Abstract
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 19 bp 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 toward the understanding of sulfur metabolism in A. caldus. This study also provided a powerful genetic tool for studies in A. caldus.
Highlights
The tetH and doxDA genes are arranged in a cluster in A. caldus and Acidithiobacillus thiooxidans, while they were separately distributed in the genomes of Acidianus and other Acidithiobacillus spp
BLAST and multiple alignment (Figure S1) results demonstrated that the two subunits DoxD and DoxA are fused as one protein in the three A. caldus strains, MTH-04, SM-1, and ATCC 51756, indicating that the doxD gene in this tetH cluster encodes a protein that has two domains corresponding to DoxD and DoxA (Müller et al, 2004)
We developed a reliable markerless gene knockout method for A. caldus and constructed RsrS-RsrR two-component system mutants
Summary
Widely distributed within the chemoautotrophic bacteria and archaea (Goebel and Stackebrandt, 1994; Friedrich, 1997; Suzuki, 1999; Kletzin et al, 2004; Friedrich et al, 2005; Frigaard and Dahl, 2008; Ghosh and Dam, 2009), have evolved a variety of sulfur redox enzymes to metabolize elemental sulfur and various reduced inorganic sulfur compounds (RISCs). Thiosulfate, a central intermediate, plays a key role in inorganic sulfur metabolism in these sulfur oxidizers (Friedrich et al, 2005; Ghosh and Dam, 2009) It is metabolized mainly through the sulfur oxidizing (Sox) enzyme system and the tetrathionate intermediate (S4I) pathway. The alternate S4I pathway is widely found in chemoautotrophic genera including Acidithiobacillus, Thermithiobacillus, Halothiobacillus, and Tetrathiobacter (Dam et al, 2007; Ghosh and Dam, 2009) This pathway is made up of a thiosulfate:quinol oxidoreductase (Tqo or DoxDA) and a tetrathionate hydrolase (TetH). The Sox and S4I pathways play important roles in the metabolism of RISCs in sulfur-oxidizing microorganisms
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