Q-179. Sequence Analysis and Characterization of Dihydrodiol Dehydrogenase Gene in Sphingomonas paucimobilis var. EPA505

J. Jalili;
Clemson Univ., Clemson, SC.

Bioremediation using certain species of microorganisms is an inexpensive and useful decontamination of soils contaminated with Polycyclic Aromatic Hydrocarbons (PAHs). To have an effective bioremediation, microorganisms must enzymatically attach the pollutants and convert them to harmless products. Sphingomonas paucimobilis EPA505 has been isolated from soils highly contaminated with coal wastes. This bacterium, which can grow on phenanthrene, naphthalene, fluoranthene, toluene and several other high molecular weight PAHs, has the ability to degrade environmental pollutants caused by PAHs. A common set of enzymes is responsible for the metabolic steps in degradation of the polyaromatic compounds. An initial dioxygenase attack results in the formation of a cis-dihydrodiol. This step of dioxygenation is followed by a dehydrogenase attack to rearomatize the ring structure in order to form a dihyroxylated product. These resultant substrates can be cleaved by another dioxygenase finally leading to tricarboxylic acid (TCA) cycle intermediate. The primary objective of this research is to clone and sequence cis-dihydrodiol dehydrogenase gene of Sphingomonas paucimobilis EPA505 strain. It should be noted that such sequence analysis of cis-dihydrodiol dehydrogenase gene has been reported in several bacteria, however, none of these studies have characterized cis-dihydrodiol dehydrogenase (ddh) gene of Sphingomonas paucimobilis EPA505 strain. Our objectives are: develop a cloning strategy and sequence analysis of ddh gene; determine overexpression of ddh and purification procedure, and perform detailed ddh enzyme assays. To achieve this purposes, primers from the conserved regions of Sphingomonas strain CHY-1 and Sphingobium yanoikuyae strain B1 were dsigned. The PCR product was sequenced and hybridized with the BAC library of the Sphingomonas paucimobilis EPA505. The clones will be sequenced and overexpressed for DNA analysis and protein characterization.