Polycyclic aromatic hydrocarbons (PAH's) constitute a class of aromatic hydrocarbons consisting of two or more fused benzene rings in linear, angular, and cluster arrangements. Since many of the aromatic hydrocarbons found in the environment have a natural pyrolytic origin, they must have been in contact with microorganisms throughout evolutionary periods of time. It is not surprising, therefore, that microorganisms capable of degrading aromatic compounds have evolved. Almost all of what is known about the metabolism of PAH's is based on fast-growing gram negative organisms and naphthalene (two rings) and phenanthrene (three rings) as model substrates. Slow-growing gram positive organisms such as Mycobacterium have been shown not only to degrade naphthalene and phenanthrene but also to degrade PAH's consisting of four or more aromatic rings. In fact, this class of organisms is perhaps the best-studied group because of their ability to mineralize high molecular weight PAH's. Catabolic pathways have been proposed based on the identification of a few intermediate compounds detected in culture supernatants. In this project molecular genetics and biochemistry will be used to investigate the ability of Mycobacterium strain PYO1 to degrade high molecular weight PAH's. Cosmid clones containing the PYO1 genes required for phenanthrene and pyrene degradation have been identified, and analysis of the 30 kb of sequence obtained so far has identified at least 21 genes that are involved in the degradation of PAH's intermingled with five insertion sequences. Putative functions have been assigned to each open reading frame and will be tested with biological analyses using expression in heterologous hosts, mRNA analyses, and knock-out mutants. These experiments are expected to result in the determination of every step in the catabolic pathway with the identification of every catabolic intermediate.
Highly recalcitrant, stable polycyclic aromatic compounds are found in the environment. Such compounds can be formed during coal gasification and liquification processes, improper waste incineration practices, and in forest fires. Sources of the compounds include oil, carbon black, coal tar, and other petroleum-derived products. Mycobacterium species of bacteria and their relatives are unique in their ability to degrade these compounds. However, little is known about the molecular and biochemical basis of these abilities. In this project, the ability of Mycobacterium strains to degrade polycyclic compounds will be investigated at the gene and enzyme levels. A thorough knowledge of how Mycobacteria degrade these compounds will aid in the design of bioremediative processes for removing polycyclic compounds from the environment.
