Master of Science (Research)
School of Biological Sciences
Wu, Shiyang, Bioinformatics analysis and preliminary functional study of proteins associated with arsenic metabolism and resistance, Master of Science (Research) thesis, School of Biological Sciences, University of Wollongong, 2007. http://ro.uow.edu.au/theses/2699
Arsenic, a naturally occurring metalloid, is widely spread in the environment and is toxic to living organisms. Its pollution, caused mostly by mining, industrial and agricultural uses, has become a global problem. To elucidate arsenic resistance mechanisms evolved in diverse organisms has been an active research area which will help combat the threatening pollutant. This study was set to investigate some proteins associated with arsenic metabolism and resistance.
Firstly, bioinformatics methods were used to analyze three kinds of such proteins. Arsenic extrusion, with its major component being arsenic pump proteins, is a common arsenic resistant pathway found in prokaryotes and eukaryotes. Although arsenic pump proteins are widely spread among the prokaryotic and eukaryotic kingdoms, the distribution of these proteins in the ArsB, YqcL and MRP1 families is different. The widespread of these arsenic pumps among the prokaryotic and eukaryotic kingdoms is probably due to horizontal gene transfer events. Biomethylation is an important arsenic metabolism in living organisms. However, the genes responsible for arsenic methyltransferases, the key enzymes involved in this process, have only been identified in human, rat and Halobacterium. Based on the available literature and the limited sequence information on arsenic methyltransferases, the major features of arsenic methyltransferases are that they are SAM-dependent methyltransferases with an arsenite binding site (two close cysteines separated within five other amino acids). Utilizing these features, three potential arsenic methyltransferases (accession numbers: NP_001031256, NP_565170 and NP_181637) from Arabidopsis thaliana and one potential arsenic methyltransferase YafS protein (accession number: BAA77884) from E. coli were identified through bioinformatics prediction. Bioinformatics approaches were also used to predict the function of ORF2 protein in the arsROrf2BC operon in Bacillus sp CDB3 strain. The results show that the ORF2 protein is a bleomycin resistance protein and is possibly associated with arsenic metabolism.
To complement the preceding bioinformatics analysis, preliminary functional studies of the ArsB homologue in Arabidopsis thaliana (named AtB protein in this study), YafS, ORF2 and YHR209w (a putative arsenic methyltransferase from yeast Saccharomyces cerevisiae) proteins, were carried out in bacterial E. coli cells. Bacterial expression plasmids for AtB, YafS and YHR209w were constructed. The functional tests showed that compared with the control, the E. coli cells harbouring AtB, yhr209w or yafS gene were more sensitive to arsenite. In addition, an arsenite resistance assay of yeast wild-type and yhr209w knock-out mutant strains demonstrated that the knock-out of yhr209w gene resulted in a slightly elevated tolerance to arsenite compared with the wild-type strain. Therefore, this result also indicated that YHR209w protein may confer slight sensitivity to arsenite. My experimental work also confirmed the bioinformatics prediction result that the function of ORF2 protein was associated with bleomycin resistance.