Open AccessReal‐time PCR quantification of a green fluorescent protein‐labeled, genetically engineered Pseudomonas putida strain during 2‐chlorobenzoate degradation in soil
Author(s) -
Wang Gejiao,
Gentry Terry J,
Grass Gregor,
Josephson Karen,
Rensing Christopher,
Pepper Ian L
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2004.tb09497.x
Subject(s) - pseudomonas putida , strain (injury) , genetically engineered , degradation (telecommunications) , green fluorescent protein , pseudomonas , pseudomonadales , bacterial strain , pseudomonadaceae , fluorescence , chemistry , biodegradation , genetically modified organism , microbiology and biotechnology , real time polymerase chain reaction , biology , bacteria , biochemistry , gene , genetics , telecommunications , physics , quantum mechanics , computer science , organic chemistry , anatomy
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.