| Selenium(Se) is a essential trace elenment for living systems. It is a component of selenocysteine and selenoenzyme and plays an important role in anti-cancer, anti-oxidation and so on, but high concentrations of selenium have a high toxicity to life. In nature, selenium exists in four mainly oxidation states:Se(-2), Se(0), Se(+4) and Se(+6). Selenite is the most toxic among the selenium oxyanions. There are already some researches showing the important role that the bacteria play in the transformation of selenium. At present, the researches on the Se(Ⅳ) resistant bacteria and Se(Ⅳ) reducing bacteria are relatively abundant and have found that many bacteria have the ability to reduce selenite to elemental selenium or grow in a high concentration of selenite. However, the mechanism of selenium metabolism is far from being elucidated. In this research, we studied the diversity of Se(Ⅳ) reducing bacteria in Se-rich soil ES2and then chose one of the Se(Ⅳ) reducing bacteria, namely Comamonas testosteroni S44, to study the mechanisms of Se(IV) resistance and Se(Ⅳ) reduction.The Se mined soil ES2was collected from Enshi City in Hubei province. The content of the total selenium is38.35mg/kg, indicating that Enshi area belongs to Se-rich area. A total of72Se(Ⅳ) reducing bacteria were isolated from the Se mined soil ES2. According to the phylogenetic analysis based on the16S rRNA gene sequences,29species belong to20genus were identified, which were affiliated with Actinobacteria, Proteobacteria and Firmicutes. This indicates that the diversity of Se(Ⅳ) reducing bacteria in the soil is very abundant. Based on the measurements of MIC and reduction rates of Se (Ⅳ), we found the Se(Ⅳ) reduction rates have no correlation to the MIC. Moreover, the MIC and Se(Ⅳ) reduction rate does not correlated to genus and species either.In order to find the genes correlated to the bacterial metabolism of Se, a Tn5insertion mutant library was constructed for the strain Comamonas testosteroni S44. Through the complementary assay of iscR and real-time RT-PCR experiment, we demonstrated iscR was relevant to both the Se(Ⅳ) resistance and reduction. By measuring the amount of GSH in the cells of wild type, mutant and complementary strains, we found that reduced glutathione may be involved in Se(Ⅳ) reduction.In conclusion, we predict that the mechanisms of IscR involved in the reduction of selenite are as follow:firstly, Se(Ⅵ) is reduced to Se(Ⅳ) by selenate reductase complex in the periplasmic space. Secondly, Se(Ⅳ) was then transported into the cytoplasm in which it is further reduced to Se. In the progress of the reduction of selenite to elemental selenium, the bacteria will produce a large scale of peroxides which can suppress the operon of isc. As a result, the bacteria will synthesize Fe-S cluster and GSH. The enzymes containing Fe-S cluster can reduce the selenite to selenium and the GSH is one of the major reducing substances of selenite. The GSH will react with Se(Ⅳ) to produce GS-Se-SG which will generate GS-Se-catalyzed by GSH reductase. The GS-Se" is volatile and it quickly forms Se and GSH then. Moreover, during the reduction the IscR could also promote expressions of other relevant genes in strain Comamonas testosteroni S44.This study focus on the diversity of Selenite reducing bacteria, MIC and bacterial species relationship, and is the first report on elucidating the important role of iscR playing in the selenite resistance and reduction as well as selenate reduction. These results also provide a new insight into the selenium metabolic mechanism of microbe. |
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