Q-180. Sequencing and Analysis of PAH Degrading Dioxygenases in Sphingomonas paucimobilis EPA505

R. Persad, T. A. Hughes;
Clemson Univ., Clemson, SC.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants which have serious impacts on our environment and can directly pose health hazards to humans and other organisms. Structurally, PAHs are very stable compounds making them difficult to breakdown. Some prokaryotes however, can use these compounds as a source of carbon and energy thereby facilitating bioremediation. Sphingomonas paucimobilis EPA505 was one of the first microorganisms known to degrade PAHs with more than three fused rings. There are several enzymes involved in the breakdown of PAHs. Dioxygenases are of particular importance however, since they are responsible for cleaving the aromatic ring. In order to study the molecular basis of PAH degradation in EPA505, a BAC library was constructed and probed for PAH degrading genes. BAC clones containing genes of interest were picked for high-throughput sequencing. BLAST searches using the partial sequences obtained revealed homology to PAH degrading genes including the small subunit of a toluene-benzoate dioxygenase of Sphingomonas sp. P2, a biphenyl- 2-3-diol 1, 2-dioxygenase of Sphingomonas paucimobilis strain Q1 and the terminal component of a PAH-hydroxylating dioxygenase from Sphingomonas sp. CHY-1. Furthermore, an open reading frame search revealed ORFs which correspond to these regions confirming the presence of functional genes. Continuing to sequence the regions flanking these areas will reveal more about the organization of these genes including whether or not they are part of an operon. In addition, these genes will be cloned into an expression vector for protein isolation and characterization.