PCR AMPLIFICATION OF CATECHOL 2,3-DIOXYGENASE GENE-SEQUENCES FROM NATURALLY-OCCURRING HYDROCARBON-DEGRADING BACTERIA ISOLATED FROM PETROLEUM HYDROCARBON CONTAMINATED GROUNDWATER

Polymerase chain reaction amplification was used to detect catechol 2, 3-dioxygenase (CD) gene sequences in the native bacterial populations present in gasoline contaminated groundwater samples. The primers used for the PCR assay were selected from the DNA sequences of the conserv ed region of the Cd gene of xyIE, nahH, and pAW313. A 30 bp DNA sequen ce (ICP313) internal to the structural gene of CD was used as a probe to identify the amplified DNA fragment. The identity of the amplified DNA fragment was confirmed by digesting the DNA fragment with StuI and Bg/I restriction enzymes which produced DNA fragments of expected siz es. The specificity of the PCR assay was tested using DNA isolated fro m a variety of bacterial isolates which include Escherichia coli ATCC 25922, Enterobacter faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 2 7853, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 292 13, Micrococcus roseus ATCC 516, Serratia marcescens ATCC 60, Klebsiel la pneumoniae ATCC 13883, Chryseobacterium gleum ATCC 29896, Chromatiu m vinosum ATCC 17899, Comamonas testosteroni ATCC 11996, Methylosinus trichosporium OB3b, Bacillus subtilis ATCC 21697, Alcaligenes eutrophu s ATCC 17698, Arthrobacter globiniformis ATCC 35698, Pseudomonas putid a mt-2 ATCC 33015, Acinetobacter calcoaceticus ATCC 31012, and Pseudom onas putida ATCC 17484. Amplification of DNA was not observed in bacte rial isolates lacking CD activity. Our primers amplified CD gene seque nces in naturally occurring hydrocarbon degrading bacteria isolated fr om groundwater contaminated with gasoline. The detection limit of the PCR amplification was 10(-2) cells of Escherichia coli (pAW313) per ml . A previously characterized hydrocarbon degrader Pseudomonas putida O U83 was used as a positive control. The usefulness of PCR amplificatio n for the detection of CD specific,genotypes in the environmental samp les was verified using DNA directly extracted fi om gasoline contamina ted groundwater samples as a template. A CD gene specific DNA fragment was amplified from 5 out of 6 gasoline contaminated groundwater sampl es. The identity and specificity of the PCR amplified DNA generated us ing the DNA directly extracted from the gasoline contaminated groundwa ter as template was confirmed by restriction enzyme digestion and Sout hern hybridization using the 30 bp internal probe (ICP313). Based on t he evidence presented in this study, we report for the first time, use fulness of PCR amplification for the detection of CD gene sequences in gasoline contaminated groundwater.