Mycobacterium vanbaalenii PYR-1 is capable of degrading a number of polycyclic aromatic hydrocarbons (PAHs) to ring cleavage metabolites via multiple pathways. Genes for the large and small subunits of a pyrene dioxygenase, nidA and nidB, respectively, were previously identified in M. vanbaalenii PYR-1 [Appl. Environ. Microbiol. 67 (2001) 3577]. A library of the M. vanbaalenii PYR-1 genome was constructed in a fosmid vector to identify additional genes involved in PAH degradation. Twelve fosmid clones containing nidA were identified by Southern hybridization. Sequence analysis of one nidA-positive clone, pFOS608, revealed a number of additional genes involved in PAH degradation. At this locus, one putative operon contained genes involved in phthalate degradation, and another contained genes encoding a putative ABC transporter(s). A number of the genes found in this region are homologous to those involved in phenanthrene degradation via the phthalic acid pathway. The majority of phenanthrene degradation genes were located between putative transposase genes. In Escherichia coli, pFOS608 converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, and converted 1-hydroxy-2-naphthoic acid into 2'-carboxybenzalpyruvate, 2-carboxybenzaldehyde, and phthalic acid. A subclone containing nidA and nidB converted phenanthrene into phenanthrene cis-3,4-dihydrodiol, suggesting that the NidAB dioxygenase is responsible for an initial attack on phenanthrene. This study is the first to identify genes responsible for the degradation of phenanthrene via the phthalic acid pathway in Mycobacterium species. Published by Elsevier Inc.