Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis

Sphingomonas spp possess unique abilities to degrade refractory contaminant s and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amp lification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingom onas aromaticivorans B0695R in three texturally distinct soil types, showin g detection limits between 1.3-2.2 x 10(3) CFU g(-1) dry soil. A sphingolip id extraction protocol was also developed to monitor Sphingomonas populatio ns in soil quantitatively, The detection limit of the assay was 20 pmol g(- 1) dry soil, equivalent to about 3 x 10(5) cells g(-1) dry soil. Survival o f S. aromaticivorans B0695R was monitored in the three different soils by a ntibiotic selective plate counting, PCR and sphingolipid analysis. All thre e approaches showed that the B0695R cells persisted in the low biomass Sequ atchie sub-soil at about 3-5 x 10(7) cells g(-1) dry soil. In comparison to the plate counting assay, both the PCR and sphingolipid analysis detected a significantly higher level of B0695R cells in the clay soil and Sequatchi e top-soil, indicating the possibility of the presence of viable but non-cu lturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas populatio n in the environment.