Doctoral Dissertations

Date of Award

12-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Sayler

Committee Members

Terry W. Schultz, Dwayne Savage, Gary Stacy

Abstract

Environmental sites contaminated by polycyclic aromatic hydrocarbons (PAHs) and heterocycles typically are characterized by the presence of approximately one-hundred different contaminants of varying ring structure and substitution patterns. The biodegradation of the majority of these contaminants by the NAH system for naphthalene degradation, which is currently the most well-studied enzyme system for the breakdown of PAHs, has not been examined in great detail. Using the bacterium Pseudomonas fluorescens 5R, and its mutant P. fluorescens 5RL, which contains a nonfunctional NAH system lower pathway, the metabolism of a suite of substituted naphthalenes and heterocyclic compounds by the NAH upper pathway was examined. Based on gas chromatography/mass spectrometry (GC/MS) analysis of metabolites, it was found that most naphthalenes substituted at the 1 and 2 position are transformed by the NAH upper pathway to their respective salicylate analogs. The heterocyclic compounds which were examined were characterized by an analogous cleavage of one aromatic ring. Substitution on both rings led to incomplete upper pathway metabolism and the formation of dead-end, hydroxylated parent compounds.

The ability of the cloned lower pathway genes for salicylate hydroxylase and catechol- 2,3-dioxygenase of P. fluorescens 5R to cleave commercially available salicylate analogs and upper pathway metabolites was also examined. It was found that the lower pathway is limited in its ability to cleave many upper pathway products, except those with methyl- or chloro- substituents. The ability of these products to act as a surrogate for salicylate and induce NAH system transcription was examined using P. fluorescens 5RL. Many compounds were found to induce transcription. In general, with respect to the ability of the NAH system to facilitate growth on these substituted PAHs and heterocycles, only those compounds which undergo both the upper and lower pathway cleavage reactions and produce an inducer of transcription served as sole carbon and energy sources.

The capacity of the NAH system to preferentially degrade a mixture of PAHs and heterocycles was examined using P. fluorescens SR and 5RL, an E. coli harboring the gene for naphthalene dioxygenase (nahA), and a number of other bacterial isolates possessing the NAH system. It was found that naphthalene dioxygenase dictates the kinetics of mixed PAH degradation. In addition, preferential rates of degradation occurred for those compounds of least substitution which most resemble naphthalene structurally.

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