003c/em\u003eYQ-N3 genes.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/322958d31e19651451e9e83b.png"},{"id":19130426,"identity":"5fae3b36-cd0e-448c-919a-8bf3c1f1a7f9","added_by":"auto","created_at":"2022-03-11 15:52:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":931002,"visible":true,"origin":"","legend":"\u003cp\u003eTrends of physicochemical indexes during Fe\u003csup\u003e2+\u003c/sup\u003eoxidation by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/a94eefa894ce304d04d5b7da.png"},{"id":19130097,"identity":"10c4c2ee-a67e-4c66-a6c4-9f313ef74437","added_by":"auto","created_at":"2022-03-11 15:49:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":290157,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of minerals generated by Fe\u003csup\u003e2+\u003c/sup\u003eoxidated by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/934c57beef17c11c2a937e40.png"},{"id":19130425,"identity":"79095a2b-33fc-43f0-a42c-5786f712376c","added_by":"auto","created_at":"2022-03-11 15:52:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":682481,"visible":true,"origin":"","legend":"\u003cp\u003eTrends of physicochemical indexes during S\u003csup\u003e0+\u003c/sup\u003eoxidation by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/6e169e46b31b367ef8999fc1.png"},{"id":19130100,"identity":"0d23831e-cdf1-4200-9103-87e899d03408","added_by":"auto","created_at":"2022-03-11 15:49:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":21824,"visible":true,"origin":"","legend":"\u003cp\u003eTrends of physicochemical indexes during FeS\u003csub\u003e2\u003c/sub\u003eoxidation by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/c0211476b8aadde877a443b7.png"},{"id":19130427,"identity":"da0e757b-4825-4b5f-bd66-bbab99916ee1","added_by":"auto","created_at":"2022-03-11 15:52:29","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":295406,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of residues after FeS\u003csub\u003e2\u003c/sub\u003e was oxidated by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/9eb114b417959f1b5eafad9b.png"},{"id":19130429,"identity":"8d89f346-3d5a-4664-b553-e115cea6ee24","added_by":"auto","created_at":"2022-03-11 15:52:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":467755,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1430566/v1/6e6c2270-0b24-4cc2-8f18-bbe63ca2fe58.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eComplete Genome Sequence and Iron-Sulfur Oxidation Characteristics of The Newly Isolated \u003cem\u003eAcidithiobacillus Ferrooxidans \u003c/em\u003eYQ-N3\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e is an aerobic, acidophilic, gram-negative chemotrophic prokaryote (Kelly and Wood 2000). This bacterium is widely present in acidic mine soils, puddles, and environments containing iron or sulfur deposits (Barreto et al. 2003). Its major energy sources are Fe\u003csup\u003e2+\u003c/sup\u003e, S, and sulfide minerals, and its metabolites include sulfate and Fe\u003csup\u003e3+\u003c/sup\u003e (Adams et al. 1947). Therefore, it plays a key role in the natural biogeochemical cycles of Fe and S (Zhang et al. 2018). \u003cem\u003eA. ferrooxidans\u003c/em\u003e participates in the oxidation of metal sulfide minerals in mining areas via the oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e and reduced sulfur compounds, resulting in acid mine drainage (AMD) in mining areas. Due to its low pH and high sulfate and heavy metal contents, AMD can severely impact the surrounding soil and groundwater (Surber and Simonton 2017). In fact, the United States Environmental Protection Agency (EPA) has reported that the environmental risks posed by AMD are second only to global warming and ozone depletion (Iakovleva et al. 2015). However, despite considerable efforts to identify the generation mechanisms of AMD and potential removal strategies, little progress has been made toward the development of effective AMD mitigation methods.\u003c/p\u003e\n\u003cp\u003eDue to its capacity to oxidize Fe\u003csup\u003e2+\u003c/sup\u003e and reduced sulfur compounds at an industrial scale, \u003cem\u003eA. ferrooxidans\u003c/em\u003e has been widely applied in bioleaching, biological desulfurization, biosynthesis, and biochemical production (Vald\u0026eacute;s et al. 2008). For instance, Lorenzo-Tallafigo \u003cem\u003eet al\u003c/em\u003e developed a new process for the recovery of lead, silver, and gold from polymetallic sulfide ores using \u003cem\u003eA. ferrooxidans\u003c/em\u003e and demonstrated that the process was cleaner than traditional hydrometallurgical methods (e.g, hot brine leaching) (Lorenzo-Tallafigo et al. 2019). The process included a bio-oxidation stage, where sulfides were oxidized in the presence of extremophiles, followed by pickling and citric acid leaching, after which lead was successfully recovered. Nie \u003cem\u003eet al\u003c/em\u003e isolated \u003cem\u003eA. ferrooxidans\u003c/em\u003e Z1 from printed circuit board waste and confirmed that \u003cem\u003eA. ferrooxidans\u003c/em\u003e Z1 was able to extract 96% copper from metal concentrates at an initial Fe\u003csup\u003e2+\u003c/sup\u003e concentration of 12 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after 7 days (Nie et al. 2015). A single \u003cem\u003eA. ferrooxidans\u003c/em\u003e bioreactor can reportedly remove 98.0% SO\u003csub\u003e2\u003c/sub\u003e and 99.0% H\u003csub\u003e2\u003c/sub\u003eS from gas (Yu et al. 2007). Biological desulfurization of coal is beneficial for its clean utilization. Rout \u003cem\u003eet al\u003c/em\u003e reported that \u003cem\u003eA. ferrooxidans\u003c/em\u003e cells catalyzed the removal of sulfur in both organic and inorganic forms in coal samples, and approximately 79% of the total sulfur was removed from coal samples during a microbiological desulfurization process in a 500 mL flask within 14 d (Rout et al. 2021). Additionally, \u003cem\u003eA. ferrooxidans\u003c/em\u003e can also be used to produce a variety of biomaterials, including schwertmannite, jarosite, iron-sulfur clusters (ISC), and magnetosomes (Mengran et al. 2021). Therefore, an in-depth study of \u003cem\u003eA. ferrooxidans\u003c/em\u003e would not only provide key insights into the mechanisms of AMD generation and potential AMD removal strategies but would also shed light on the metabolic processes that enable microbes to adapt to harsh environments. In turn, this would broaden our understanding of redox metabolism in bacteria and its value in industrial applications. Currently, most studies on \u003cem\u003eA. ferrooxidans\u003c/em\u003e have focused on the model strain ATCC23270, whereas newly discovered \u003cem\u003eA. ferrooxidans\u003c/em\u003e strains have remained largely uncharacterized. Therefore, examining novel \u003cem\u003eA. ferrooxidans\u003c/em\u003e strains may provide new insights into the involvement of \u003cem\u003eA. ferrooxidans\u003c/em\u003e in biogeochemical cycles and its industrial applicability.\u003c/p\u003e\n\u003cp\u003eIn this study, a novel \u003cem\u003eA. ferrooxidans\u003c/em\u003e strain was isolated from sediments of a river polluted by the AMD of an abandoned mine in Shanxi, China. The oxidation features of Fe\u003csup\u003e2+\u003c/sup\u003e, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e were studied and the whole genome of the newly isolated strain was sequenced. Additionally, the capacity of the newly discovered strain to oxidize iron and sulfur and its adaptability to the environment were analyzed from a functional gene perspective. Therefore, our study provides a theoretical basis for studying the formation and processing of AMD, as well as for the development and optimization of industrial applications using \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation, purification, and identification\u003c/h2\u003e \u003cp\u003ePrevious studies have demonstrated that \u003cem\u003eA. ferrooxidans\u003c/em\u003e is ubiquitous in acid mining sites. This bacterium is strictly aerobic, acidophilic, chemoautotrophic, and can oxidize metallic sulfides. Specifically, \u003cem\u003eA. ferrooxidans\u003c/em\u003e can use Fe\u003csup\u003e2+\u003c/sup\u003e and reduced sulfur as energy sources. This bacterium grows at 20\u0026ndash;40\u0026deg;C with a pH of 1.5\u0026ndash;3.5 and can use CO\u003csub\u003e2\u003c/sub\u003e as a carbon source and NH\u003csup\u003e4+\u003c/sup\u003e as a nitrogen source (Rawlings \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). According to the growth characteristics of \u003cem\u003eA. ferrooxidans\u003c/em\u003e, Leathen or 9K medium are often used during the isolation and purification of this bacterium (Lavalle et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In this study, 9K medium was selected for isolation and purification of \u003cem\u003eA. ferrooxidans\u003c/em\u003e. 9K liquid medium is composed of solutions A and B. In turn, solution A is composed of 3.00 g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e∙SO\u003csub\u003e4\u003c/sub\u003e, 0.10 g/L KCl, 0.655 g/L K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO, 0.50 g/L MgSO\u003csub\u003e3\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, and 0.01 