Rother Dagmar, Orawski Grazyna, Bardischewsky Frank, Friedrich Cornelius G
Lehrstuhl für Technische Mikrobiologie, Fachbereich Bio- und Chemieingenieurwesen, Universität Dortmund, Emil-Figge-Strasse 66, D-44221 Dortmund, Germany.
Microbiology (Reading). 2005 May;151(Pt 5):1707-1716. doi: 10.1099/mic.0.27724-0.
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 homogenate mutant GBOmegaS carrying a disruption of soxS by the Omega-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 His6-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.
嗜糖假单胞菌GB17需要硫代硫酸盐来诱导硫氧化（Sox）酶系统。soxRS基因与soxVWXYZA-H基因呈反向排列。soxR预测为ArsR家族的转录调节因子，soxS预测为周质硫氧还蛋白。携带编码Ω-卡那霉素抗性的插入序列破坏soxS的匀浆突变体GBΩS在异养和兼养生长条件下表现出低硫代硫酸盐氧化活性。用soxR互补可抑制该活性，表明SoxR起阻遏作用，而SoxS对完全表达至关重要。序列分析揭示了soxS-soxV和soxW-soxX基因间区域的操纵子特征。通过引物延伸分析在每个区域鉴定出一个转录起始位点。将这两个区域都克隆到载体pRI1中并转移到嗜糖假单胞菌中。携带含有soxS-soxV或soxW-soxX的pRI1的菌株在异养条件下以低速率表达sox基因，表明阻遏物被滴定。SoxR的序列分析表明在87-108位有一个螺旋-转角-螺旋（HTH）基序，并在C末端发现一个不变的Cys-80和一个半胱氨酸残基。SoxR在大肠杆菌中以N端His6标签过量表达并纯化至近乎同质。用SoxR进行的电泳凝胶迁移率变动分析使soxS-soxV区域作为一条单带迁移受阻，而soxW-soxX区域显示至少两个蛋白质-DNA复合物。这些数据证明了SoxR与相关DNA的结合。这被认为是关于化学营养型硫氧化在分子水平调控的首次报道。
