| Abstract:Because of the distinct physiological and biochemical properities and function, leaching microbacteria are widely used in the industrial recovery of copper (bioleaching or biomining). Recently, the lack of sequenced genomes has restricted the fundamental research and further utilization. As one of the main members of bio-leaching system, Acidit-hiobacillus thiooxidans(A. thiooxidans) grows and survives by utilizing energy autotrophically derived from the oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs), whilst sulfate, which is the metabolic products, plays a key role in the process of regulating pH in this system. Based on the development of high-throughput sequencing technology, the whole-genome sequencing of A. thiooxidans A01was performed, and then bioinformatics analysis of genome sequence was carried out, providing guidance to the future research.The genomic DNA of A. thiooxidans A01was sequenced by BGI-Shenzhen (Beijing Genomics Institute) using Illumina HiSeq2000, and then the raw sequences were filtered and assembled. According to our sequencing data, the draft genome of A. thiooxidans A01contains3,820,158bp with GC content of53.08%distributed in213contigs. Based on previous data, coding regions detection and potential genes identification were performed using Glimmer. Subsequently, each putative gene was annotated using the BLASTx program against the Non-redundant protein database, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Groups of proteins (COG). As a result,3,660protein-coding sequences (CDSs) were predicted. As to the3,660CDSs,2,537were assigned a putative function in the current databases,136were conserved hypothetical protein, and987were hypothetical proteins. In addition,111tRNA and one5S-16S-23S operon were predicted using tRNAscan-SE and RNAmmer, respectively. The draft genome sequence was deposited at GenBank under the accession number AZMO00000000.Further research reveals that the genome of A. thiooxidans A01contains key sulfur metabolism enzymes involved in the oxidation of elemental sulfur and RISCs, such as sulfur dioxygenase (SDO), sulfide quinone reductase (SQR), thiosulfate:quinone oxidoreductase (TQO), tetrathionate hydrolase (TetH), sulfur oxidizing protein (Sox) system and their associated electron transport components. In addition, another putative pathway was found in the cytoplasm of A. thiooxidans, which catalyzes sulfite to sulfate as the final product by phosphoadenosine phosphosulfate (PAPS) reductase and adenylylsulfate (APS) kinase. This differs from its closest relative Acidithiobacillus caldus, which is performed by sulfate adenylyltransferase (SAT).Draft genome sequence of A. thiooxidans A01indicated the presence of sulfur oxygenase reductase gene (sor), which was first detected in this species. The complementary DNA fragment was speculated to encode a putative311-aa full-length protein sulfur oxygenase reductase (SOR). Based on the crystal structure of SOR from Acidianus tengchongensis (3BXV) as the modeling template, the three-dimensional (3D) model of SOR from A. thiooxidans was simulated using the software Discovery Studio version2.5. Structural analysis of SOR showed that three cysteines located in the two conserved domains, C32at V-G-P-K-V-C32as well as C102and C105at C102-X-X-C105, might form the substrate activation and binding site. It was proposed that conserved motif H87-X3-H9I-X23-E115acted as ligands might combine with iron atom to constitute a mononuclear non-heme iron center, catalyzing the oxidation reaction of substrate.Primers were designed relied on gene sequences involved in the sulfur oxidation metabolism, and then real-time quantitative PCR analysis was performed. Results showed that most of sulfur oxidation genes were highly expressed in the S0medium than that in the Na2S2O3medium at the mid-log phase. Herein, sulfur oxidation model of A. thiooxidans A01has been constructed based on previous studies from other sulfur oxidizing strains and its genome sequence analyses. |
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