g/L Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO. After the preparation of solution A, the pH of the solution was adjusted to 1.8 with 1:1 concentrated sulfuric acid and sterilized with high temperature and humidity at 121\u0026deg;C. Solution B contains FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO and was sterilized using a microporous membrane (d\u0026thinsp;=\u0026thinsp;0.22 \u0026micro;m). Upon cooling down to room temperature, solution A was evenly mixed with solution B before use. Furthermore, 9K solid medium contains solution A, solution B, and solution C. Solutions A and B were prepared as described above, whereas solution C was a 7.5 g/L agarose solution. After solution C was heated and dissolved, it was sterilized with high temperature and humidity at 121\u0026deg;C. Once solution A was cooled to approximately 60\u0026deg;C, it was mixed with solution C. When the pH was adjusted to 2.0, solution B was quickly added to the mixture to make a solid medium (Kai et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe strains used in this study were isolated from sediments of a river polluted by AMD from an abandoned mine in Shanxi, China. Following aseptic procedures, the sediment samples were collected and stored at 4\u0026deg;C and then transported to the laboratory. Next, 10 g of sediment sample was added to 100 mL of sterile water and the mixture was incubated at 30\u0026deg;C and 180 r/min for three days. The samples were then filtered to obtain a bacterial solution, which was stored at 4\u0026deg;C. The bacteria-containing solution was inoculated into an Erlenmeyer flask containing 9K liquid medium at a 10% inoculum volume. Next, the flask was covered with a perforated sealing film and transferred to a constant temperature incubator at 30\u0026deg;C and 180 r/min for continuous enrichment culture. Once the bacteria had multiplied until the medium turned reddish-brown, the culture was inoculated into a new 9K liquid medium at a 10% inoculum volume rate, and isolation and purification were performed after 5\u0026ndash;6 consecutive enrichments. The enrichment experiments were conducted in triplicate, including blank controls. An alternate solid-liquid culture method was used for the isolation and purification of \u003cem\u003eA. ferrooxidans\u003c/em\u003e (Feng et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The gradient dilution method was adopted during the isolation. Sterile sulfuric acid solution (pH\u0026thinsp;=\u0026thinsp;1.8) was used to dilute the enriched bacterial liquid samples, and each sample was sequentially diluted from the initial concentration 10, 10\u003csup\u003e2\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e5\u003c/sup\u003e, and 10\u003csup\u003e6\u003c/sup\u003e times. Next, 0.2 mL of bacterial solutions at different concentrations were inoculated into the solidified 9K solid medium and evenly coated using a sterile coating rod. After coating, the Petri dish was placed upright in a constant temperature incubator at 30\u0026deg;C for 10 h, after which the Petri dish was incubated upside down to prevent contamination caused by the backflow of water to the 9K solid medium. Isolation and purification were performed when the liquid medium turned reddish-brown. This process was repeated until pure strains were obtained.\u003c/p\u003e \u003cp\u003eA small amount of bacterial solution was taken after isolation and purification, after which a 1-1.5 cm diameter bacterial smear was evenly spread on a glass slide with a sterile inoculation loop, and ammonium oxalate crystal violet staining solution was added in a dropwise fashion to the smeared area, which was then stained for 1 min, and washed with water. After blotting with absorbent paper, anhydrous ethanol was also added in a dropwise fashion to cover the entire smear area and then washed with water for 30 s. After blotting with absorbent paper, safranin dye solution was added in a dropwise fashion and stained for 1 min. The samples were then washed with water, blot dried with absorbent paper, and placed under a light microscope to observe bacterial morphology and Gram staining. Afterward, 100 mL of bacterial liquid cultured to the log phase was frozen and centrifuged at high speed, fixed with 2.5% glutaraldehyde overnight, and centrifuged to store the bacteria. The bacteria were subjected to gradient dehydration with 30%, 50%, 75%, 90%, and 100% Ethanol (10 min per step). The samples were then freeze-dried at -20, -40, -60, and \u0026minus;\u0026thinsp;80\u0026deg;C and kept at each temperature for 12 h. After sample loading and gold spraying, the samples were examined via scanning electron microscopy (SEM).\u003c/p\u003e \u003cp\u003eBacterial cultures at the logarithmic phase were frozen and centrifuged to obtain the bacterial cells for bacterial species identification. The amplicons were sequenced using a 3730 first-generation sequencer (paired-end sequencing) to obtain ABI sequencing peak map files.\u003c/p\u003e \u003cp\u003eThe bacteria obtained in the experiment were identified as pure \u003cem\u003eA. ferrooxidans\u003c/em\u003e, after which their growth conditions were studied, including the optimum growth temperature and inoculum size. \u003cem\u003eA. ferrooxidans\u003c/em\u003e was inoculated in 9K liquid medium with 10% of the inoculum, and the pH of the medium was adjusted to 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2. The samples were then placed and cultured in a 30 ℃ incubator at 180 r/min. The bacterial concentration of the culture medium was calculated using a hemocytometer to explore the optimal initial pH for growth, and 10% of the inoculum was inoculated into 9K liquid medium. The culture temperature was set to 15, 20, 25, 30, 35, and 40\u0026deg;C, and the initial pH of the medium was 1.8. A hemocytometer was used to calculate the bacterial concentration to study the optimal growth temperature. To determine the optimal inoculum volume, \u003cem\u003eA. ferrooxidans\u003c/em\u003e was inoculated into 9K liquid medium at a 5%, 10%, 15%, and 20% inoculum volume rate, after which the samples were incubated at 30\u0026deg;C and 180 r/min. The initial pH of the medium was also 1.8, and the bacterial concentration of the culture medium was calculated using a hemocytometer to study the optimal inoculum amount.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWhole-genome sequencing\u003c/h2\u003e \u003cp\u003eThe bacteria collected above were sent to Beijing BMK Biological Co, Ltd, and whole-genome sequencing was carried out using PacBio sequencing technology. The experimental process was performed according to the standard protocol provided by PacBio, including sample quality detection, library construction, library quality detection, and library sequencing. PacBio sequencing technology consists of using a SMRT chip as the sequencing carrier. In the nanopore inside the SMRT chip, DNA polymerase is combined with the template, and four kinds of bases (dNTPs) are labeled with 4 different fluorescent dyes. In the base-pairing stage, the addition of different bases will emit different wavelengths, and the type of bases entering can be determined according to the wavelength and peak value of the emitted light (Rhoads and Au \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Low-quality and short fragments with a length of less than 2,000 bp were removed from the reads obtained after sequencing, and clean reads were obtained for genome assembly and functional annotation. To fully display the features of the genome, a genome circle diagram of a single sample was generated using the Circos software and a variety of information was displayed in the diagram to provide a comprehensive and intuitive understanding of the characteristics of the strain genome. Upon comparing the 16S rRNA sequences in the NCBI database, the 19 strains that were closest to the species level were selected, and the NJ (Neighbor-Joining) method was used to construct a phylogenetic tree using the MEGA 6.0 software to visualize the evolutionary relationship of the sample and the near-source species. Chromosome genes were predicted using Glimmer (Delcher et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and plasma genome was predicted using GeneMarkS (Besemer and Borodovsky \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The predicted genome was compared with the COG and KEGG databases based on protein sequence, and functional annotation was performed to obtain the corresponding gene function information.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOxidation characteristics of Fe2+, S0, and FeS2 by\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eA. ferrooxidans\u003c/span\u003e \u003cb\u003eYQ-N3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs the dominant microbe in metallic sulfide mining areas, \u003cem\u003eA. ferrooxidans\u003c/em\u003e plays a vital role in Fe and S biogeochemical cycling (Akcil et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, studies on the Fe\u003csup\u003e2+\u003c/sup\u003e, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e oxidation capacities of novel A. \u003cem\u003eferrooxidans\u003c/em\u003e strains are essential, specifically due to their theoretical and practical value.