Molecular Analysis of Polycyclic Aromatic Hydrocarbon Degradation by Mycobacteria

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9723921 Zylstra Polycyclic aromatic hydrocarbons (PAHs) 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 have evolved that are capable of degrading aromatic compounds. Almost all of what is known about the metabolism of PAHs 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 PAHs consisting of four or more aromatic rings. In fact, this class of organisms is perhaps the best studied group for their ability to mineralize high molecular weight PAHs. Unfortunately, however, very little is known about the ability of these organisms to metabolize PAHs. Catabolic pathways have been proposed but these are based on the identification of only a few intermediate compounds that have been identified in culture supernatants. This project involves the use of molecular genetics and biochemistry to investigate the ability of Mycobacterium strains PYO1 and PYO5C to degrade high molecular weight PAHs in more detail. A clone for the initial dioxygenase in the catabolic pathway(s) has been obtained. Assuming that the genes for PAH degradation are clustered, the remaining genes in the pathway can be located by nucleotide sequencing. Sequence analysis coupled with biological analyses with expressed, cloned genes and with knock-out mutants will identify the function of each identified open reading frame. These experiments will result in the determination of every step in the catabolic pathway with the identification of every catabolic intermediate. In the case that the genes fo r PAH degradation are not linked in Mycobacterium then genes will be identified through transposon mutagenesis of the Mycobacterium strain. This will be aided by the fact that an insertion sequence has been identified in and cloned from the target Mycobacterium strain. Mycobacterium species of bacteria and their relatives are unique in their ability to degrade highly-recalcitrant, stable polycyclic aromatic compounds in the environment. However, little is known about the molecular and biochemical basis of this activity. This project is designed to investigate the ability of Mycobacterium strains to degrade polycyclic compounds at the gene and enzyme level. A thorough knowledge of how Mycobacterium degrade these compounds will aid in the design of bioremediative processes for removing polycyclic compounds from the environment.