Real-time PCR quantification of a green fluorescent protein-labeled, genetically engineered Pseudomonas putida strain during 2-chlorobenzoate degradation in soil.
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.
Wang
Gejiao
G
Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ 85721, USA.
Gentry
Terry J
TJ
Grass
Gregor
G
Josephson
Karen
K
Rensing
Christopher
C
Pepper
Ian L
IL
D002723:Chlorobenzoates / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D012989:Soil Pollutants / Q000378:metabolism*
D002723:Chlorobenzoates; D008164:Luminescent Proteins; D012989:Soil Pollutants; D049452:Green Fluorescent Proteins; C060415:2-chlorobenzoic acid