A. ferrooxidans YNTRS–40 is a gram-negative non-endospore-forming chemolithoautotrophic aerobic bacteria in the order Acidithiobacillales of the class Acidithiobacillia (Additional file 1: Table S1). It possesses resistance against heavy metal and oligotrophic conditions. Although several Acidithiobacillus species with the capacity of iron and sulfur oxidization were identified from acidic environments, their genetic features associated with the resistance to extreme environment were ambiguous [4]. In this study, A. ferrooxidans YNTRS–40 was isolated and its genome was sequenced to analyze the stress resistance. Microscopically, YNTRS–40 cells displayed rod-shaped and were 0.28–0.40 μm in width and 1.00–1.68 μm in length (Fig. 1). This strain grew to a logarithmic stage fastly after 48 hours under aerobic conditions at pH 1.75, 28oC and 120 rpm in a shaker with modified 9K medium. The complete genome sequences have been submitted to GeneBank under the accession number CP040511 (Chromosome) and CP040512 (Plasmid).
The genome size of A. ferrooxidans YNTRS–40 is 3,257,037-bp, and the genome contains one circular chromosome of 3,209,933-bp with 58.54% GC content and one circular plasmid (47,104-bp with 56.43% GC content). The circular chromosome comprised 3349 predicted CDS genes, 6 rRNAs, 52 tRNAs and 6 ncRNAs (Table 1) and the circular plasmid contained 70 predicted CDS genes. The statistics and properties of the genome were summarized in Table 1. Total 2015 genes identified from chromosome were classified into 26 functional categories based on Cluster of Orthologous Groups (COG) (Table 2) [37]. Among all categories, the inorganic ion transport and metabolism category (P, 6.70%), the energy production and conversion category (C, 6.40%) and the defense mechanisms category (V, 4.47%) indicated that the strain YNTRS–40 can grow in the environment with high concentrations of metal ion.
Based on 16S rRNA gene sequence analysis, it can be seen that all strains clustered separately into different clades, such as A. ferriphilus (Clade Ⅰ), A. ferrivorans (Clade Ⅰ), A. ferridurans (Clade Ⅱ), A. ferrooxidans (Clade Ⅲ), A. thiooxidans (Clade Ⅳ), A. albertensis (Clade Ⅳ), and A. caldus (Clade Ⅴ). This finding was similar to a study by Zhang et al. [38] and slightly different from a literature, in which A. ferridurans, A. thiooxidans and A. albertensis clustered into the common clade [14]. The strain YNTRS–40 appeared to represent a coherent group with Acidithiobacillus ferrooxidans ATCC 11821 and Acidithiobacillus ferrooxidans ATCC 53993 (Fig. 3).
Genomic features related to adaptation to diverse stresses
Acidithiobacillus spp. possess extreme environmental resistance, and they can adjust to extremely acidic conditions (grow optimally at pH 2.0 and survive in pH 1.0–4.5) and their survival, colonization, growth and development [39, 40]. To balance the extracellular and intracellular environment heterogeneity in extremely acidic habitats containing heavy metal ions, these acidophilic microorganisms diverge and evolve to possess the acid and metal resistance [38].
Genome analysis using the Rpsblast and the Interproscan v5.30–69.0 [41] revealed that several functional genes involved in the adaptation of strain YNTRS–40 to extreme environments (Table 2 and Additional file 1: Table S3). The expression of proteins such as oxidoreductase and transferase in the cell should be regulated under the environment containing sulfur and metal ions. It has been documented that the oxidoreductase and transferase not only participate in energy generation but also enhance tolerance to environmental stress [42, 43]. Based on the category of biological process in Additional file 1: Table S3, the gene function of strain YNTRS–40 such as response to extracellular stimulus, cellular response to stress and response to oxidative stress indicated that it could cope with extreme environmental stress.
Based on the COG analysis (Table 2), the functional genes related to defense mechanisms (V), cell wall/membrane/envelope biogenesis (M), amino acid transport and metabolism (E), inorganic ion transport and metabolism (P) and general function prediction only (R) were slightly more than the other genes. These revealed that this strain exhibited excellent environmental adaptability and has potential applications in ecological industry such as sulfur removal from gases, mental extraction from electronic waste [44]. Additionally, the genes associated with function unknown (S) indicated that strain YNTRS–40 might possess some new genes [45].
The plasmid usually contains the genes related to the secondary metabolism according to the characteristics of microorganisms [46]. There were 70 CDSs in the circular plasmid of strain YNTRS–40, and 39 CDSs of them were predicted as hypothetical proteins, and the rest were found to involve in metabolism and defense (Additional file 1: Table S4). The RAST annotation results showed that the plasmid comprised all kinds of secondary metabolism-related genes, transcriptional regulatory genes, transposase-related genes, mobile element protein-related genes and stress-tolerance genes. These indicated that the primary metabolism-related genes were not present in this plasmid, and the presence of plasmid might favor the adaptation of this strain to environmental stress.
Genomic features related to the oxidation of ferrous iron and sulfur
A. ferrooxidans can gain energy from the oxidation of Fe2+ for growth and survival [5, 20]. During the oxidation of Fe2+, most of the electrons are transferred to O2 along the potential gradient which called downhill potential gradient, while a small part of electrons is transmitted conversely along the potential gradient which named uphill potential gradient [47]. In the latter process, the NAD(P)H is generated and involve in CO2 fixation and aerobic metabolism [20, 47, 48]. These two electron transfer pathways, namely the downhill and the uphill potential gradient are interrelated [47].
The KEGG analysis showed that several coding genes related to the downhill and the uphill electron transfer pathway existed in the strain YNTRS–40. Among them, the rus operon, which is consist of cyc2, cyc1, cup, coxB, coxA,coxC, coxD and rus genes, were found to involve in electron transfer in the downhill electron pathway [49]. The previous study suggested that the expression and regulation of the rus operon are associated with the substrate electron donor in environment [49]. The operon might be activated persistently when bacteria use Fe2+ as an electron donor, but only expressed transitorily during the period of early logarithmic growth when cells take S as an electron donor [50, 51]. Additionally, the petⅠ, petⅡ and res operon were found in genome of the strain YNTRS–40. Among them, petⅠencodes a bc1 complex that participates in the inverse electron transfer when the strain uses Fe2+ as a substrate, and petⅡ encodes another bc1 complex, which is responsible for forwarding electron transfer when S is used as substrate [52]. The res operon near the pet operon encodes ResB and ResC protein which might serve as a molecular chaperone in the maturation process of the c1 cytochrome of the bc1 complex [53].
A. ferrooxidans also can obtain the energy required for growth by oxidizing reduced sulfur. Sulfur was found to be more favorable energy source than Fe2+ due to it can provide more ATP than Fe2+ at the same molar level [54, 55]. The KEGG results indicated that some genes involved in sulfur oxidation, including sqr, doxDA, cydAB and cyoABCD genes which code sulfide quinone reductase, thiosulfate quinone oxidoreductase and thiosulfate dehydrogenase, respectively. These genes might be upregulated in strain YNTRS–40 when sulfur is used as substrate. Additionally, the enzymes encoded by these genes were coupled to a respiratory chain and occurred at different nodes in the respiratory chain [56]. These results suggested that the strain YNTRS–40 has potential for industrial application through iron and sulfur oxidizing such as metal bioleaching, gas desulfurization, and bioremediation.