\u003c/p\u003e \u003cp\u003eThis study characterized \u003cem\u003eA. ferrooxidans\u003c/em\u003e strain YQ-N3 isolated in the previous stage, and its ability to oxidize FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e as an energy source was discussed. The preserved YQ-N3 strain was transferred to 9K medium containing FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e with 10% of the inoculum, then placed in a constant temperature culture shaker at 30\u0026deg;C and 180 r/min for multiple activations. The bacterial density was determined using a hemocytometer, and acclimated log-phase bacteria were collected. The domesticated strains were inoculated into 9K medium containing FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e at a 10% inoculum volume proportion, and the bacterial concentration and Fe\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, pH, and Eh in the culture system were monitored thereafter. The specific experimental design is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Three parallel groups and one blank control were set for each group of experiments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy design for the assessment of Fe\u003csup\u003e2+\u003c/sup\u003e, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e oxidation by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnergy source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacterial inoculum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.f\u003c/em\u003e Oxidation Fe\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (8.95 g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (8.95 g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.f\u003c/em\u003e Oxidation S\u003csup\u003e0\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003csup\u003e0\u003c/sup\u003e (0.5 g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003csup\u003e0\u003c/sup\u003e (0.5 g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eA.f\u003c/em\u003e Oxidation FeS\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFeS\u003csub\u003e2\u003c/sub\u003e (5 g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFeS\u003csub\u003e2\u003c/sub\u003e (5 g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote : All experiments were conducted with 200 mL of 9K medium without Fe.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAt the end of the experiment, mineral samples generated by oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e and FeS\u003csub\u003e2\u003c/sub\u003e before and after the reaction were collected. After dust removal, the samples were ground and screened for mineral composition analysis. The phases and components of the collected samples were determined by X-ray diffraction (XRD) using an X-ray diffractometer (Bruker D8 ADVANCE, Germany) with Cu Kα radiation operated at 40 kV and 30 mA. The samples were scanned at a 0.02 \u0026deg;/s rate to record the patterns within a 2θ range of 3-105\u0026deg;, the mineral composition of the sample was then analyzed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGrowth characteristics of\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eA. ferrooxidans\u003c/span\u003e \u003cstrong\u003eYQ-N3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, sediment was collected from a river polluted by the AMD of an abandoned mine in Shanxi, China. The sediment samples were treated with sterile water and inoculated in 9K-Fe\u003csup\u003e2+\u003c/sup\u003e liquid medium for enrichment culture. After cultivation, all three replicate cultures exhibited the same characteristics. The color of the medium gradually changed from light blue-green to turbid, after which it became clear red-brown after continued cultivation. After culturing on 9K-Fe\u003csup\u003e2+\u003c/sup\u003e solid medium for 30 days via the dilution coating method, colonies as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e-A appeared on the plate. The colonies were round, with a prominent center, neat edges, and yellow surrounding. After repeated solid-liquid alternating culture, the bacteria were collected by high-speed freezing and centrifugation, and DNA was extracted for gene sequencing. After gene amplification, there was a single clear target band, which was tentatively named \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 according to the comparison between the sequencing results and BLAST. The collected bacteria were diluted several times with normal saline and then observed by Gram staining, thus confirming that the bacteria were gram-negative. Optical microscope observations showed that the bacteria were rod-shaped (both as single cells and aggregates) and were able to swim rapidly. SEM analyses indicated that the bacteria had a short rod shape with blunt rounded ends, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e-B, with a length of approximately 0.8\u0026ndash;1.2 \u0026micro;m and a width of 0.2\u0026ndash;0.5 \u0026micro;m.\u003c/p\u003e\n\u003cp\u003eUpon comparing the 16S rRNA sequences in the GenBank database, the 19 strains that were closest at the species level were selected, and the NJ (Neighbor-Joining) method was used to construct the phylogenetic tree of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 using the MEGA 6.0 software, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Phylogenetic tree analyses demonstrated that \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 was distinct from \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eOceanicoccus\u003c/em\u003e, \u003cem\u003eLuteimonas\u003c/em\u003e, and \u003cem\u003eXanthomonas\u003c/em\u003e, but appeared to be related to \u003cem\u003eA. thiooxidans\u003c/em\u003e, \u003cem\u003eA. ferridurans\u003c/em\u003e, and \u003cem\u003eA. ferrivorans\u003c/em\u003e. Particularly, the 16S rDNA sequence of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 was more than 99.93% similar to that of \u003cem\u003eA. ferrooxidans\u003c/em\u003e ATCC23270, and it was therefore concluded that the isolated strains were \u003cem\u003eA. ferrooxidans\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAfter screening and obtaining pure \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 cultures, the optimum growth conditions were explored. The results showed that \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 could grow at a pH range of 1.6\u0026ndash;2.4 and a temperature range of 20\u0026ndash;35 ℃, which was consistent with previous studies. The strain achieved optimal growth at an initial pH of 1.8, a 30 ℃ temperature, and an inoculum volume of 10%. Under these conditions, \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 could reach a density of up to 2.3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cells/mL after 16 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome overview of\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eA. ferrooxidans\u003c/span\u003e \u003cstrong\u003eYQ-N3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon sequencing the whole genome of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3, short-length circular consensus sequencing (ccs) reads were filtered out from the raw data as a quality control measure. The filtered ccs reads were \u003cem\u003edenovo\u003c/em\u003e assembled, and the assembled genome was corrected for errors. Once the assembly was completed, genome analysis and functional annotation were performed. Whole-genome analysis indicated that the genome size of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 was 3,217,720 bp, including one circular chromosome and five circular plasmids (plasmid A, plasmid B, plasmid C, plasmid D, and plasmid E). The sequence length of the circular chromosome was 3,043,496 bp and the GC content was 58.64%. To fully demonstrate the genome features of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3, the genome circle was drawn using the Circos software (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The coding sequences (CDs) in the genome were predicted using the glimmer, GeneMarkS, and prodigal software. A total of 3,200 predicted CD genes, 6 rRNAs, 46 tRNAs, and 18 sRNAs were predicted. The sequence length of the circular Plasmid A was 79,659 bp and the GC content was 61.64%. The length of Plasmid B was 34,460 bp and the GC content was 60.54%. The sequence length of Plasmid C was 29,178 bp and the GC content was 62.67%. The sequence length of plasmid D was 23,017 bp and the GC content was 60.69%. The sequence length of plasmid E was 7,910 bp and the GC content was 52.40%. Among them, the Plasmid E sequence had not been annotated in the reference database. The complete genome sequences for the main chromosome and plasmids A-E of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 were submitted to the GenBank database under accession numbers CP084172.1, CP084173.1, CP084174.1, CP084175.1, CP084176.1, and CP084177.1, respectively.