Disrupting ROS-protection mechanism allows hydrogen peroxide to accumulate and oxidize Sb(III) to Sb(V) in Pseudomonas stutzeri TS44

Dan Wang, Fengqiu Zhu, Qian Wang, Christopher Rensing, Peng Yu, Jing Gong, Gejiao Wang

Microbial antimonite [Sb(III)] oxidation converts toxic Sb(III) into less toxic antimonate [Sb(V)] and plays an important role in the biogeochemical Sb cycle. Currently, little is known about the mechanisms underlying bacterial Sb(III) resistance and oxidation. In this study, Tn5 transposon mutagenesis was conducted in the Sb(III)-oxidizing strain Pseudomonas stutzeri TS44 to isolate the genes responsible for Sb(III) resistance and oxidation. An insertion mutation into gshA, encoding a glutamate cysteine ligase involved in glutathione biosynthesis, generated a strain called P. stutzeri TS44-gshA540. This mutant strain was complemented with a plasmid carrying gshA to generate strain P. stutzeri TS44-gshA-C. The transcription of gshA, the two superoxide dismutase (SOD)-encoding genes sodB and sodC as well as the catalase-encoding gene katE was monitored because gshA-encoded glutamate cysteine ligase is responsible for the biosynthesis of glutathione (GSH) and involved in the cellular stress defense system as are superoxide dismutase and catalase responsible for the conversion of ROS. In addition, the cellular content of total ROS and in particular H2O2 was analyzed. Compared to the wild type P. stutzeri TS44 and TS44-gshA-C, the mutant P. stutzeri TS44-gshA540 had a lower GSH content and exhibited an increased content of total ROS and H2O2 and increased the Sb(III) oxidation rate. Furthermore, the transcription of sodB, sodC and katE was induced by Sb(III). A positive linear correlation was found between the Sb(III) oxidation rate and the H2O2 content (R (2) = 0.97), indicating that the accumulated H2O2 is correlated to the increased Sb(III) oxidation rate. Based on the results, we propose that a disruption of the pathway involved in ROS-protection allowed H2O2 to accumulate. In addition to the previously reported enzyme mediated Sb(III) oxidation, the mechanism of bacterial oxidation of Sb(III) to Sb(V) includes a non-enzymatic mediated step using H2O2 as the oxidant.