Design and optimization of real time PCR assays for the quantification of aromatic ring-hydroxylating dioxygenase genes in marine sediments

LOZADA, M.; MARCOS, M. S.; RIVA MERCADAL. J. P.; FERRERO. M.; DIONISI, H. M.

Rosario

Congreso; V Congreso Argentino de Microbiología General; 2008

Sociedad Argentina de Microbiología General (SAMIGE)

In aquatic ecosystems, sediments can serve both as reservoirs and as potential sources of contaminants to the water column. In many sites along the Patagonian coast, we detected between one and eleven different polycyclic aromatic hydrocarbons (PAHs), some of them in concentrations exceeding those recommended by sediment quality guidelines to protect aquatic life. Using culture independent methods, we detected in these sediments eight different types of aromatic ring-hydroxylating dioxygenase (ARHD) genes, which code for enzymes involved in the first step of PAH biodegradation pathways. Of these, five were classified as novel gene types, as they form deeply rooted branches (less than 70% identity at the amino acid level) with previously described ARHD sequences. In accordance, we have designed eight Real Time PCR (qPCR) assays, including primers and TaqMan probes, for the quantification of these genes in environmental samples. The design took into account annealing and thermodynamic properties of the primers-probe system aiming to increase assays performance. Given the high cost of the TaqMan probes, the assays were first tested in conventional assays with the DNA-binding fluorophore EvaGreen. Annealing and plate-read temperatures and magnesium ion concentrations were optimized for each assay. Sensitivity and linearity were tested with standard curves built with plasmids carrying the appropriate gene fragments previously obtained by cloning and confirmed by sequencing. Specificity was tested using environmental DNA as template by running melting curves against the corresponding standards and by agarose gel electrophoresis. Preliminary assays were performed using DNA samples directly extracted from coastal sediments with different levels of PAH contamination. The number of ARHD gene copies per microliter of extracted DNA was estimated for each assay at two DNA concentrations. Similarly, 16S rRNA gene copy number was estimated using universal eubacterial primers. These values were used to estimate the relative abundance of the different ARHDs in the microbial community, with respect to 16S rRNA gene abundance. In certain chronically polluted sites, values for an individual gene type could reach up to 1800 (±200) copies per microliter of extracted DNA, representing the 0.07% of bacterial 16S rRNA genes detected in the same sample. Our results indicate that the assays are sensitive enough to detect genes present at very low frequencies in the community, and will be fundamental to analyze the relative abundance of these genes in different coastal environments. These qPCR assays will also allow to analyze the dynamics of bacterial populations carrying these functionally relevant genes in microcosm experiments, in order to identify the ecological role and environmental needs of these populations present in the sediments. In addition, these ARHD qPCR assays will be used to identify clusters of catabolic genes in a metagenomic fosmid library.