Culture independent detection of Sphingomonas sp. EPA 505 related strains in soils contaminated with polycyclic aromatic hydrocarbons (PAHs)

Abstract

The Sphingomonas genus hosts many interesting pollutant-degrading strains. Sphingomonas sp. EPA505 is the best studied polycyclic aromatic hydrocarbon (PAH)-degrading Sphingomonas strain. Based on 16S rRNA gene sequence analysis, Sphingomonas sp. strain EPA505 forms a separate branch in the Sphingomonas phylogenetic tree grouping exclusively PAH-degrading isolates. For specific PCR detection and monitoring of Sphingomonas sp. EPA505 and related strains in PAH-contaminated soils, a new 16S rRNA gene-based primer set was designed. The new primer set was shown to be highly selective for Sphingomonas sp. strain EPA505 as it only amplified DNA from strain EPA505 and not from other tested Sphingomonas strains or soil bacteria not belonging to the Sphingomonas genus. Using DNA extracts of a variety of inoculated PAH-contaminated soils, the primer pair was able to detect EPA505 in concentrations as low as 10(2) cells per gram of soil. Applying the new primer set, 16S rRNA gene fragments which were 99-100% similar to the corresponding gene of strain EPA505 were amplified from four of five PAH-contaminated soils. On the other hand, no PCR products were obtained from any of five tested uncontaminated soils. The preferential presence of EPA505 related Sphingomonas strains in PAH-contaminated soils with very different contamination profiles and different origin suggests an important role of this type of Sphingomonas in the natural Sphingomonas community colonizing PAH-contaminated sites.