Cloning and Expression of Thiocyanate Resistance Genes in Iron/Sulphur Oxidising Bacteria

摘要

Microbial oxidation of refractory sulphide ores (particularly gold and copper ores) are well-established techniques that arc increasingly employed because of their low capital and operating costs and environmental benefits. CSIRO has established a multidivisional project, the 'BioMineral Processing Initiative', with the goal of generating new and improved high temperature micro-organisms for biological oxidation of these refractory ores. The project is currently focused on the discovery/generation of novel hyperthermophiles for the bioleaching of copper from chalcopyrite. As part of this project we are investigating the practicalities of genetic modification of iron/sulphur oxidising micro-organisms. The molecular biology of iron/sulphur oxidising acidophiles is still in its infancy. This work is aimed at improving our knowledge of vectors, promoters, selectable markers, gene transfer and expression in these organisms. The Acidithiobacillus ferrooxidans genome is currently available and genomics is proceeding at a rate where in the next few years we can expect at least one or two relevant microbial genomes to be sequenced each year. New genes that impart useful characteristics to iron/sulphur oxidising acidophiles will be available from published genomes and the study of newly isolated hyperthermophiles. We need to be in a position to clone and express those genes. This paper will describe the isolation, transfer and expression of thiocyanate hydrolase genes in Escherichia coli and At. ferrooxidans. Most common antibiotics are not effective against iron/sulphur oxidising acidophiles. Also, the use of antibiotics or their resistance markers outside of a laboratory setting would not be appropriate. Resistance to thiocyanate should be a useful selectable marker, without implications for human or animal health. Additionally, thiocyanate resistant iron/sulphur oxidising acidophiles might have application in gold bio-oxidation plants in arid areas where reuse of contaminated tailings water is desirable. The cloning of thiocyanate genes in broad host range plasmids under the control of E. coli and At. ferrooxidans promoters and the expression and phenotypic effects in both species will be described.