Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD_α) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD_α gene amplicons from soil DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHD_α genes of Gram-positive and Gram-negative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHD_α genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHD_α gene-carrying soil bacteria to phenanthrene spiking.Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic compounds composed of two or more fused aromatic rings. Although PAHs are ubiquitous in the environment (from natural oil seeps, brush fires, and plant derivatives), anthropogenic activities, such as disposal of coal-processing waste, mining accidents, petroleum wastes, and vehicle exhaust, have drastically increased their occurrence in the environment. The fate of PAHs in soil is of great interest due to their potential for bioaccumulation, persistence, transport, and toxicity. Microbe-driven aerobic degradation of PAHs is well documented (15-17). The diversity of PAH-degrading genes in soils is assumed to be huge, but the extent of diversity and how it is influenced by different soil types or their history and type of pollution are not yet fully explored. Knowledge of the genes coding for dioxygenase enzymes that catalyze the primary step of PAH degradation by incorporating molecular oxygen into the aromatic nucleus is an essential prerequisite to unraveling the contributions of microbial population networks to transformation, assimilation, and degradation of organic chemicals in soil. Recently, the complete genomes of several PAH-degrading bacteria became available and allowed new insights into degradative pathways (6, 18, 36). Organic pollutants also serve as nutrients for those microbes that have the appropriate genetic makeup to utilize them, resulting in their increased metabolic activity and abundance (4, 14). In the last decade, impressive progress was seen in techniques that allow cultivation-independent analysis of microbial communities and thus overcome the most severe limitations in studying microbial communities in natural habitats, namely, that only a rather small portion of microbes are accessible to standard cultivation conditions (1, 29). For more than a decade, cultivation-independent approaches have also been employed to unravel the responses of microbial communities in soils and sediments to PAH pollution. In all these studies, PCR amplification of PAH-degrading gene fragments from nucleic acids directly extracted from environmental samples was used to explore the abundance and diversity of PAH ring-hydroxylating dioxygenase (PAH-RHD_α) genes (4, 8, 9, 13, 14, 22, 34, 37). Despite the known biases of PCR amplification from mixed templates, these techniques allow highly sensitive and specific detection even from minute amounts of nucleic acids. In order to select suitable primer systems, previously published primer systems were analyzed for their ranges of target sequences. The existing primer systems were found to have limitations, as they often target only a rather narrow range of sequences, e.g., nahAc- or phnAc-type sequences (21, 34) or only PAH-RHD_α genes from Gram-negative bacteria (3, 13). In other studies, two-primer systems were used to target PAH-RHD_α genes of both Gram-positive and Gram-negative bacteria (4, 37). Only one primer system targeting the Rieske gene fragment was described that amplified a small fragment from PAH-RHD_α genes from both Gram-negative and Gram-positive bacteria (24). However, the amplicon size was only 78 bp and the primer might also target genes coding for dioxygenases that attack nonpolar aromatic compounds, such as benzene, toluene, and xylene. Therefore, this work aimed to design an improved primer system that targets PAH-RHD_α genes from both Gram-positive and Gram-negative bacteria and provides larger amplicon sizes. The novel primer system was tested in silico and validated by sequencing cloned PAH-RHD_α genes amplified from total-community (TC) DNA and was used in endpoint and quantitative real-time PCR (qPCR) formats. The primer system was also applied to study the responses of soil microbial communities in two different soils (a Cambisol and a Luvisol representing typical arable soils in Central Europe with different texture compositions) to artificial phenanthrene pollution.