Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAM-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from acti vated sludge or soil samples. Sequence analysis of PCR products from severa l Pseudomonas strains showed that alpha subunits (nahAc allele) of this gen us are highly conserved. PCR primers for the specific detection of alpha su bunit genes of initial PAH dioxygenases from Pseudomonas strains were not s uitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived fro m the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAM-degradation gene cluster from C. testosteroni strain H. Parts of th is gene cluster containing three subunits of the initial PAH dioxygenase we re isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and be ta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5 alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignme nt of alpha subunits from C. testosteroni H and various PAM-degrading bacte ria permitted the design of new primers and oligonucleotide probes which ar e useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.