\u003c/p\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eGenes associated with iron and sulfur metabolism\u003c/h2\u003e\n\u003cp\u003eIn-depth characterization of its iron and sulfur metabolism system from a functional gene perspective would not only provide key insights into the physiology of these microorganisms but could establish a theoretical basis for the efficient application of these bacteria in industry. According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation results of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3, the genes related to iron and sulfur metabolism are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Our findings demonstrated that this strain possesses multiple genes related to Fe\u003csup\u003e2+\u003c/sup\u003e oxidation metabolism, including cyc2, rus, petA, petB, coxA, coxB, and coxC. \u003cem\u003eA. ferrooxidans\u003c/em\u003e can oxidize various reductive inorganic sulfides, including sulfur, sulfides, thiosulfates, tetrathionates, and sulfites. \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 possesses multiple genes related to sulfur metabolism, including sqr, doxDA, moaD, cysD, hdrA2, hdrB2, hdrC2, and thiS.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eGeneral features and genomic comparison between A. ferrooxidans YQ-N3 and selected representatives.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eStrain\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGeographic origin\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBioProject\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGenome size (Mb)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGC%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCDS\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePlasmids\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eScaffolds\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e \u003cstrong\u003eYQ-N3\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eShanxi, China\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543567\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.21772\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.7316%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3132\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e YNTRS-40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543563\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.25704\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.4696%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3168\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e ATCC23270\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBituminous coal\u003c/p\u003e\n\u003cp\u003emine effluent\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543564\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.9824\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2927\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e BYM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBaiyin, China\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543565\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.2555\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.4696%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3134\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e NFP31\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVolcanic ash deposits on Miyake-jima, Japan\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543566\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.24985\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.4724%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3193\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e ATCC53993\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543568\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.88504\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.9%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2811\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e CCM4253\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMine waters, Czech Republic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA5435690\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.19656\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.6%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3073\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e DSM16786\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWudalianchi Heilongjiang, China\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543570\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.67579\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.4%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3636\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e YQH-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eWudalianchi volcano water, China\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543571\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.11122\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.60%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e96\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e Hel18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFlue dust\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543572\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.10916\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.6%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3065\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e123\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e PQ506\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSantiago, Chile\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543573\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.37146\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.3%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3342\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e277\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e PQ50\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSantiago, Chile\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543574\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.51657\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.4%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3534\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e305\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e CF3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSantiago, Chile\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543575\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.01139\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.7%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3057\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e310\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e F221\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543576\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.00995\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.7%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3006\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e360\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e BY0502\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543577\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.97667\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e56.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3026\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e295\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e BY-3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGansu, China\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543578\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.83234\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e57.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3777\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e194\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e RVS1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAndacollo gold mining area, Argentina\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543579\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.82631\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2705\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e49\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e DLC-5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543580\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.18422\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e57.6%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2090\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e COP1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543581\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.008\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3855\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1561\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e S10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSantiago, Chile\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePRJNA543582\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.953\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.8%\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3998\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1827\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"8\"\u003eNote: \u0026ldquo;-\u0026rdquo; indicates no data.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eList of selected genes identified in strain YQ-N3 strain via KEGG annotation, including genes for iron and sulfur metabolism.