Molecular cloning, nucleotide sequence, and expression of genes encoding apolcyclic aromatic ring dioxygenase from Mycobacterium sp strain PYR-1

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hy drocarbons (PAHs) primarily through the introduction of both atoms of molec ular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PA H degradation, two-dimensional (2D) gel electrophoresis of PAH-induced prot eins from cultures of Mycobacterium sp. strain PYR-1 was used to detect pro teins that increased after phenanthrene, dibenzothiophene, and pyrene expos ure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide prob e designed from this protein sequence was used to screen dioxygenase-positi ve clones from a genomic library of Mycobacterium sp. strain PYR-1. Three c lones, each containing a 5,288-bp DNA insert with three genes of the dioxyg enase system, were obtained. The genes in the DNA insert, from the 5' to th e 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (a)-subunit genes, arranged in a sequence differ ent from those of genes encoding other bacterial dioxygenase systems. Phylo genetic analysis showed that the large a subunit did not cluster with most of the known a-subunit sequences but rather with three newly described alph a subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of t he genes for PAH degradation was confirmed in a phagemid clone containing a ll three genes, as well as in plasmid subelones containing the two genes en coding the dioxygenase subunits.