Department of Microbiology, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata - 700054, India.
Department of Microbiology, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata - 700054, India.
Microbiol Res. 2017 Dec;205:1-7. doi: 10.1016/j.micres.2017.08.002. Epub 2017 Aug 12.
Molecular mechanisms of chemolithotrophic tetrathionate oxidation are not clearly understood. Here we used transposon(Tn5-mob)-insertion mutagenesis to search for novel tetrathionate oxidation genes in the facultatively chemolithoautotrophic betaproteobacterium Advenella kashmirensis that not only oxidizes tetrathionate, but also produces the same as an intermediate during thiosulfate oxidation. Genome-wide random insertion of Tn5-mob occurred at a frequency of one per 10 donor E. coli cells. A library of 8000 transconjugants yielded five tetrathionate-oxidation-impaired mutants, of which, the one named Ak_Tn_16 was studied here in detail. When grown chemolithoautotrophically on thiosulfate, Ak_Tn_16 converted the total thiosulfate supplied to equivalent amount of tetrathionate, exactly in the same way as the wild type. It could not, however, oxidize the intermediary tetrathionate to sulfate; Ak_Tn_16 could not also oxidize tetrathionate when it was supplied as the starting chemolithotrophic substrate. In the Ak_Tn_16 genome, Tn5-mob was found to have transposed in a novel soxO gene, located just-upstream of soxB, within the sox gene cluster. SoxO was predicted, via iterative threading assembly simulation, to be a glutathione-disulfide (GSSG) reductase. When Ak_Tn_16 was grown in tetrathionate-based chemolithoautotrophic medium supplemented with reduced glutathione (GSH) its tetrathionate-oxidation deficiency, remarkably, was ameliorated. Implications for a key role of GSH in tetrathionate oxidation are discussed in the light of other molecular evidences available for A. kashmirensis.
化能自养四硫代硫酸盐氧化的分子机制尚不清楚。本研究采用转座子（Tn5-mob）插入诱变，在兼性化能自养β变形菌 Advenella kashmirensis 中寻找新的四硫代硫酸盐氧化基因，该菌不仅能氧化四硫代硫酸盐，还能在硫代硫酸盐氧化过程中产生同样的中间产物。Tn5-mob 在供体大肠杆菌中的随机插入频率为每 10 个细胞一个。8000 个转导子文库产生了 5 个四硫代硫酸盐氧化缺陷突变体，其中一个命名为 Ak_Tn_16，在此详细研究。当以硫代硫酸盐为唯一自养碳源和能源生长时，Ak_Tn_16 将提供的总硫代硫酸盐转化为等量的四硫代硫酸盐，与野生型完全相同。然而，它不能将中间产物四硫代硫酸盐氧化为硫酸盐；当提供四硫代硫酸盐作为起始化能自养底物时，Ak_Tn_16 也不能氧化四硫代硫酸盐。在 Ak_Tn_16 基因组中，Tn5-mob 被发现转座到位于 soxB 上游的新 soxO 基因中，该基因位于 sox 基因簇内。通过迭代线程组装模拟预测，SoxO 是一种谷胱甘肽二硫化物（GSSG）还原酶。当 Ak_Tn_16 在基于四硫代硫酸盐的化能自养培养基中生长并补充还原型谷胱甘肽（GSH）时，其四硫代硫酸盐氧化缺陷显著改善。根据 A. kashmirensis 现有的其他分子证据，讨论了 GSH 在四硫代硫酸盐氧化中的关键作用。
