Differential detection of key enzymes of polyaromatic-hydrocarbon-degrading bacteria using PCR and gene probes

Bacteria with ability to degrade polyaromatic hydrocarbons (PAHs), isolated from wastewater and soil samples, were investigated for their taxonomic, p hysiological and genetic diversity. Eighteen isolates able to metabolize na phthalene or phenanthrene as sole carbon source were taxonomically affiliat ed to different subclasses of the Proteobacteria (Sphingomonas spp., Acidov orax spp., Comamonas spp. and Pseudomonas spp.) and to phyla of Cram-positi ve bacteria with low and high DNA G+C content (Paenibacillus sp. and Rhodoc occus spp., respectively). Representatives of the genera Pseudomonas and Sp hingomonas formed a remarkably high fraction of these isolates; 9 out of 18 strains belonged to these groups. Tests for enzyme activities showed that the majority of the isolates growing with PAHs as sole sources of carbon an d energy had an active catechol 2,3-dioxygenase (C230). C230 specific activ ities were very diverse, ranging from 0.1 to 650 mU (mg protein)(-1). Pseud omonas and Sphingomonas strains showed considerably higher activities than the other isolates. All PAH degraders were examined for the presence of an initial PAH dioxygenase and C230, which catalyse key steps of PAH degradati on, by PCR amplification of gene fragments and subsequent hybridization. PC R primers and internal oligonucieotide probes were developed for the specif ic detection of the genes of Pseudomonas and Sphingomonas strains.