Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil

Gejiao Wang, Terry J. Gentry, Gregor Grass, Karen Josephson, Christopher Rensing, Ian L. Pepper

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted.

Original languageEnglish (US)
Pages (from-to)307-314
Number of pages8
JournalFEMS Microbiology Letters
Volume233
Issue number2
DOIs
StatePublished - Apr 15 2004

Keywords

  • Bioaugmentation
  • Chlorobenzoate
  • GFP
  • Plasmid
  • Real-time PCR
  • cbdA

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Genetics

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