Data_Sheet_2_New Insights Into Acidithiobacillus thiooxidans Sulfur Metabolism Through Coupled Gene Expression, Solution Chemistry, Microscopy, and Spectroscopy Analyses.docx

Here, we experimentally expand understanding of the reactions and enzymes involved in Acidithiobacillus thiooxidans ATCC 19377 S0 and S2⁢O32- metabolism by developing models that integrate gene expression analyzed by RNA-Seq, solution sulfur speciation, electron microscopy and spectroscopy. The A. thiooxidansS2⁢O32- metabolism model involves the conversion of S2⁢O32- to SO42-, S0 and S4⁢O62-, mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr), and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S0 to SO32-, S2⁢O32- and polythionates in the A. thiooxidans S0 metabolism model. Our combined results shed light onto the important role specifically of TetH in S2⁢O32- metabolism. Also, we show that activity of Hdr proteins rather than Sdo are likely associated with S0 oxidation. Finally, our data suggest that formation of intracellular S2⁢O32- is a critical step in S0 metabolism, and that recycling of internally generated SO32- occurs, through comproportionating reactions that result in S2⁢O32-. Electron microscopy and spectroscopy confirmed intracellular production and storage of S0 during growth on both S0 and S2⁢O32- substrates.