Construction And Characterization Of TetH Overexpressed And Knockout Strains Of Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans is a chemolithoautotrophic bacterium and a major participant in consortia of microorganisms used for the bioleaching of low-grade sulfide ores. It gains energy from the oxidation of ferrous iron （Fe2+）, sulfur （S0） and various reduced inorganic sulfur compounds （RISCs）. Study on the mechanism of Fe2+oxidation has made significant progress, however, multiple states of sulfur and some nonenzymetic reactions made the study of the RISCs metabolism more complicated. Tetrathionate is a key intermediate during RISCs oxidation, and it also can be used as the sole energy source for the growth of At. ferrooxidans. Tetrathionate can be hydrolyzed to thiosulfate, sulfate and sulfur by tetrathionate hydrolase （TetH）. However, most of the studies on the tetH gene and TetH were derived from bioinformatics analysis, lacking related experimental statistics. In this study, we constructed a tetH overexpression strain and a tetH knockout mutant by markerless exchange, which not only provided substantial base for studying the tetH gene and its function, but also experimental guidance for future research on other interested genes involved in sulfur oxidation in this organism.In this study, we first replaced the tetH gene promoter with tac due to its high transcription competence, and the recombinant plasmid pMSDl-tac-tetH was constructed, which was then introduced into At. ferrooxidans ATCC23270through conjugation. At. ferrooxidans ATCC23270（pMSD1-tac-tetH） was successfully obtained on the Sm selecting plate.Also, we constructed a tetH gene knockout mutant by markerless exchange. A suicide vector pKIT-KtetH was firstly constructed and conjugated into At. ferrooxidans ATCC23270to produce the single recombinants, which were screened on Km selecting plates. Then recombinant plasmid pMSD1-I-SceI was conjugated into the identified single recombinant to promote the occurrence of the second recombination event. The double cross-over tetH gene knockout mutant was initially screened from colonies of single recombinant carrying plasmid pMSD1-I-Scel on the Sm selecting plates by using PCR with designed primers, and was finally determined by Southern blotting analysis.To investigate the characteristics of At. ferrooxidans ATCC23270wild type, the tetH knockout mutant and the tetH overexpression strain, growth curves of different strains grown in9K-S0,9K-K2S4O6and9K-FeSO4liquid media were detected respectively, together with the utilization of tetrathionate, TetH activities and transcriptional analysis of related genes involved in sulfur-oxidation by real time quantitative PCR. The results indicated that （1） The tetH overexpression strain grew best in sulfur and tetrathionate media compared to the wild type and the tetH knockout mutant, and possessed a highest utilization rate of tetrathionate and TetH activity. However, the cell yields in tetrathionate medium were lower than that in sulfur medium.（2） The tetH knockout mutant couldn’t grow with tetrathionate as the sole energy source, but could survive in sulfur medium with a lower growth rate, which indicated that tetH gene is unique involved in tetrathionate metabolism, and alternative sulfur oxidation pathways might exist to bypass the production of tetrathionate.（3）The tetH knockout mutant could survive in ferrous medium with a higher decreasing rate of ferrous iron compared to the wild type, while the tetH overexpression strain possessed a lowest rate, which was consistent with the reduction activities of ferric iron to ferrous iron by TetH.（4） qRT-PCR results showed that tetH and doxD₂act synergistically, and the latter was considered important in thiosulfate metabolism. Of both sqr genes, AFE₀267seemed to play a more important role in sulfide oxidation than AFE₁792, which was suggested for the first time. Anyway, the work on transcriptome analysis is being underway which will provide us more information about the tetH mutant and the tetH overexpression strain, as well as the information on whole sulfur-oxidizing mechanism in At. ferrooxidans ATCC23270.