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

doi:10.21203/rs.3.rs-1430566/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-1430566/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Acidithiobacillus ferrooxidans (A. ferrooxidans) is a chemoautotroph that can simultaneously oxidize Fe²⁺, 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 FeSO₄·7H₂O, S⁰, and FeS₂ 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 Fe²⁺ and S⁰ and enhance the hydrophilicity of S⁰. Moreover, this strain can accelerate FeS₂ 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

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 Fe²⁺, S, and sulfide minerals, and its metabolites include sulfate and Fe³⁺ (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 Fe²⁺ 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 Fe²⁺ 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 Fe²⁺ concentration of 12 g L^− 1 after 7 days (Nie et al. 2015). A single A. ferrooxidans bioreactor can reportedly remove 98.0% SO₂ and 99.0% H₂S 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 Fe²⁺, S⁰, and FeS₂ 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.

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 Fe²⁺ and reduced sulfur as energy sources. This bacterium grows at 20–40°C with a pH of 1.5–3.5 and can use CO₂ as a carbon source and NH⁴⁺ 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 (NH₄)₂∙SO₄, 0.10 g/L KCl, 0.655 g/L K₂HPO₄·3H₂O, 0.50 g/L MgSO₃·7H₂O, and 0.01 g/L Ca(NO₃)₂·4H₂O. 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 FeSO₄·7H₂O 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, 10², 10³, 10⁴, 10⁵, and 10⁶ 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 Fe²⁺, S⁰, and FeS₂ 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 FeSO₄·7H₂O, S⁰, and FeS₂ as an energy source was discussed. The preserved YQ-N3 strain was transferred to 9K medium containing FeSO₄·7H₂O, S⁰, and FeS₂ 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 FeSO₄·7H₂O, S⁰, and FeS₂ at a 10% inoculum volume proportion, and the bacterial concentration and Fe²⁺, Fe³⁺, SO₄²⁻, 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 Fe²⁺, S⁰, and FeS₂ oxidation by *A. ferrooxidans* YQ-N3
Group	Energy source	Bacterial inoculum
A.f Oxidation Fe²⁺	FeSO₄·7H₂O (8.95 g)	10%
Control	FeSO₄·7H₂O (8.95 g)	0%
A.f Oxidation S⁰	S⁰ (0.5 g)	10%
Control	S⁰ (0.5 g)	0%
A.f Oxidation FeS₂	FeS₂ (5 g)	10%
Control	FeS₂ (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 Fe²⁺ and FeS₂ 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.

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-Fe²⁺ 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-Fe²⁺ 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 × 10⁸ 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 Fe²⁺ 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 PQ505	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⁺, S⁰, and pyrite by A. ferrooxidans YQ-N3

During Fe²⁺ 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, Fe²⁺ content, and total iron content of the medium during Fe²⁺ oxidation. According to Fig. 1a, 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 Fe²⁺ into Fe³⁺ (Offeddu et al. 2015). Fe³⁺, SO4²⁻, and other cations (e.g, K⁺, Na⁺, NH₄⁺) begin to react as the Fe³⁺ content increases, which results in the production of H⁺ and the minerals jarosite (KFe₃(SO₄)₂(OH)₆) and ammoniojarosite ((NH₄)₂Fe₆(SO₄)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 1b shows that the Fe²⁺ content in the experiment involving A. ferrooxidans YQ-N3 continued to decrease rapidly, and the Fe²⁺ 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% Fe²⁺ 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 Fe²⁺, and the Fe³⁺ generated by the reaction leads to a decrease in the total iron content in the system. Fe²⁺ is naturally oxidized and generates free Fe³⁺ in the blank control group during the reaction, and therefore the total iron content remained unchanged in the system.

During oxidation of S⁰ 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 S⁰ by A. ferrooxidans YQ-N3, the pH value of the medium decreased from 2.2 to 1.74, whereas the SO₄²⁻ 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 S⁰ to SO₄²⁻.

