Degenerate primers as biomarker for gene-targeted metagenomics of the catechol 1, 2-dioxygenase-encoding gene in microbial populations of petroleum-contaminated environments

Catechol 1, 2-dioxygenase (1, 2-CTD) plays an important role in the biodegradation of most aromatic pollutants (e.g. phenol) by cleaving the bond between the phenolic hydroxyl groups. In this study, we analysed microbial populations in petroleum-contaminated soil samples by the culture-dependent approach and chose one population as a sample location for metagenomic studies. This population showed the largest number of phenol-degrading isolates and the most phylogenetic diversity based on repetitive sequence-based PCR and 16S rDNA analyses. The isolates were assigned mainly to the bacterial genera Acinetobacter and Pseudomonas. To enable maximum recovery of 1, 2-CTD coding sequence from the genomic pool of bacterial populations, we extracted the substrate-induced RNA, reverse-transcribed it to the specific cDNA using primers designed for this purpose and constructed a metagenomic library. Alignment analysis of one recombinant sequence indicated a significant divergence, with the 1, 2-CTD gene of Pseudomonas aeruginosa DK2 as the closest species according to sequence similarity. This result indicates that such specifically designed primers are likely suitable candidates as biomarkers to construct 1, 2-CTD gene-targeted metagenomic libraries. By sequencing the larger clones of this metagenomic library, we hope to obtain novel and unpredicted 1, 2-CTD genes that can contribute to our knowledge of microbial diversity of these genes.