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene name\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene length\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKEGG gene ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKEGG orthology description\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003efdxA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e327bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr: AFE_0014\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eferredoxin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003efeoA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e288bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_2523\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eferrous iron transport protein A\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003efeoB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2349bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_2524\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eferrous iron transport protein B\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ehemH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1014bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_0179\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eprotoporphyrin/coproporphyrin ferrochelatase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecyc2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1458bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3153\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiron: rusticyanin reductase [EC:1.16.9.1]\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eresB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1845bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3112\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecytochrome c biogenesis protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ehyaC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e756bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_2429\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNi/Fe-hydrogenase 1 B-type cytochrome subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecoxA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1884bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe:Lferr_2747\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecytochrome c oxidase subunit I\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecoxB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e765bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe:Lferr_2748\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecytochrome c oxidase subunit II\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecoxC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e555bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3148\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecytochrome c oxidase subunit III\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eporA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e984bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etti:THITH_06955\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epyruvate ferredoxin oxidoreductase alpha subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eporB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1164bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etig:THII_3692\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epyruvate ferredoxin oxidoreductase beta subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eporC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e603bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etti:THITH_06960\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epyruvate ferredoxin oxidoreductase gamma subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2340bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe:Lferr_1935\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiron complex outermembrane receptor protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003efdxA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e621bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_1844\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eferredoxin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003efdx\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e306bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_1541\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eferredoxin, 2Fe-2S\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003erus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e564bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3146\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003erustycanin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1035bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe:Lferr_1212\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiron complex transport system substrate-binding protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epetA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e621bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe:Lferr_2707\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eubiquinol-cytochrome c reductase iron-sulfur subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epetB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1209bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe:Lferr_2708\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eubiquinol-cytochrome c reductase cytochrome b subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epetC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e729bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3111\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eubiquinol-cytochrome c reductase cytochrome c1 subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eerpA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e372bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafj:AFERRID_10140\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiron-sulfur cluster insertion protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emoaD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e243bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_0975\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esulfur-carrier protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiscA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e324bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_0675\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiron-sulfur cluster assembly protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003edoxDA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1083bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe: Lferr_0045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ethiosulfate dehydrogenase (quinone)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003edoxA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1083bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafj:AFERRID_13680\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ethiosulfate dehydrogenase (quinone) small subunit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecysN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1353bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3125\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esulfate adenylyltransferase subunit 1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecysD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e939bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafe:Lferr_2723\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esulfate adenylyltransferase subunit 2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecysJ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1767bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3121\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esulfite reductase (NADPH) flavoprotein alpha-component\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecysI\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1692bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3122\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esulfite reductase (NADPH) hemoprotein beta-component\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecysH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e738bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_3123\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ephosphoadenosine phosphosulfate reductase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esqr\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1140bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_2601\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esulfide:quinone oxidoreductase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ehdrA2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1056bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_2553\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eheterodisulfide reductase subunit A2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ehdrB2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e912bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_2550\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eheterodisulfide