Sulfur oxidation is an essential component of the earth’s sulfur cycle. Acidithiobacillus spp. can oxidize various reduced inorganic sulfur compounds (RISCs) with high efficiency to obtain electrons for their autotrophic growth. Strains in this genus have been widely applied in bioleaching and biological desulfurization. Diverse sulfur-metabolic pathways and corresponding regulatory systems have been discovered in these acidophilic sulfur-oxidizing bacteria. The sulfur-metabolic enzymes in Acidithiobacillus spp. can be categorized as elemental sulfur oxidation enzymes (sulfur dioxygenase, sulfur oxygenase reductase, and Hdr-like complex), enzymes in thiosulfate oxidation pathways (tetrathionate intermediate thiosulfate oxidation (S₄I) pathway, the sulfur oxidizing enzyme (Sox) system and thiosulfate dehydrogenase), sulfide oxidation enzymes (sulfide:quinone oxidoreductase) and sulfite oxidation pathways/enzymes. The two-component systems (TCSs) are the typical regulation elements for periplasmic thiosulfate metabolism in these autotrophic sulfur-oxidizing bacteria. Examples are RsrS/RsrR responsible for S₄I pathway regulation and TspS/TspR for Sox system regulation. The proposal of sulfur metabolic and regulatory models provide new insights and overall understanding of the sulfur-metabolic processes in Acidithiobacillus spp. The future research directions and existing barriers in the bacterial sulfur metabolism are also emphasized here and the breakthroughs in these areas will accelerate the research on the sulfur oxidation in Acidithiobacillus spp. and other sulfur oxidizers.

Acidithiobacillus caldus is a chemolithoautotrophic sulfur-oxidizing bacterium that is widely used for bioleaching processes. Acidithiobacillus spp. are suggested to contain sulfur dioxygenases (SDOs) that facilitate sulfur oxidation. In this study, two putative sdo genes (A5904_0421 and A5904_1112) were detected in the genome of A. caldus MTH-04 by BLASTP searching with the previously identified SDO (A5904_0790). We cloned and expressed these genes, and detected the SDO activity of recombinant protein A5904_0421 by a GSH-dependent in vitro assay. Phylogenetic analysis indicated that A5904_0421and its homologous SDOs, mainly found in autotrophic bacteria, were distantly related to known SDOs and were categorized as a new subgroup of SDOs. The potential functions of genes A5904_0421 (termed sdo1) and A5904_0790 (termed sdo2) were investigated by generating three knockout mutants (Δsdo1, Δsdo2 and Δsdo1&2), two sdo overexpression strains (OE-sdo1 and OE-sdo2) and two sdo complemented strains (Δsdo1/sdo1’ and Δsdo2/sdo2’) of A. caldus MTH-04. Deletion or overexpression of the sdo genes did not obviously affect growth of the bacteria on S⁰, indicating that the SDOs did not play an essential role in the oxidation of extracellular elemental sulfur in A. caldus. The deletion of sdo1 resulted in complete inhibition of growth on tetrathionate, slight inhibition of growth on thiosulfate and increased GSH-dependent sulfur oxidation activity on S⁰. Transcriptional analysis revealed a strong correlation between sdo1 and the tetrathionate intermediate pathway. The deletion of sdo2 promoted bacterial growth on tetrathionate and thiosulfate, and overexpression of sdo2 altered gene expression patterns of sulfide:quinone oxidoreductase and rhodanese. Taken together, the results suggest that sdo1 is essential for the survival of A. caldus when tetrathionate is used as the sole energy resource, and sdo2 may also play a role in sulfur metabolism.

Acidithiobacillus ferrooxidans is an acidophilic and chemolithotrophic sulfur- and iron-oxidizing bacterium that has been widely used in the bioleaching process for extracting metals. Extracellular polymeric substances (EPS) are essential for bacteria-ore interactions, and the regulation of EPS synthesis could be an important way of influencing the efficiency of the bioleaching process. Therefore, exploring and utilizing the regulatory pathways of EPS synthesis to improve the bacterial bioleaching capability have posed a challenge in the study and application of bioleaching bacteria. Here, several engineering strains were constructed using genetic manipulation methods. And we revealed the regulatory function of the AfeI/R quorum sensing (QS) system in EPS synthesis and biofilm formation of A. ferrooxidans, and the AfeI/R-mediated EPS synthesis could influence bacteria-substrate interactions and the efficiency of bioleaching. Finally, an AfeI/R-mediated bioleaching model was proposed to illustrate the role of QS system in this process. This study provided new insights into and clues for developing highly efficient bioleaching bacteria and modulating the bioleaching process.

Acidithiobacillus caldus (A. caldus) is a common bioleaching bacterium that possesses a sophisticated and highly efficient inorganic sulfur compound metabolism network. Thiosulfate, a central intermediate in the sulfur metabolism network of A. caldus and other sulfur-oxidizing microorganisms, can be metabolized via the tetrathionate intermediate (S4I) pathway catalyzed by thiosulfate:quinol oxidoreductase (Tqo or DoxDA) and tetrathionate hydrolase (TetH). In A. caldus, there is an additional two-component system called RsrS-RsrR. Since rsrS and rsrR are arranged as an operon with doxDA and tetH in the genome, we suggest that the regulation of the S4I pathway may occur via the RsrS-RsrR system. To examine the regulatory role of the two-component system RsrS-RsrR on the S4I pathway, ΔrsrR and ΔrsrS strains were constructed in A. caldus using a newly developed markerless gene knockout method. Transcriptional analysis of the tetH cluster in the wild type and mutant strains revealed positive regulation of the S4I pathway by the RsrS-RsrR system. A 19bp inverted repeat sequence (IRS, AACACCTGTTACACCTGTT) located upstream of the tetH promoter was identified as the binding site for RsrR by using electrophoretic mobility shift assays (EMSAs) in vitro and promoter-probe vectors in vivo. In addition, ΔrsrR and ΔrsrS strains cultivated in K2S4O6- medium exhibited significant growth differences when compared with the wild type. Transcriptional analysis indicated that the absence of rsrS or rsrR had different effects on the expression of genes involved in sulfur metabolism and signaling systems. Finally, a model of tetrathionate sensing by RsrS, signal transduction via RsrR, and transcriptional activation of tetH-doxDA was proposed to provide insights towards the understanding of sulfur metabolism in A. caldus. This study also provided a powerful genetic tool for studies in A. caldus.

Chromium contamination has been an increasing threat to the environment and to human health. Cr(VI) and Cr(III) are the most common states of chromium. However, compared with Cr(III), Cr(VI) is more toxic and more easily absorbed, therefore, it is more harmful to human beings. Thus, the conversion of toxic Cr(VI) into Cr(III) is an accepted strategy for chromium detoxification. Here, we isolated two Bacillus cereus strains with a high chromium tolerance and reduction ability, named B. cereus D and 332, respectively. Both strains demonstrated a strong pH and temperature adaptability and survival under 8 mM Cr(VI). B. cereus D achieved 87.8% Cr(VI) removal in 24 h with an initial 2 mM Cr(VI). Cu(II) was found to increase the removal rate of Cr(VI) significantly. With the addition of 0.4 mM Cu(II), 99.9% of Cr(VI) in the culture was removed by B. cereus 332 in 24 h. This is the highest removal efficiency in the literature that we have seen to date. The immobilization experiments found that sodium alginate with diatomite was the better method for immobilization and B. cereus 332 was more efficient in immobilized cells. Our research provided valuable information and new, highly effective strains for the bioremediation of chromium pollution.