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Applied and Environmental Microbiology, April 2004, p. 1944-1955, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.1944-1955.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Occurrence and Phylogenetic Diversity of Sphingomonas Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons

Natalie M. E. J. Leys,1,2,{dagger} Annemie Ryngaert,1 Leen Bastiaens,1 Willy Verstraete,2 Eva M. Top,2,{ddagger} and Dirk Springael1,3*

Environmental Technology, Flemish Institute for Technological Research, 2400 Mol,1 Laboratory of Microbial Ecology and Technology, University of Ghent, 9000 Ghent,2 Laboratory for Soil and Water Management, Catholic University of Leuven, 3001 Heverlee, Belgium3

Received 3 September 2003/ Accepted 5 December 2003

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 104 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.

* Corresponding author. Present address: Catholic University of Leuven (KUL), Laboratory for Soil and Water Management, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium. Phone: 32 (16) 321604. Fax: 32 (16) 321997. E-mail: dirk.springael{at}agr.kuleuven.ac.be.

{dagger} Present address: Belgian Nuclear Research Centre (SCK/CEN), Laboratory of Microbiology, 2400 Mol, Belgium.

{ddagger} Present address: University of Idaho, Department of Biological Sciences, Moscow, ID 83844-3051.

Applied and Environmental Microbiology, April 2004, p. 1944-1955, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.1944-1955.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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