Browsing by Author "Abewickrama, J.H.G."

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Chromium accumulation in Cr (VI) resistant bacteria
(Book of Abstracts, Annual Research Symposium 2014, 2014) Abewickrama, J.H.G.; Jayarathne, D.L.; Rathnayake, I.V.N.; Gunawardane, M.M.
Accumulation of Cr(VI) is one of the methods used in bacteria to overcome elevated chromium concentrations. These hyperaccumulators of Cr(VI) are important in bioremediation of Cr (VI) containing wastes. Accumulation of Cr(VI) has been previously reported in Alcaligenes eutrophus CH34, Bacillus subtilis and some Pseudomonas sp. During this study Chromium accumulation of a Cr (VI) resistant Psuedomonas sp., Escherichia coli JM109 and the transformant E.coli JM109 with plasmids extracted from the Cr(VI) resistant Pseudomonas sp. were compared. Cr (VI) accumulation was estimated at 0, 5, 10, 15 mg/L Cr (VI) concentrations after 72 hour exposure time. The cells were subjected to acid lysis and the chromium concentrations were estimated using Atomic Absorption Spectrometry. Cr (VI) accumulation percentages of Cr (VI) resistant Pseudomonas sp. under 5, 10, 15 mg/L Cr (VI) concentrations were 51.38%, 37.46% and 43.13% respectively. Those percentages were 2.6%, 1.54%, and 1.12% for E.coli JM109 while they were 35.58%, 20.06% and 34.03% for the transformant. There is a possibility of chromium accumulation being regulated by Cr (VI) resistant genetic determinants found on plasmids. In addition, the nature of the host that bears the plasmid is also important for the Cr (VI) accumulation mechanism.These hyperaccumulators of Cr (VI) showed potential use in bioremediation of Cr(VI) containing wastes.
Determining plasmid mediated heavy metal resistance of bacteria
(2016) Abewickrama, J.H.G.
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.
Plasmid mediated Chromium resistance of bacteria
(Book of Abstracts, Annual Research Symposium 2014, 2014) Abewickrama, J.H.G.; Jayarathne, D.L.; Rathnayake, I.V.N.; Gunawardane, M.M.
Plasmid-mediated Cr(VI) resistant bacteria are naturally found in environments contaminated with chromium releasing industrial effluents. These envirnments may contain microorganisms those have genetically regulated mechanisms to overcome elevated Cr(VI) levels. Such mechanisms could be regulated by genes found either in chromosomal DNA or plasmid DNA. In order to understand the exact mechanism and for the possible use of such mechanisms in monitoring and control of heavy metal pollution, it is important to determine whether the resistance is plasmid borne or controlled by chromosomal DNA. There are certain plasmids which contain genes to resist highly toxic hexavalent chromium (chromates and dichromates). Resistance to chromate is determined by decreased chromate transport by the resistant cells. The genes for a hydrophobic polypeptide, ChrA, have been identified in Cr(VI) resistance plasmids of Pseudomonas aeruginosa and Alcaligenes eutrophus.