Dynamic changes in nahAc gene copy numbers during degradation of naphthalene in PAH-contaminated soils

Many bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) contain the nahAc gene that encodes a component of multimeric naphthalene dioxygenases. Because the nahAc gene is highly conserved, this gene serves as a potential biomarker for PAH degradation activity. The aim of this research was to examine the relationship between the rate of naphthalene degradation and the copy number of the nahAc gene in soils using conventional and real-time PCR. Four sets of degenerate primers for real-time PCR were designed based on the nahAc DNA sequences of 33 bacterial species. Before addition of naphthalene, copy numbers of the nahAc gene were below the detection limits of the assay at 5x10(3) copy numbers per gram of soil, but increased by over a thousand fold to 10(7) copies after 6 days of exposure to naphthalene vapors (approximately 30 ppm soil water concentration). Two unreported naphthalene dioxygenase homologs were found in the naphthalene-spiked soil by cloning and sequencing of the PCR products from the nahAc primers. Results of these experiments demonstrate the highly dynamic changes that occur in soil microbial communities after exposure to naphthalene and suggest that there is a direct relationship between gene copy numbers and degradation rates for naphthalene in PAH-contaminated soils.