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

Abstract

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.

FIG. 1.

Sphingomonas species differentiation by DGGE analysis of DNA fragments amplified with primers Sphingo108f/GC40 and Sphingo420r. The separate lanes represent the different species-specific DGGE melting profiles of different tested Sphingomonas strains. Lanes: 1, Sphingomonas sp. strain VM0506; 2, Sphingomonas sp. strain LB126; 3, S. macrogolitabida DSM8826^T; 4, S. natatoria DSM3183^T; 5, S. mali DSM10565^T; 6, S. terrae DSM8831^T; 7, S. yanoikuyae DSM7462^T; 8, S. suberifaciens DSM7465^T; 9, S. asaccharolytica DSM10564^T; 10, S. pruni DSM10566^T; 11, S. capsulata DSM30196^T; 12, S. rosa DSM7285^T; 13, S. aromaticivorans DSM12444^T; 14, S. xenophaga DSM6383^T; 15, Zymomonas mobilis LMG448^T; 16, Erythrobacter litoralis DSM8509^T; 17, Sphingomonas sp. strain LH227; 18, S. wittichii DSM6014^T; 19, Sphingomonas sp. strain EPA505; 20, S. paucimobilis DSM1098^T; 21, Sphingomonas sp. strain LH128; 22, S. subarctica DSM10700^T; 23, S. subarctica DSM10699; 24, S. paucimobilis LMG2239; 25, S. parapaucimobilis DSM7463^T; 26, S. sanguis LMG2240; 27, S. trueperi DSM7225^T; 28, S. flava DSM6824; 29, S. adhaesiva DSM7418^T. Lanes were ordered with Bionumerics software to group and compare several DGGE profiles.

FIG. 2.

DGGE analyses of indigenous Sphingomonas communities in natural soil samples using primers Sphingo108f and GC40-Sphingo420r in PCR. The separate lanes indicate the DGGE fingerprints of the indigenous Sphingomonas community of PAH-contaminated soils AndE, Barl, TM, B101, and K3840. Cloned bands are indicated within the soil fingerprint based on the comparison of migration profiles of pure clones and the soil profile. A mixture of six strains was used as a marker during DGGE analysis.

FIG. 3.

Phylogenetic analyses of Sphingomonas sequences retrieved from soil DNA extract with primers Sphingo108f and GC40-Sphingo420r in PCR. The phylogenetic relationships of cloned sequences are indicated in a character-based evolutionary tree based on the total length of the 16S rRNA gene sequences and constructed using the neighbor-joining algorithm. An out-group of the closely related genera Rhizobium andRhodospirillum was included to root the tree. The bar at the top indicates the percent similarity, with 1% indicating 1 nucleotide substitution per 100 positions. The tree was tested for branching order confidence by maximum-parsimony analysis and a round of 500 bootstraps. Bootstrap values are indicated at branch points, and values above 70% indicate reliable branches. Extended branches were collapsed to form smaller blocks. Most important representative strains are indicated per block, with the accession numbers of the sequences indicated between parentheses. Species harboring PAH-degrading isolates are indicated with an asterisk. The positions of the clone sequences retrieved from soil are indicated on the right of the tree. Species are grouped based on their 16S rRNA gene sequence similarity. Species groups resembled the clustering previously described by Takeuchi et al. (46), who divided the Sphingomonas genus into four new genera based on the 16S rRNA gene dendrogram. Later, this division of the Sphingomonas genus was reconsidered by Yabuuchi et al. (54) due to the lack of phenotypic and biochemical evidence. The clusters in the figure indicated as I to IV represent the phylogenetic clusters previously assigned to the genera “Sphingomonas sensu stricto,” “Sphingobium,” “Novosphingobium,” and “Sphingopyxis,” respectively (46).