Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons

• Published in: Applied and Environmental Microbiology Vol. 70; no. 4; pp. 1944 - 1955
• Main Authors: Leys, N.M.E.J, Ryngaert, A, Bastiaens, L, Verstraete, W, Top, E.M, Springael, D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-04-2004
• Subjects: Bacteria; Base Sequence; Biodegradation, Environmental; Biological and medical sciences; denaturing gradient gel electrophoresis; DNA primers; DNA Primers - genetics; DNA, Bacterial - genetics; Environmental Microbiology; Fundamental and applied biological sciences. Psychology; gel electrophoresis; genes; Genetic diversity; Microbiology; Molecular Sequence Data; nucleotide sequences; phylogeny; polluted soils; Polycyclic aromatic hydrocarbons; Polycyclic Aromatic Hydrocarbons - metabolism; Polymerase Chain Reaction; Quantitative genetics; ribosomal RNA; RNA, Bacterial - genetics; RNA, Ribosomal, 16S - genetics; Sequence Homology, Nucleic Acid; soil bacteria; Soil Microbiology; Soil Pollutants - metabolism; soil pollution; species diversity; Sphingomonas; Sphingomonas - classification; Sphingomonas - genetics; Sphingomonas - isolation & purification; Sphingomonas - metabolism; strains; Hydrocarbon; Diversity; Bacteria; Sphingomonas; Phylogeny; Polycyclic aromatic compound; Soil pollution
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.70.4.1944-1955.2004

Bacterial strains of the genus Sphingomonas are 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 of Sphingomonas strains 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 the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity 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 family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains 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 10(4) CFU g of soil-1. The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences 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 designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.