Figure 9 illustrates the trends of pH, ORP, SO₄²⁻, Fe²⁺, 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 SO₄²⁻ concentration increased to 5433.8 mg/L after 45 d, the Fe²⁺ 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 SO₄²⁻ concentration increased to 563 mg/L after 45 d, the Fe²⁺ 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 S⁰ into SO₄²⁻ exceeded the levels consumed by Fe²⁺ oxidation. A. ferrooxidans YQ-N3 can accelerate the oxidation of Fe²⁺ to Fe³⁺ and pyrite oxidation can be carried out continuously and rapidly. The dissolved Fe mainly exists in the form of Fe³⁺ in this system, and therefore ORP continues to increase.

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 Fe²⁺ 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 Fe²⁺ 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 Fe²⁺ oxidation pass along the downhill potential gradient. Specifically, electrons generated by Fe²⁺ 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 O₂, where they finally combine with protons to generate H₂O (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 Fe²⁺ (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 Fe²⁺ 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 Cu²⁺ is directly transferred from protein to protein in the cus system during ion transport, thereby reducing the potential toxicity of free Cu²⁺ 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 (Fe²⁺, S⁰, and pyrite) by A. ferrooxidans YQ-N3 was investigated. The results indicate A. ferrooxidans YQ-N3 can accelerate the oxidation of Fe²⁺ and contributes to the formation of minerals such as jarosite and ammoniojarosite while also accelerating the oxidation of S⁰ and enhancing its hydrophilicity. A. ferrooxidans YQ-N3 can also achieve rapid and continuous oxidation of pyrite by accelerating Fe²⁺ oxidation, thereby increasing the ORP and the SO₄²⁻ 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.

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 Fe²⁺, S⁰, and FeS₂, 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.

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).

Conflicts Of Interest

There are no conflicts of interest to declare.