reductase subunit B2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ehdrC2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e720bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_2551\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eheterodisulfide reductase subunit C2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ethiS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e201bp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eafr:AFE_0642\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esulfur carrier protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eGene traits related to environmental adaptability\u003c/h2\u003e\n\u003cp\u003eBased on protein sequence alignments, the coding genes predicted by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 were compared with the COG (Clusters of Orthologous Groups of proteins) and KEGG databases for functional annotation, and the corresponding functional annotations were obtained Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e shows the COG annotation classification statistics of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3. Our findings indicated that \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 has a total of 2,571 genes annotated in COG, accounting for approximately 80.34% of the total number of genes, and these genes are classified into 22 COG types. Among these genes, a total of 728 with unknown function were identified. The proportion of genes related to replication, recombination and repair (L), cell wall/membrane/envelope biogenesis (M), energy production and conversion (C), and inorganic ion transport and metabolism (P) was slightly higher than that of genes with other functions, accounting for 9.45%, 6.76%, 7.0%, and 6.2% of all annotated genes, respectively. Additionally, defense mechanisms accounted for 5.98% of all annotated genes, suggesting that this strain has a strong ability to self-repair and resist harsh environments (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe results of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 genome annotation using the KEGG pathway database indicated that the gene functions of this bacterium could be mainly divided into six categories, including Metabolism, Cellular Processes, Human Diseases, Genetic Information Processing, Organismal Systems, and Environmental Information Processing, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. Among these classifications, 1,353 functional genes related to Metabolism could be divided into 12 types, 81 functional genes related to Cellular Processes could be divided into 3 types, 124 genes related to Human Diseases could be divided into 10 types, 183 functional genes related to Genetic Information Processing could be divided into 6 categories, 56 genes related to Organic Systems could be divided into 8 categories, and 177 genes related to Environmental Information Processing could be divided into two categories.\u003c/p\u003e\n\u003cp\u003eGene island prediction of the \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 genome was performed using IslandViewer (Bertelli et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). The prophage prediction was performed using Phage_Finder (Fouts \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). CRISPR-Cas (Clustered Regularly Interspersed Short Palindromic Repeats) prediction was performed using Minced (Bland et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). The predicted results indicated that the chromosome of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 contained 10 gene islands, whereas Plasmid A contained one gene island, and these gene islands contained a total of 181 CDs. Interestingly, our analyses indicated that a prophage genome had integrated into the chromosome of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3. The sequence length of the prophage was 17,089 bp, and had 19 CDs, and a 58.64% GC content. The chromosome of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 was predicted to contain four CRISPRs with average repeat lengths of 27, 28, 23, and 27 bp, respectively. The average lengths of the spacer sequences were 27, 32, 43, and 45 bp, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidation of Fe\u003c/strong\u003e \u003csup\u003e \u003cstrong\u003e2+\u003c/strong\u003e \u003c/sup\u003e, \u003cstrong\u003eS\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/sup\u003e, \u003cstrong\u003eand pyrite by\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003eA. ferrooxidans\u003c/span\u003e \u003cstrong\u003eYQ-N3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring Fe\u003csup\u003e2+\u003c/sup\u003e oxidation by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3, the color of the medium gradually shifted from clear light green to yellow-green at first, and after 16 h, the medium completely changed to reddish-brown. Then, a yellow precipitate gradually appeared in the reaction system. The blank control group did not exhibit any obvious color change. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows the trends of pH, ORP, Fe\u003csup\u003e2+\u003c/sup\u003e content, and total iron content of the medium during Fe\u003csup\u003e2+\u003c/sup\u003e oxidation. According to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, the pH in the experiment involving \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 increased from 1.8 to 2.16 after 48 h, and then rapidly decreased to 1.7. ORP also increased throughout the experiment but this trend decelerated slightly after 48 h. In the blank control group, pH and ORP showed a slight upward trend throughout the experiment. This can be attributed to H\u003csup\u003e+\u003c/sup\u003e consumption by oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e into Fe\u003csup\u003e3+\u003c/sup\u003e (Offeddu et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Fe\u003csup\u003e3+\u003c/sup\u003e, SO4\u003csup\u003e2\u0026minus;\u003c/sup\u003e, and other cations (e.g, K\u003csup\u003e+\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) begin to react as the Fe\u003csup\u003e3+\u003c/sup\u003e content increases, which results in the production of H\u003csup\u003e+\u003c/sup\u003e and the minerals jarosite (KFe\u003csub\u003e3\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(OH)\u003csub\u003e6\u003c/sub\u003e) and ammoniojarosite ((NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eFe\u003csub\u003e6\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e4\u003c/sub\u003e(OH)\u003csub\u003e12\u003c/sub\u003e) (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e), which is consistent with previous studies (Nazari et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, the pH rapidly decreased and the ORP increase rate slowed down in the later stage of the experimental group (Li et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb shows that the Fe\u003csup\u003e2+\u003c/sup\u003e content in the experiment involving \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 continued to decrease rapidly, and the Fe\u003csup\u003e2+\u003c/sup\u003e content reached 0 mg/L when the reaction reached 60 h. Total iron concentration decreased by approximately 2700 mg/L during the whole reaction period, whereas only 14.5% Fe\u003csup\u003e2+\u003c/sup\u003e was oxidized and the total iron concentration in the blank control group remained unchanged for 96 h. This can be attributed to the fact that \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 accelerates the oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e, and the Fe\u003csup\u003e3+\u003c/sup\u003e generated by the reaction leads to a decrease in the total iron content in the system. Fe\u003csup\u003e2+\u003c/sup\u003e is naturally oxidized and generates free Fe\u003csup\u003e3+\u003c/sup\u003e in the blank control group during the reaction, and therefore the total iron content remained unchanged in the system.