A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, some of which are highly carcinogenic, genotoxic, and a threat to public health (5). Many PAHs, including phenanthrene, benzofluoranthene, pyrene, benz [a] anthracene, benzo[a]pyrene, and chrysene, are therefore listed on the U.S. Environmental Protection Agency's priority-pollutant list (13). Degradation of PAHs by indigenous microorganisms seems to play an important role in the removal of contaminants from the environment, particularly from subsurface material and groundwater (4,23,25). Due to the high cost associated with trapping, incinerating, and physically removing toxic chemicals from the environment, bioremediation technologies are being developed to clean up PAH-contaminated environments (19). Especially, nocardioform actinomycetes, including Mycobacterium spp., seem to be involved in the degradation of high-molecular-weight PAHs in soil and sediments (2,6,8).Studies of the molecular basis of PAH degradation mechanisms in Mycobacterium species are lacking but extremely important for better understanding and application of these organisms for bioremediation. In previous studies, Mycobacterium sp. strain PYR-1 has been shown to mineralize anthracene, fluoranthene, pyrene, 1-nitropyrene, phenanthrene, and benzo[a]pyrene (8-10, 14, 15, 20, 24). Mycobacterium sp. strain PYR-1 is known to have an inducible system for PAH degradation (8). Our initial approach to identifying genes involved in PAH degradation in strain PYR-1 was to recover proteins produced upon exposure of the strain to pyrene during pyrene metabolism. In this article, we report the cloning, expression, and characterization of a PAH-inducible catalase-peroxidase gene, katG, from Mycobacterium sp. strain PYR-1.
MATERIALS AND METHODSStrains, chemicals, and culture medi...