Acosta M, Beard S, Ponce J, et al (2005) Identification of putative sulfurtransferase genes in the extremophilic A. ferrooxidans ATCC 23270 genome: structural and functional characterization of the proteins. Omics J Integr Biol 9: 13-29.
Adams DJ, Pennington P, Mc Lemore VT (1947) The role of microorganisms in acid mine drainage: a preliminary report. Science 106(2751): 253-256.
Akcil A, Ciftci H, Deveci H (2007) Role and Contribution of Pure and Mixed Cultures of Mesophiles in Bioleaching of a Pyritic Chalcopyrite Concentrate. Minerals Engineering 20(3): 310-318.
Almárcegui RJ, Navarro CA, Paradela A, et al (2014) Response to copper of A. ferrooxidans ATCC 23270 grown in elemental sulfur. Res Microbiol 165(9): 761-772.
Amouric A, Appia-Ayme C, Yarzabal A, et al (2009) Regulation of the iron and sulfur oxidation pathwayways in the acidophilic A. Ferrooxidans. Advanced Materials Research 71-73(6): 163-166.
Andrés Y, Appia-Ayme C, Ratouchniak J, et al (2004) Regulation of the expression of the A. ferrooxidansrus operon encoding two cytochromes c, a cytochrome oxidase and rusticyanin. Microbiology 150(7): 2113-2123.
Barreto M, Quatrini R, Bueno S, et al (2003) Aspects of the predicted physiology of A. ferrooxidans deduced from an analysis of its partial genome sequence. Hydrometallurgy 71(1-2): 97-105.
Bertelli C, Laird MR, Williams KP, et al (2017) IslandViewer 4: Expanded prediction of genomic islands for larger-scale datasets. Nucleic Acids Res 45 (Web Server issue).
Besemer J, Borodovsky M (2005) GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Research 33(Web Server): W451-W454.
Bird LJ, Bonnefoy V, Newman DK (2011) Bioenergetic challenges of microbial iron metabolisms. Trends Microbiol 19(7): 330-340.
Bland C, Ramsey TL, Sabree F, et al (2007)CRISPR Recognition Tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics 8(1): 209-210.
Bonnefoy V, Holmes DS (2012) Genomic insights into microbial iron oxidation and iron uptake strategies in extremely acidic environments. Environmental Microbiology 14(7): 1597-1611.
Cady KC, Bondy-Denomy J, Heussler GE, et al (2012) The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J Bacteriol 194(21): 5728-5738.
Chacón KN, J Perkins, Mathe Z, et al (2018) Trapping intermediates in metal transfer reactions of the CusCBAF export pump of Escherichia coli. Commun Biol 1(1).
Coutinho TJD, Franco GR, Lobo FP (2015) Homology-independent metrics for comparative genomics. Comput Struct Biotec 132(5): 352-357.
Delcher AL, Bratke KA, Powers EC, et al (2007) Identifying bacterial genes and endosymbiont DNA with GLIMMER. Bioinformatics 23(6): 673-679.
Dominguez-Mirazo M, Jin R, Weitz JS (2019) Functional and comparative genomic analysis of integrated prophage-like sequences in " candidatusliberibacter asiaticus". mSphere 4(6): 409-419.
Fabian HM, Tiago MB, Tim U, et al (2004) Coupling of the pathwayway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate: quinone oxidoreductase. Mol Microbiol 53 (4): 1147-1160.
Feng GZ, Liao LM, Liu ZY, et al (2017) Identification and characterization of A. ferrooxidans with high activity and resistance isolated from ancient mine area. Journal of Microbiology.
Fouts DE (2006) Phage_Finder: automated identification and classification of prophage regions in complete bacterial genome sequences. Nucleic Acids Res 34(20): 5839-5851.
Gonzáleztoril E, Llobetbrossa E, Casamayor EO, et al (2003) Microbial Ecology of an Extreme Acidic Environment, the Tinto River. Appl Environ Microb 69(8): 4853-4865.
Hallberg KB, Elena G, Johnson DB. (2010) A. ferrivorans, sp. nov.; facultatively anaerobic, psychrotolerant iron-, and sulfur-oxidizing acidophiles isolated from metal mine-impacted environments. Extremophiles 14(1): 9-19.
Iakovleva E, Makila E, Salonen J, et al (2015) Acid mine drainage (AMD) treatment: Neutralization and toxic elements removal with unmodified and modified limestone. Ecological Engineering 81: 30-40.
Janiczek O, Zemanova J, Mandl M (2007) Purification and some properties of thiosulfate dehydrogenase from A. ferrooxidans. Prep BiochemBiotechnol 37(2): 101-111.
Johnson BD (2008) Carbon, Iron and Sulfur Metabolism in Acidophilic Micro-Organisms. Adv Microb Physio 201-255.
Kai M, Yano T, Fukumori Y, et al (1989) Cytochrome oxidase of an acidophilic iron-oxidizing bacterium, Thiobacillus ferrooxidans, functions at pH 3.5. BiochemBioph Res Co 160(2): 839-843.
Kelly DP, Wood AP (2000) Reclassification of Some Species of Thiobacillus to the Newly Designated Genera A. Gen. Nov, Halothiobacillus Gen. Nov. And Thermithiobacillus Gen. Nov. International. Int J Syst EvolMicr 50(2): 511-516.
Knickerbocker C, Nordstrom DK, Southam G (2000) The role of “blebbing” in overcoming the hydrophobic barrier during biooxidation of elemental sulfur by thiobacillus thiooxidans. Chemical Geology 169 (3): 425-433.
