Occurrence and Phylogenetic Diversity ofSphingomonasStrains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons

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Abstract
Bacterial strains of the genusSphingomonasare often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection ofSphingomonasstrains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting theSphingomonas16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assessSphingomonasdiversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the familySphingomonadaceae. Single-band DGGE profiles were obtained for mostSphingomonasstrains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 104CFU g of soil−1. The analysis ofSphingomonaspopulation structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowestSphingomonasdiversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA geneSphingomonassequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designedSphingomonas-specific PCR-DGGE detection technique successfully analyzed theSphingomonascommunities from polluted soils at the species level and revealed differentSphingomonasmembers not previously detected by culture-dependent detection techniques.

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