SoxRS-mediated regulation of chemotrophic sulfur oxidation in Paracoccus pantotrophus

Lehrstuhl für Technische Mikrobiologie, Fachbereich Bio- und Chemieingenieurwesen, Universität Dortmund, Emil-Figge-Strasse 66, D-44221 Dortmund, Germany Correspondence Cornelius G. Friedrich cornelius.friedrich{at}udo.edu Paracoccus pantotrophus GB17 requires thiosulfate for induction of the sulfur-oxidizing (Sox) enzyme system. The soxRS genes are divergently oriented to the soxVWXYZA–H genes. soxR predicts a transcriptional regulator of the ArsR family and soxS a periplasmic thioredoxin. The homogenote mutant GB S carrying a disruption of soxS by the -kanamycin-resistance-encoding interposon expressed a low thiosulfate-oxidizing activity under heterotrophic and mixotrophic growth conditions. This activity was repressed by complementation with soxR , suggesting that SoxR acts as a repressor and SoxS is essential for full expression. Sequence analysis uncovered operator characteristics in the intergenic regions soxS–soxV and soxW–soxX . In each region a transcription start site was identified by primer extension analysis. Both regions were cloned into the vector pRI1 and transferred to P. pantotrophus . Strains harbouring pRI1 with soxS–soxV or soxW–soxX expressed the sox genes under heterotrophic conditions at a low rate, indicating repressor titration. Sequence analysis of SoxR suggested a helix–turn–helix (HTH) motif at position 87–108 and uncovered an invariant Cys-80 and a cysteine residue at the C-terminus. SoxR was overproduced in Escherichia coli with an N-terminal His 6 -tag and purified to near homogeneity. Electrophoretic gel mobility shift assays with SoxR retarded the soxS–soxV region as a single band while the soxW–soxX region revealed at least two protein–DNA complexes. These data demonstrated binding of SoxR to the relevant DNA. This is believed to be the first report of regulation of chemotrophic sulfur oxidation at the molecular level. Abbreviations: HTH, helix–turn–helix; Lac, lactose; Sox, sulfur oxidation; TBE, Tris/borate/EDTA