Molecular analysis of polycyclic aromatic hydrocarbon degradation by Mycobacterium sp. strain PYO1.

摘要

Polycyclic aromatic hydrocarbons (PAHs) are pervasive environmental pollutants. Sources of aromatic hydrocarbon pollution include the incomplete pyrolysis of organic materials, by-products of the petroleum industry, coal liquification and coal gasification. These pollutants persist in the environment and once biologically activated can have toxic effects on living organisms. Research on aromatic hydrocarbon catabolism focuses on efficient methods to monitor biological conversion of aromatics in the environment, biological protocols for disposing of these pollutants, and more recently, some enzymes in PAH catabolic systems are being recognized for their potential in the production of pharmaceuticals. Mycobacterium sp. strain PYO1 is capable of utilizing the PAHs pyrene and phenanthrene as sole sources of carbon and energy for growth. Bacteria utilize ring hydroxylating oxygenase enzymes to initiate aromatic hydrocarbon oxidation. Through comparative sequence analysis of oxygenase enzymes a universal primer based PCR protocol was developed to detect and identify oxygenase gene fragments. Application of this protocol to DNA isolated from a fuel oil contaminated site resulted in amplicons with a high degree of similarity to Mycobacteria oxygenases. Furthermore when compared to nearby uncontaminated soil a shift in the makeup of the microbial community relative to oxygenase genes present was observed.; The universal PCR primer protocol was also applied to Mycobacterium sp. strain PYO1 resulting in the isolation of three oxygenase gene fragments. Using these gene fragments as probes in Southern hybridization experiments produced a 6000 kb clone that contained one dioxygenase gene cluster. In an attempt to isolate the complete PAH pathway in PYO1 the dioxygenase gene cluster was used to probe a PYO1 cosmid library. Partial subcloning and sequence analysis of the cosmid clones isolated identified two additional multicomponent dioxygenase gene clusters. Messenger RNA analysis showed two of these dioxygenase genes are transcribed in the presence of pyrene while the third dioxygenase gene is transcribed in the presence of phenanthrene. The nucleotide sequence of 29,505 bp of PYO1 DNA including and encompassing these dioxygenase genes was determined. According to sequence analysis the 29,505 bp of PYO1 sequence contains many of the genes required for PAH degradation in PYO1, arranged in at least four operons.; A 1-hydroxy-2-naphthoate dioxygenase gene (pcdG) located within the putative phenanthrene pathway was placed in an expression vector and an E. coli host. Functional analysis of the PcdG enzyme was confirmed by an ultraviolet assay in which conversion of 1-hydroxy-2-naphthoic acid to 2′-carboxybenzalpyruvic acid was observed. Though several other compounds were tested as potential substrates for this enzyme none were biochemically acted upon by PcdG. All of the tested alternate substrates except phthalate could inactivate PcdG to various degrees.