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  • Springer  (2)
  • 1
    ISSN: 1476-5535
    Keywords: Keywords: Sphingomonas; sphingolipid analysis; PCR; soil
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: 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 × 103 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 × 105 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 × 107cells 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.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    World journal of microbiology and biotechnology 13 (1997), S. 305-313 
    ISSN: 1573-0972
    Keywords: NMR ; pcpB ; pcpC ; pentachlorophenol degradation ; Pseudomonas spp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Eighty-nine bacterial isolates obtained by enrichment from pentachlorophenol (PCP)-contaminated soil samples were tested for PCP dechlorination activity and hybridization to pcpB (encoding PCP-4-monooxygenase) and pcpC (encoding tetrachlorohydroquinone reductive dehalogenase) gene probes synthesized by polymerase chain reaction from Flavobacterium sp. ATCC 39723 genomic DNA. Seven isolates were able to dechlorinate PCP, hybridize to both pcpB and pcpC DNA probes, and mineralize sodium pentachlorophenate (NaPCP) at an initial concentration of 100μg/ml. Although the seven PCP-mineralizing isolates possessed DNA sequences homologous to the Flavobacterium pcpB and pcpC genes, restriction analysis revealed sequence differences between the isolates and the Flavobacterium PCP dechlorination genes. Two isolates, designated UG25 and UG30, with the fastest onset and highest extent of PCP mineralization were selected for further study. Both isolates were tentatively identified as Pseudomonas spp. and exhibited stoichiometric release of Cl− ions as PCP was degraded. The release of Cl− began concomitantly with PCP disappearance from the medium. Both UG25 and UG30 degraded NaPCP at a concentration of 250 μg/ml in a minimal salt medium. Supplementation of the medium with glutamate increased the NaPCP degradation threshold of UG25 to a concentration of 300 μg/ml but did not affect that of UG30. 31P-NMR spectra of UG25 and UG30 cell suspensions exposed to PCP showed lower intracellular ATP levels and a more acidic cytoplasmic pH relative to untreated cells. This de-energization may explain the lack of cell growth in the presence of high PCP concentrations.
    Type of Medium: Electronic Resource
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