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Three strains of Comamonas testosteroni were isolated from river sediment for the ability to degrade phenanthrene; two of the strains also grew on naphthalene, and one strain also grew on anthracene. The homology of the genes for polycyclic aromatic hydrocarbon degradation in these strains to the classical genes (nah) for naphthalene degradation from Pseudomonas putida NCIB 9816-4 was determined. The three C. testosteroni strains showed no homology to the nah gene probe even under low-stringency conditions. The genes for naphthalene and phenanthrene degradation were cloned from one of the three C. testosteroni strains. Two cosmid clones expressing polycyclic aromatic hydrocarbon dioxygenase activity were identified from a library prepared with genomic DNA from C. testosteroni GZ39. The genes coding for the first two enzymes in the catabolic pathway, phenanthrene dioxygenase and cis-phenanthrene dihydrodiol dehydrogenase, were localized to a 5.4-kb NcoI-PstI fragment by subcloning and gene expression experiments. Further subcloning and analysis revealed a novel organization of the genes, with the gene for cis-phenanthrene dihydrodiol dehydrogenase located between the genes for the individual phenanthrene dioxygenase components. A Southern blot with the cloned genes from C. testosteroni GZ39 confirmed that these genes are distinct from those found in P. putida NCIB 9816-4. Southern blots also demonstrated that C. testosteroni GZ38A possesses genes for phenanthrene degradation that are similar to those cloned from C. testosteroni GZ39. However, C. testosteroni GZ42 possesses genes for phenanthrene degradation that are not homologous to those cloned from C. testosteroni GZ39. This suggests that there are at least two different sets of genes for the degradation of phenanthrene among the three C. testosteroni strains.