Konishi Y, Asai S, Yoshida N (1995) Growth kinetics of thiobacillus thiooxidans on the surface of elemental sulfur. Appl Environ Microbiol 61 (10): 3617-3622.
Kuang JL, Huang LN, Chen LX, et al (2013) Contemporary environmental variation determines microbial diversity patterns in acid mine drainage. Isme Journal 7(5): 1038-1050.
Kucera J, Bouchal P, Lochman J, et al (2013) Ferrous iron oxidation by sulfur-oxidizing A. ferrooxidans and analysis of the process at the levels of transcription and protein synthesis. Antonie Van Leeuwenhoek 103(4): 905-919.
Lavalle L, Chiacchiarini P, Pogliani C, et al (2005) Isolation and characterization of acidophilic bacteria from Patagonia, Argentina. Process Biochemistry 40(3/4): 1095-1099.
Li W, Feng Q, Liang H, et al (2021) Passive treatment test of acid mine drainage from an abandoned coal mine in Kaili Guizhou, China Water science and technology 84(8): 1981-1996.
Liu Y, Ji J, Yu R, et al (2012) Expression, Purification and Molecular Modeling of Another HdrC from A. ferrooxidans Which Binds Only One [4Fe-4S] Cluster. Curr Microbiol 65(4): 416-423.
Lorenzo-Tallafigo J, N Iglesias-González, Mazuelos A, et al (2019) An alternative approach to recover lead, silver and gold from black gossan (polymetallic ore). Study of biological oxidation and lead recovery stages. J Clean Prod 207: 510-521.
Mealman TD, Blackburn NJ, Mcevoy MM (2012) Metal export by CusCFBA, the periplasmic Cu(I)/Ag(I) transport system of Escherichia coli. Curr Top Membr 69: 163-196.
Mengran Y, Yue Z, Shuang Z, et al (2021) Biological materials formed by A. ferrooxidans and their potential applications Electronic. J Biotechnol 52: 45-51.
Nan W, Zhi D, Leng F, et al (2011) Quorumsensing system in A. ferrooxidans involved in its resistance to Cu²⁺. Lett Appl Microbiol 53(1): 84-91.
Nazari B, Jorjani E, Hani H, et al (2014) Formation of jarosite and its effect on important ions for A. ferrooxidans bacteria. Transactions of Nonferrous Metals Society of China.
Nie H, Yang C, Zhu N, et al (2015) Isolation of A. ferrooxidansstrain Z1 and its mechanism of bioleaching copper from waste printed circuit boards. J Chem Technol Biot 90(4): 714-721.
Offeddu, FG, Cama, J, Soler, JM, et al (2015) Processes affecting the efficiency of limestone in passive treatments for AMD: Column experiments. J Environ Chem Eng 3, 304-316.
Ponce JS, Moinier D, Byrne D, et al (2012) A. ferrooxidansoxidizes ferrous iron before sulfur likely through transcriptional regulation by the global redox responding RegBA signal transducing system. Hydrometallurgy 127-128.
Rawlings DE (2005) Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microbial Cell Factories 4(1): 1-15.
Rhoads A, Au KF (2015) PacBio sequencing and its applications. GenomProteomBioinf 13(5):278-289.
Rout PG, Mohanty AK, Pradhan N, et al (2021) Study on the Reaction Mechanism of Oxidative Microbial Desulfurization of Organic Sulfur-Rich Coal. Geomicrobiology (12): 1-9.
Surber SJ, Simonton DS (2017) Disparate impacts of coal mining and reclamation concerns for West Virginia and central Appalachia. Resources Policy 54: 1-8.
Umanskii AB, Klyushnikov AM (2013) Bioleaching of low grade uranium ore containing pyrite using A. ferrooxidans and A. thiooxidans. J RadioanalNucl Ch 295(1): 151-156.
Valdés J, Pedroso I, Quatrini R, et al (2008) A. ferrooxidans metabolism: from genome sequence to industrial applications. Bmc Genomics 9(1): 597-597.
Valdés JH, Holmes DS (2009) Genomic Lessons from Biomining Organisms: Case Study of the A. Genus. Advanced Materials Research 71-73: 215-218.
Wakai S, Tsujita M, Kikumoto M, et al (2007) Purification and characterization of sulfide:quinone oxidoreductase from an acidophilic iron-oxidizing bacterium, A. ferrooxidans. Journal of the Agricultural Chemical Society of Japan 71(11): 2735-2742.
Yu Z, Huang B, Wang Y (2007) Studying advance in flue gas desulfurization by Thiobacillus ferroxidans. Acta Agric Jiangxi 19(6): 121-124.
Zhang S, Yan L, Xing W, et al (2018) A. ferrooxidans and its potential application. Extremophiles 22(21): 563-579.
Zhang Y, Zhang S, Zhao D, et al (2019) Complete Genome Sequence of A. FerrooxidansYNTRS-40, a Strain of the Ferrous Iron- and Sulfur-Oxidizing Acidophile. Microorganisms 8(1): 2.

No competing interests reported.

Download PDF

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

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

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Isolation, purification, and identification

Whole-genome sequencing

Results

Genes associated with iron and sulfur metabolism

Gene traits related to environmental adaptability

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1