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

Thesis (M.Sc.) - Asian Institute of Technology, 2006

Agrohacterum tumefaciens possesses multiple system to ensure proper copper handling under varying environmental condition. Searching A. tumefaciens C58 genome revealed that the bacteria had three loci (two on circular and one on linear chromosome) contained genes involved in copper resistance. Here, three genes, namely cueR (Atu0938), copA (Atu0937) and CueO (Atu3991) from two loci were functionally evaluated in term of copper resistance. CueR is transcriptional regulator in a MerR family while CopA and CueO are copper transporting P-type APpase and multicopper oxidase, respectively. A. tumefaciens cueR, copA and cueO. Mutants were constructed by gene knockout using pKNOCK system. cueO mutant was sensitive to Cu (II) and Cd (II) as tested by inhibition zone assays however all the MIC values against Cu (II) in all the mutant was reduced comparing to the wild type's level. The altered phenotypes could be restored by a plasmidborne expression of individual gene. Northern blot analysis revealed that the expression of cueO could be induced only by Cu (II) in an CueR-independent manner. Promoter analysis using lacZ fusion revealed that copA expression was controlled by CueR and could be hypothesized that when A. tumefaciens exposes to increase level of copper (as well as Ag) ions. Two transcription regulators, CueR and the unknown regulator, respond to this change by promote transcription of copA encoding efflux protein and cueO encode multicopper oxidase. High expression of these two protein makes A. tumefaciens more resistant to copper