Date of Award

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

V. R. Srinivasan

Abstract

Microbial degradation of diphenyl ethers was studied utilizing several different approaches. The first approach was to isolate mutants from a ligninolytic Erwinia sp. by conventional mutagenesis and selection. A copper-resistant mutant, designated as Erwinia sp. CU3614, was isolated and characterized to be capable of degrading diphenyl ethers as well as one dibenzo-p-dioxin in the presence of copper ions. Isolation of Tn5 transposon-induced mutants of Erwinia sp. CU3614, which lost degradation ability, demonstrated that in all probability a copper-associated activity in the biotransformation of aromatic ethers was involved. In order to study the degradation at the molecular biological level, a 2.1 kb Hind III fragment containing Erwinia sp. dpe gene was cloned into pUC19 and introduced into Escherichia coli. The dpe gene encoded the diphenyl ether cleavage activity. The expression of dpe was detected in E. coli directly on a specially designed medium, LTFN, containing 4-nitrodiphenyl ether. Mutations generated by transposon mini-Mu dI (lacZ Km$\sp{\rm r})$ were used to define the dpe coding region, as well as the determination that the lacZ promoter of pUC19 was initiating transcription into the dpe. A peptide of molecular weight of 21 kDa was detected as the product of dpe gene in the plasmid encoded proteins. To study the activities encoded by the dpe gene, resting cell assays were used to determine the degradation of diphenyl ethers, utilizing dpe plasmid transformants of E. coli and of a salicylic acid-utilizing bacterium, designated as PE7. Bacterium PE7 was isolated from a petroleum waste pit in Louisiana, and identified to be most likely an Acinetobacter sp. A broad-host range expression plasmid, pDPE2388, constructed from subcloning dpe gene into plasmid pKT230 was used to introduce Erwinia sp. dpe gene into this bacterial isolate PE7. Expression of dpe gene in E. coli and PE7 displayed the specificity: 4-chlorodiphenyl ether $>$ 4-nitrodiphenyl ether $>$ 4-hydroxydiphenyl ether.

Pages

144

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