A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Engineering and Management Inter-University Program on Environmental Toxicology, Technology and Management

Thesis (M.Sc.) - Asian Institute of Technology - Chulabhorn Research Institute - Mahidol University, 2007

The cop operon of Agrobacterium tumefaciens involving in copper resistance is consisted of three genes that encode CPx-type copper ATPase, CopA, a repressor, CopR and a copper chaperone, CopZ. The expression of cop operon is induced by the presence of copper and silver thereby protecting A. trmrefaciens from particular metal ion toxicity. Primer extension analysis defined the consensus sequence 5'CTTCCCATnATGGGAAG3' as the repressor binding clement. Using in vivo copARZ promoter analysis, it was demonstrated that copA expression is regulated by copR while CopZ copper chaperone played no role on the function of CopR to sensing copper. Gene disruption of copA and copZ by using the suicide plasmid pKNOCK caused reduction of bacterial ability to cope with copper and silver toxicity. Unfortunately, such reduction could not be complemented by a trans expression of copA or copZ respectively resulting from the polar effect from inactivation of one gene on the expression of other genes in the operon. Taken together, the results suggest that under copper-limiting condition CopR binds to the copARZ promoter and represses the expression of the operon. In high copper, CopR is activated to an active form of transcription activator thereby, enhancing copARZ transcript. A copper chaperone, CopZ then binds excess free Cu(I) and transfers to CopA copper export to detoxify copper