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

Abstract

Sphingomonas spp possess unique abilities to degrade refractory contaminants and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amplification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingomonas aromaticivorans B0695R in three texturally distinct soil types, showing detection limits between 1.3-2.2 × 10 3 CFU g −1 dry soil. A sphingolipid extraction protocol was also developed to monitor Sphingomonas populations in soil quantitatively. The detection limit of the assay was 20 pmol g −1 dry soil, equivalent to about 3 × 10 5 cells g −1 dry soil. Survival of S. aromaticivorans B0695R was monitored in the three different soils by antibiotic selective plate counting, PCR and sphingolipid analysis. All three approaches showed that the B0695R cells persisted in the low biomass Sequatchie sub-soil at about 3-5 × 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 Sequatchie top-soil, indicating the possibility of the presence of viable but non-culturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas population in the environment.

Ethylbenzene,o-Xylene, and BTEX Removal bySphingomonas sp. D3K1 in Rock Wool-Compost Biofilters

“…Ethylbenzene was found to be degraded by a pure culture of Stenotrophomonas maltophilia T3-c (Lee et al, 2002). On the other hand, the genus Sphingomonas has been receiving a lot of attention due to its diverse metabolic capability, with many isolated members reported to possess broad catabolic capabilities towards recalcitrant organic pollutants including single-ring aromatic and polyaromatic hydrocarbons, chlorinated biphenyls, polychlorophenol, and halogenated diphenyl ethers; although limited literature is currently available on its direct use in biofilters (Fredrickson et al, 1995;Gabriel et al, 2005;Kim and Zylstra, 1999;Leung et al, 1999;Leys et al, 2004).…”

Abundance and diversity of Sphingomonas in Shenfu petroleum-wastewater irrigation zone, China

“…To our knowledge, two degenerate Sphingomonas genus-specific PCR primers (Leung et al 1999) have been designed for Sphingomonas identification, but they were improper to directly analyze the diversity of Sphingomonas species because they could not cover the total Sphingomonas genus. One nondegenerate Sphingomonas genus-specific primer set (Leys et al 2004) was not able to exclude the detection of other Sphingomonadaceae genera, and another non-degenerate Sphingomonas genus-specific primer set was only tested using sequence databases (Murakami et al 2010).…”

Linking of Microorganisms to Phenanthrene Metabolism in Soil by Analysis of 13 C-Labeled Cell Lipids

“…The 16S DNA primer Spf-190, specific for Sphingomonas sp. (26), and the universal 16S DNA primer Univ-16S-536-A-18 (P. A. Willumsen and B. M. Hansen, unpublished data) were used to identify isolates belonging to the genus Sphingomonas sensu lato. Identification of the Sphingomonas strains was further verified by PCR amplification with the 16S DNA primers Sphingo-108F and Sphingo-429R, which are specific for the genera Sphingobium, Novosphingobium, Sphingopyxis, and Sphingomonas belonging to the Sphingomodaceae family (N. Leys, A. Reyngaert, L. Bastiaens, E. Top, and D. Springael, unpublished data).…”