\u003c/p\u003e\n\u003cp\u003eDuring oxidation of S\u003csup\u003e0\u003c/sup\u003e by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3, the medium gradually changed from clear and colorless to light yellow, the particle size of sulfur powder gradually became smaller, and its hydrophobicity gradually weakened. However, no obvious change was observed during the reaction of the blank control group. This phenomenon was mainly because \u003cem\u003eA. ferrooxidans\u003c/em\u003e adsorbed on the surface of S when oxidizing it and secreted hydrophilic organic substances that covered the surface of S to enhance its hydrophilicity (Konishi et al. \u003cspan class=\"CitationRef\"\u003e1995\u003c/span\u003e; Knickerbocker 2000). During the oxidation of S\u003csup\u003e0\u003c/sup\u003e by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3, the pH value of the medium decreased from 2.2 to 1.74, whereas the SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e concentration of the medium increased to 3896.66 mg/L after 45 d (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). This can be attributed to the release of H\u003csup\u003e+\u003c/sup\u003e through the oxidation of S\u003csup\u003e0\u003c/sup\u003e to SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates the trends of pH, ORP, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, and Fe\u003csub\u003et\u003c/sub\u003e during pyrite oxidation by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3. In the experimental group, the pH value decreased from 1.9 to 1.2 after 45 d, the ORP increased from 353 to 632 mV after 45 d, the SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e concentration increased to 5433.8 mg/L after 45 d, the Fe\u003csup\u003e2+\u003c/sup\u003e content decreased drastically from 43.6 mg/L to a negligible level after 23 d, and the Fe\u003csub\u003et\u003c/sub\u003e content increased to 3,853 mg/L after 45 d. Characterization by XRD revealed that pyrite oxidized by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 contains jarosite and FeOOH, in addition to quartz impurities (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). In the blank control group, the pH value decreased slightly, the ORP decreased and then slightly increased, the SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e concentration increased to 563 mg/L after 45 d, the Fe\u003csup\u003e2+\u003c/sup\u003e content increased to 178.2 mg/L after 45 d, and the Fe\u003csub\u003et\u003c/sub\u003e concentration increased to 197.7 mg/L after 45 d. This could be because the H\u003csup\u003e+\u003c/sup\u003e released by the oxidation of S\u003csup\u003e0\u003c/sup\u003e into SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e exceeded the levels consumed by Fe\u003csup\u003e2+\u003c/sup\u003e oxidation. \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 can accelerate the oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e to Fe\u003csup\u003e3+\u003c/sup\u003e and pyrite oxidation can be carried out continuously and rapidly. The dissolved Fe mainly exists in the form of Fe\u003csup\u003e3+\u003c/sup\u003e in this system, and therefore ORP continues to increase.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eA. ferrooxidans\u003c/em\u003e can grow in a variety of extreme environments and have great potential applications in industries such as bioleaching and biological desulfurization (Gonz\u0026aacute;leztoril et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Vald\u0026eacute;s and Holmes \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Umanskii and Klyushnikov \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, there are few published genomes of \u003cem\u003eA. ferrooxidans\u003c/em\u003e strains (Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and therefore the whole genome sequencing and analysis of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 could provide key insights into its genetic properties and potential applicability.\u003c/p\u003e \u003cp\u003eTo understand the differences between the genomes of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 and other \u003cem\u003eA. ferrooxidans\u003c/em\u003e strains, the genome information of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 and \u003cem\u003eA. ferrooxidans\u003c/em\u003e that have been published in GenBank were selected for tabulation analysis, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The genome size distribution of \u003cem\u003eA. ferrooxidans\u003c/em\u003e was 2.82631\u0026ndash;4.18422 Mb, the GC content was 56.8%-58.9%, and the CD number distribution was 2,705-3,998. The genome size, GC%, and number of CDs of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 are similar to those of other \u003cem\u003eA. ferrooxidans\u003c/em\u003e strains. However, the assembled \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 genome in the published database included no more than five plasmids.\u003c/p\u003e \u003cp\u003e\u003cem\u003eA. ferrooxidans\u003c/em\u003e is a chemoautotrophic bacterium with a complex energy metabolism pathway, which obtains energy through the oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e and reducing sulfur compounds, and generates energy to sustain its growth by fixing carbon and nitrogen in the air (Hallberg et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bonnefoy and Holmes \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The oxidative metabolism of ferrous and sulfur compounds by \u003cem\u003eA. ferrooxidans\u003c/em\u003e makes this bacterium especially well-suited for industrial applications (Ponce et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Previous studies have demonstrated that Fe\u003csup\u003e2+\u003c/sup\u003e oxidation by \u003cem\u003eA. ferrooxidans\u003c/em\u003e in the electron transport chain has two patterns, including the downhill potential gradient and the uphill potential gradient (Johnson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Most electrons generated by Fe\u003csup\u003e2+\u003c/sup\u003e oxidation pass along the downhill potential gradient. Specifically, electrons generated by Fe\u003csup\u003e2+\u003c/sup\u003e oxidation enter the cytoplasm from the outer cell membrane, then pass to the cytochrome protein cyc2, after which they are received by rus, a ceruloplasmin, and passed to cyc2, cytochrome protein. Afterward, they are received by cytochrome oxidase aa3 and then passed to O\u003csub\u003e2\u003c/sub\u003e, where they finally combine with protons to generate H\u003csub\u003e2\u003c/sub\u003eO (Bird et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In this process, the oxidation process of the rus operon plays a dominant role, and the rus operon is composed of multiple genes including cyc2, coxA, coxB, coxC, and rus (Andr\u0026eacute;s et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Furthermore, studies on the regulation of the expression of key rus operon genes in model microorganisms show that the expression of the rus operon is induced by Fe\u003csup\u003e2+\u003c/sup\u003e (Amouric et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kucera et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The sqr gene, which encodes the sulfide quinone reductase enzyme, has been previously identified in various \u003cem\u003eA. ferrooxidans\u003c/em\u003e strains (Wakai et al. 2017). doxDA encoding thiosulfate quinone reductase was also identified in ATCC23270 (Fabian et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) and CCM4253 (Janiczek et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). When elemental sulfur was used as an energy source, the transcription level of the heterodisulfide reductase (HdrABC) complex encoded by ten genes including hdrB, hdrA, hdrC2, and hdrB2 was significantly up-regulated, indicating that this complex was related to sulfur metabolism in \u003cem\u003eA. ferrooxidans\u003c/em\u003e (Liu t al. 2012). Therefore, YQ-N3 can oxidize Fe\u003csup\u003e2+\u003c/sup\u003e and sulfur and could thus have industrial applications. However, its metabolic pathways remain to be further explored.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. ferrooxidans\u003c/em\u003e is widely found in metal mines, high-sulfate coal mines, and other extreme environments due to its high environmental resistance, as well as its tolerance to metal ions and high acidity (Acosta et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The characterization of genes associated with environmental resistance in \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 would provide insights into the mechanisms that mediate its adaptability, which also lays the foundation for future research on its survival state and industrial application value in extreme environments. In this study, the environmental resistance-related gene signatures in \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 were investigated from the perspective of gene annotation and mobile genetic elements. The results of COG annotation show that the proportion of genes with uncharacterized functions was much higher than that of the other categories, suggesting that this strain likely possesses several novel genes. In addition, YQ-N3-gene-1491 and YQ-N3-gene-2286 located in the chromosome were annotated as ENOG410ZI6F and their COG description was \u0026ldquo;heavy metal transport detoxification protein,\u0026rdquo; which further suggested that this strain was resistant to heavy metals.