Q-188. Investigation of the Molecular Mechanisms for Phenanthrene Degradation in Burkholderia sp. strain Ch1-1

J. Zhao, W. J. Hickey;
Univ. of Wisconsin, Madison, WI.

Burkhoderia are ubiquitous, environmentally important bacteria that can degrade recalcitrant environmental pollutants including polynuclear aromatic hydrocarbons (PAHs). However, to date, the phenanthrene-degrading Burkholderia sp. strain 007 is the only representative of this group for which the molecular basis for PAH degradation has been determined. The current study focused on Burkholderia sp. strain Ch1-1, which was isolated from PAH-contaminated soil based on its growth on phenanthrene (PHN). The objective of the studies was to determine if the molecular basis of PHN degradation in strain Ch1-1 was similar or different from that in strain 007. Proteome analysis of PHN-grown Ch1-1 by liquid chromatography-tandem mass spectrometry revealed three enzymes involved in phenanthrene pathway, including 1-hydroxy-2-naphthoate dioxygenase (PhnG), 1-hydroxy-2-naphthoaldehyde dehydrogenase (PhnF) and PhnA, a ring-hydroxylating dioxygenase (RHD) that would initiate PHN degradation. Genes encoding the PhnA large subunit (phnAc) and small subunit (phnAd) were cloned by PCR, and had the greatest nucleotide sequence identity (96 %) to phnAcd of Alcaligenes faecalis AFK2. Phylogenetic analysis of PAH RHD sequences showed that PhnAcd of strains AFK2 and Ch1-1 formed a separate branch, which was divergent from the RHD of strain 007 and Nah-like RHD. Heterologous expression of PhnAcd confirmed its activity on phenanthrene as well as pyrene as substrates. Substrate utilization tests indicated that strain Ch1-1 metabolized PHN through protocatechuate as central intermediate, not salicylate as does strain 007. Burkholderia sp. strain Ch1-1 possesses a novel PAH-RHD, encoded by phnAcd that is divergent from the RHD of Burkholderia sp. strain 007 and other classical nah-like genes. The lower pathways for PHN metabolism in strain Ch1-1 and strain 007 were also divergent. This research expands the current knowledge of PAH-oxidizing RHD in proteobacteria, provides insights into diversity of molecular mechanisms for PAH degradation in Burkholderia.