Construction of recombinant bacteria to elucidate catabolic regulation and critical catabolic reactions of phenanthrene metabolism by the NAH system

Author

Bruce Michael Applegate

Date of Award

8-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Sayler

Committee Members

Gary Stacey, Riggsby, Peter Gresshoff

Abstract

Pseudomonas putida strain PpG7 harbors the catabolic plasmid NAH7 which mediates the biotransformation of naphthalene. The plasmid contains the NAH system consisting of two distinct operons coding for two sets of enzymes: The first operon, nah, codes for the enzymes that metabolize naphthalene to salicylate (upper pathway) which is initiated by a dioxygenase attack on one ring followed by a dehydrogenation forming 1,2 dihydroxynaphthlene; The second operon, sal, codes for a set of enzymes (lower pathway) responsible for subsequent conversion of salicylate to TCA cycle intermediates. Both sets of enzymes are controlled by a LysR regulatory protein which induces both operons in the presence of salicylate. NAH7 and other NAH-type plasmids have also been shown to mediate mineralization of 9-¹⁴C labeled phenanthrene producing ¹⁴CO2. However, this study shows strains harboring these plasmids can not utilize phenanthrene as a sole carbon and energy source. A transposon mutagenized NAH-type plasmid pUTK21 which can no longer mediate the transformation of salicylate to TCA cycle intermediates has been shown to mediate the biotransformation of phenanthrene to 1-hydroxy-2-naphthoic acid, a compound with a structure similar to salicylate. The strain harboring the plasmid pUTK21 designated 5RL will convert phenanthrene to 1-hydroxy-2-naphthoic acid but fails to produce ¹⁴CO2. This failure to produce ¹⁴CO2 is consistent with the production of the dead end metabolite 1-hydroxy-2-naphthoic acid. The absence of ¹⁴CO2 directly links the enzymes responsible for salicylate metabolism to the further metabolism of 1-hydroxy- 2-naphthoic acid. Hybridization results using a nahG (the first enzyme in the lower pathway) gene probe showed a direct connection of a negative result with lack of mineralization. The further metabolism of 1-hydroxy-2-naphthoic acid was thought to proceed by the conversion of 1-hydroxy-2-naphthoic acid to 1,2-dihydroxynaphthalene by a simultaneous hydroxylation/decarboxylation mediated by the NahG. If this is correct 1,2-dihydroxynaphthalene would be further metabolized through the upper pathway to salicylate and ultimately TCA cycle intermediates, nah-lux bioluminescent reporter bacteria showed that inducing intermediates were not produced to allow induction of the enzymes. Since the hybridization results indicated that the nahG gene product was correlated with the ability to mineralize this gene was cloned and expressed in Eschericia coli When cells expressing the nahG gene product were incubated with 1-hydroxy-2-naphthoic acid a characteristic orange color indicative of a ring cleavage product was produced. Results from this study show that the further metabolism of 1- hydroxy-2-naphthoic acid appears to consist of a ring cleavage of the 1-hydroxy-2- naphthoic acid and a subsequent aldolase reaction to form phthallic acid. These results show that the NAH system can only partially metabolize phenanthrene.

Recommended Citation

Applegate, Bruce Michael, "Construction of recombinant bacteria to elucidate catabolic regulation and critical catabolic reactions of phenanthrene metabolism by the NAH system. " PhD diss., University of Tennessee, 1997.
https://trace.tennessee.edu/utk_graddiss/9427