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Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences

Abstract

A species-specific PCR technique to detect an oil-degrading bacterium, Corynebacterium sp. IC10, released into sand microcosms is described. PCR primers, specific to strain IC10, were designed based on 16S rRNA gene sequences and tested against both closely and distantly related bacterial strains using four primer combinations involving two forward and two reverse primers. Two sets of them were specific to the strain IC10 and Corynebacterium variabilis and one set was selected for further analysis. The PCR amplification was able to detect 1 pg template DNA of strain IC10 and 1.2×104 c.f.u. of IC10 ml wet sand−1 in the presence of 3×108 Escherichia coli cells. In non-sterile sand microcosms seeded with the strain IC10, the sensitivity of detection decreased to 9.6×105 c.f.u. ml wet sand−1. The detection sensitivity thus depends on the complexity of background heterogeneous DNA of environmental samples. The assay is suitable for detection of Corynebacterium sp. IC10 in laboratory microcosms, however, cross reaction with non-oil degrading coryneforms may prohibit its use in uncharacterized systems.

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References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25: 3389–3402.

    Article  PubMed  Google Scholar 

  • Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identifi-cation and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: 143–169.

    PubMed  Google Scholar 

  • Bogardt AH, Hemmingsen BB (1992) Enumeration of phenanthrene-degrading bacteria by an overlayer technique and its use in evaluation of petroleum-contaminated sites. Appl. Environ. Microbiol. 58: 2579–2582.

    PubMed  Google Scholar 

  • Boivin-Jahns V, Bianchi A, Ruimy R, Garcin J, Daumas S, Christen R (1995) Comparison of phenotypical andmolecular methods for the identification of bacterial strains isolated from a deep subsurface environment. Appl. Environ. Microbiol. 61: 3400–3406.

    Google Scholar 

  • Felsenstein J (1993) PHYLIP (phylogeny inference package), Version 3.5. Seattle: University of Washington.

    Google Scholar 

  • Fogel GB, Collins CR, Li J, Brunk CF (1999) Prokaryotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population. Microbiol. Ecol. 38: 93–113.

    Google Scholar 

  • Giovannoni SK (1991) The polymerase chain reaction. In: Stackebrandt E, Goodfellow M, eds. Nucleic Acid Techniques in Bacterial Systematics. New York: John Wiley & Sons, pp. 177–201.

    Google Scholar 

  • Kim TH, Lee JH, Oh YS, Bae KS, Kim S-J (1999) Identification and characterization of an oil-degrading yeast, Yarrowia lipolytica 180. J. Microbiol. 37: 128–135.

    Google Scholar 

  • Ko SH, Lee HK, Kim SJ (1998) Effect of hydrocarbon uptake modes on oil degradation rate by mixed cultures of petroleum degraders. Kor. J. Biotechnol. Bioeng. 13: 606–614.

    Google Scholar 

  • Kuske CR, Banton KL, Adorada DL, Stark PC, Hill KK, Jackson PJ (1998) Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl. Environ. Microbiol. 64: 2463–2472.

    PubMed  Google Scholar 

  • Lee JH, Shin HH, Lee HK, Kwon KK, Kim S-J (1998) Efficient removal of humic substances in preparing DNA extract from marine sediments. Kor. J. Microbiol. 34: 132–136.

    Google Scholar 

  • Leser TD, Boye M, Hendriksen NB (1995) Survival and activity of Pseudomonas sp. strain B13 (FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton. Appl. Environ. Microbiol. 61: 1201–1207.

    PubMed  Google Scholar 

  • Lichtenstein AV, Moiseev VL, Zaboikin MM (1990) A procedure for DNA and RNA transfer to membrane filters avoiding weightinduced gel flattening. Anal. Biochem. 191: 187–191.

    PubMed  Google Scholar 

  • Maidak BL, Cole JR, Parker Jr CT, Garrity GM, Larsen N, Li B, Lilburn TG, McCaughey MJ, Olsen GJ, Overbeek R, Pramanik S, Schmidt TM, Tiedje JM, Woese CR (1999) A new version of the RDP (Ribosomal Database Project). Nucl. Acids Res. 27: 171–173.

    PubMed  Google Scholar 

  • Oh YS, Choi WY, Lee YH, Choi SC, Kim SJ (2000) Biological treatment of oil-contaminated sand: comparison of oil degradation based on thin-layer chromatography/flame ionization detector and respirometric analysis. Biotechnol. Lett. 22: 595–598.

    Google Scholar 

  • Shin SK, Oh YS, Kim S-J (1999) Biodegradation of phenanthrene by Sphingomonas sp. strain KH3–2. J. Microbiol. 37: 185–192.

    Google Scholar 

  • Tsai Y-L, Olson BH (1992) Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Appl. Environ. Microbiol. 58: 754–757.

    PubMed  Google Scholar 

  • Widmer F, Seidler RJ, Gillevet PM, Watrud LS, Giovanni GDD (1998) A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples. Appl. Environ. Microbiol. 64: 2545–2553.

    PubMed  Google Scholar 

  • Zhou J, Palumbo AV, Tiedje JM (1997) Sensitive detection of a novel class of toluene degrading denitrifiers, Azoarcus tolulyticus, with small-subunit rRNA primers and probes. Appl. Environ. Microbiol. 63: 2384–2390.

    PubMed  Google Scholar 

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Correspondence to Sang-Jin Kim.

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Lee, JH., Jung, SY. & Kim, SJ. Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences. Biotechnology Letters 23, 1741–1748 (2001). https://doi.org/10.1023/A:1012440232320

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  • DOI: https://doi.org/10.1023/A:1012440232320

  • 16S rDNA
  • Corynebacterium sp.
  • oil-degradation
  • species specific-PCR