Abstract

Isolation and Transformation of Plasmid DNA of Streptococcus spp. (MTCC No. 9724) and Assessment of Mercuric Reductase Activity for Mercury (II) Detoxification

Author(s): Subarna Bhattacharyya, Srabanti Basu, Punarbasu Chaudhuri and Subhas Chandra Santra

The objective of the present study was to find out the molecular basis for and mercury resistance and mechanism of mercury detoxification by isolated Streptococcus sp. (MTCCNo. 9724). For this purpose a bacterial strain was isolated from the soil of solid waste dumping site of Kolkata, India and identified at IMTEC Chandigarh, India. Plasmid DNA of the strain was isolated and was detected by horizontal electrophoresis in 0.7% agarose gel using TAE buffer (1X). Transformation of isolated plasmid DNA of Streptococcus sp. (MTCC No. 9724) was carried out at room temperature with heating at 42oC(shock therapy). E.coli HB101 which was used as competent cells for isolated plasmid and the transformation efficiency was measured by dividing colonies observed with mass of plasmid introduced to the competent cells. Both bacterial cells i.e. isolated Streptococcus sp. (MTCC No. 9724) and transformed E.coli HB101 used for assaymercuric reductase enzyme. Here NADPH was used as substrate of the test enzyme and magnesium acetate, EDTA, and Alpha-mercaptaethanol was added for enzymatic reaction. The whole experimental set up was kept in dark place for one hour and reading was taken spectrophotometrically at 340 nm wavelength. A single band of plasmid DNA with molecular weight of 3000 base pair was isolated from Streptococcus sp. (MTCC No. 9724). The transformation efficiency was 20%and the MIC for mercury of transformed E.coli HB101 was 44.5 mg/l. This study ultimately identifies the molecular basis of the mercury removal mechanism of isolated Streptococcus sp. (MTCCNo. 9724). The responsible genetic material and mercuric reductase enzyme activity of the wild organism was confirmed through this study.