Determining plasmid mediated heavy metal resistance of bacteria

Abstract

Bacteria isolated from tannery effluent waters and soil samples contaminated with wastes of brassware industry were exposed to different concentrations of Chromium and Copper. Their tolerance levels were measured. Seven morphologically different bacteria, isolated from tannery effluent water, were tested for Cr (VI) tolerance. Among the isolates, three bacteria were selected as Cr (VI) resistant based on their MICs, percentage survival and ECso values. Chromium resistant bacteria were identified by biochemical methods as Pseudomonas sp. and Bacillus sp., while Psuedomonas sp., Alcaligenes sp. and Aeromonas sp. were Cu2+ resistant. All heavy metal resistant bacterial isolates were subjected to genomic DNA extraction and plasmid DNA extraction. Their plasmid sizes were also determined. Out of all the bacterial isolates, two Cr (VI) resistant bacteria and one Cu2+ resistant bacterium contained plasmids. In order to determine whether their heavy metal resistant mechanism is plasmid borne, the plasmids and genomic DNA of heavy metal resistant bacteria were introduced into competent E.coli JM I 09, using chemical transformation methods. Transformants were tested for Cr (VI) and Cu2+ resistance. One transformant could tolerate Cr (VI) in a level close to the resistance level of original Cr (VI) resistant bacterium. The transformant was found to pocess the newly introduced plasmid. There were no transformants which could tolerate Cu2+ as much as the original Cu2+ resistant bacterium In order to determine genes responsible for the heavy metal resistance, the plasmid DNA of the transform ant was subjected to PCR amplification using oligonucleotide primers designed for chrB, merA and nccA genes that are known as genes those encode heavy metal resistance. It was found that the isolated plasmid contained merA gene confirming that the plasmid bear mercury resistance. The absence of amplified chrB product showed that the Cr (VI) resistant gene carried by the particular plasmid is different from the chrB gene, for which the particular primer was designed. Further investigations are needed to determine the exact sequence of Cr (VI) resistant gene elements of the plasmid. The transformant together with the original bacterium were tested for Hg2+ and confirmed that it is Hg2+ resistant.