Molecular analysis of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHs

A PCR-based molecular tool was developed to estimate the diversity of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHS. A degenerate primer pair specific to catA sequences was designed by multiple alignment of known sequences coding a key intermediate of the β-ketoadiapate pathway degrading catechol, namely catechol 1,2-dioxygenase. The specificity of this primer pair was assessed in 21 pure strains by PCR and sequencing. Comparison of the 16S rDNA and catA phylogenies revealed an absence of congruence between these two genes. The primer set was able to amplify catA sequences in DNA extracts from an industrial soil highly contaminated with heavy metals and polycyclic aromatic hydrocarbons (PAHs). RFLP screening of the catA library (95 clones) yielded 32 RFLP families. All of the 43 clone sequences obtained exhibited 86% identity on average to known CatA. Phylogenetic analysis revealed that these CatA sequences were related to Actinobacteria, α-, β- and γ-Proteobacteria phyla and confirmed the absence of congruence with 16S rDNA sequences, which implies horizontal gene transfer of the cat gene cluster between soil microbiota. Our results suggest that the diversity of the catA bacterial community is maintained in highly contaminated soil.