\u003c/p\u003e \u003cp\u003eThe results of KEGG annotation show that the majority of the gene types in the \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 genome summary were associated with Metabolism, followed by Environmental Information Processing. The genes related to Environmental Information Processing were divided into signal transduction and membrane transport, among which multiple genes related to signal transduction were annotated, including cusA, cusB, cusR, and cusS. Previous studies have demonstrated that the cus proton pump system is widely distributed in gram-negative bacteria and is associated with copper and silver resistance in \u003cem\u003eA. ferrooxidans\u003c/em\u003e (Mealman et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In fact, cusA, cusB, and cusF possess metal-binding sites (Chac\u0026oacute;n et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and Cu\u003csup\u003e2+\u003c/sup\u003e is directly transferred from protein to protein in the cus system during ion transport, thereby reducing the potential toxicity of free Cu\u003csup\u003e2+\u003c/sup\u003e to cells (Alm\u0026aacute;rcegui et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chac\u0026oacute;n et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nan \u003cem\u003eet al\u003c/em\u003e linked the adaptability of \u003cem\u003eA. ferrooxidans\u003c/em\u003e to chemotactic movement and quorum sensing (QS), both of which allow this bacterium to grow, develop, and reproduce in extreme environments (Nan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMobile genetic elements refer to some exogenous gene fragments that can be incorporated into bacterial genomes to adapt to environmental changes or improve the likelihood of survival. These fragments generally contain genes that encode enzymes of other proteins with specific functions to help bacteria overcome adverse conditions or take advantage of resources that otherwise could not be exploited. The gene islands often carry functional genes or contain integrase, plasmid conjugation-related factors, etc. (Coutinho et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The prophage sequence often contained some functional genes, such as antibiotic resistance genes and virulence genes, among others, all of which enhanced the adaptability of this bacterium to its environment (Dominguez-Mirazo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The CRISPR/Cas system is a prokaryotic immune system designed to resist the invasion of exogenous genetic material (Cady et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In summary, our findings suggested that \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 may have strong adaptability to a variety of extreme environments, making it a promising candidate for various industrial applications such as biological desulfurization, bioleaching, metal processing, and others.\u003c/p\u003e \u003cp\u003eStudying the oxidation features of Fe and reduced sulfur compounds by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 is helpful to further exploit its industrial application potential. In this study, the oxidation of different energy substances (Fe\u003csup\u003e2+\u003c/sup\u003e, S\u003csup\u003e0\u003c/sup\u003e, and pyrite) by \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 was investigated. The results indicate \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 can accelerate the oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e and contributes to the formation of minerals such as jarosite and ammoniojarosite while also accelerating the oxidation of S\u003csup\u003e0\u003c/sup\u003e and enhancing its hydrophilicity. \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 can also achieve rapid and continuous oxidation of pyrite by accelerating Fe\u003csup\u003e2+\u003c/sup\u003e oxidation, thereby increasing the ORP and the SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e concentration, decreasing the pH value, and generating minerals (e.g, jarosite and FeOOH), all of which are hallmarks of the AMD generation process (Kuang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Thus, \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 can accelerate the production of AMD.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, a novel \u003cem\u003eA. ferrooxidans\u003c/em\u003e strain was isolated from sediment from a river polluted by the AMD of an abandoned mine in Shanxi, China. Our whole-genome sequencing analyses revealed that the genome size of \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 is 3,217,720 bp. This genome includes one circular chromosome and five circular plasmids (Plasmid A, Plasmid B, Plasmid C, Plasmid D, and Plasmid E), among which Plasmid E had not been previously annotated in public databases. The sequence length and GC content of the circular chromosome were 3,043,496 bp and 58.64%, respectively. Additionally, 3,200 CD genes, six rRNAs, 46 tRNAs, and 18 sRNAs were predicted. This newly identified strain possesses multiple genes related to iron and sulfur metabolism and environmental resistance. However, additional studies are required to characterize its energy metabolism pathways. \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 can accelerate the oxidation of Fe\u003csup\u003e2+\u003c/sup\u003e, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e, as well as the generation of secondary minerals during the oxidation process. This study not only establishes a theoretical foundation for future research on the generation and treatment of AMD but also provides key insights into the role and potential of \u003cem\u003eA. ferrooxidans\u003c/em\u003e in biogeochemistry and industrial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (Grant No. 41977159); Open fund project of Key Laboratory of coal measures mineral resources of China Coal Geology administration (KFKT-2020-3).\u003c/p\u003e\n\u003cp\u003eConflicts Of Interest\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcosta M, Beard S, Ponce J, et al (2005) Identification of putative sulfurtransferase genes in the extremophilic \u003cem\u003eA. ferrooxidans\u003c/em\u003e ATCC 23270 genome: structural and functional characterization of the proteins. 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Extremophiles 22(21): 563-579.\u003c/li\u003e\n\u003cli\u003eZhang Y, Zhang S, Zhao D, et al (2019) Complete Genome Sequence of \u003cem\u003eA. Ferrooxidans\u003c/em\u003eYNTRS-40, a Strain of the Ferrous Iron- and Sulfur-Oxidizing Acidophile. Microorganisms 8(1): 2.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acidithiobacillusferrooxidans, Complete genome, iron-sulfur oxidation, environmental adaptability","lastPublishedDoi":"10.21203/rs.3.rs-1430566/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1430566/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAcidithiobacillus ferrooxidans\u003c/em\u003e (\u003cem\u003eA. ferrooxidans\u003c/em\u003e) is a chemoautotroph that can simultaneously oxidize Fe\u003csup\u003e2+\u003c/sup\u003e, S, and reduced sulfur compounds. Therefore, this bacterium plays a key role in the natural cycles of Fe and S. In this study, a novel \u003cem\u003eA. ferrooxidans\u003c/em\u003e strain was isolated from sediments of a river polluted by the acid mine drainage (AMD) of an abandoned mine in Shanxi, China, after which it was characterized via whole-genome sequencing. Furthermore, its functional genes related to iron and sulfur metabolism and response to environmental stress were analyzed, and its capacity to oxidize FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, S\u003csup\u003e0\u003c/sup\u003e, and FeS\u003csub\u003e2\u003c/sub\u003e as an energy source was preliminarily discussed. The whole-genome sequencing results revealed that \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 has a 3,217,720 bp genome, which is comprised of one circular chromosome and five circular plasmids (Plasmid A, Plasmid B, Plasmid C, Plasmid D, Plasmid E). Among these, Plasmid E had not been previously described in this species, and its genome contains various functional genes related to iron and sulfur, drug resistance, and heavy metal resistance. \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 can increase the oxidation rate of Fe\u003csup\u003e2+\u003c/sup\u003e and S\u003csup\u003e0\u003c/sup\u003e and enhance the hydrophilicity of S\u003csup\u003e0\u003c/sup\u003e. Moreover, this strain can accelerate FeS\u003csub\u003e2\u003c/sub\u003e oxidation and the formation of secondary minerals. The present study demonstrated that the newly isolated \u003cem\u003eA. ferrooxidans\u003c/em\u003e YQ-N3 could bio-oxidize iron and sulfur under acidic conditions, which was supported by our genome analysis results. Collectively, our findings provide important insights into the role and potential of \u003cem\u003eA. ferrooxidans\u003c/em\u003e in biogeochemistry and industrial applications.\u003c/p\u003e","manuscriptTitle":"Complete Genome Sequence and Iron-Sulfur Oxidation Characteristics of The Newly Isolated Acidithiobacillus Ferrooxidans YQ-N3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-11 15:49:27","doi":"10.21203/rs.3.rs-1430566/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"273af22b-b761-478a-bd64-e0b4eba2e672","date":"2023-02-13T09:09:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-03-21T07:13:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-11T16:29:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-03-09T04:57:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2022-03-08T10:04:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f5ea5979-573d-463a-abba-29b72c637e28","owner":[],"postedDate":"March 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2023-03-09T00:29:15+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-11 15:49:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1430566","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1430566","identity":"rs-1430566","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}