Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain.
Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered micro-organism (GEM) and its recombinant DNA in environmental samples. This broad host-range, mobilizable plasmid contained chlorocatechol (clc) degradative genes, antibiotic resistance genes (ampicillin and kanamycin) and a fragment of eukaryotic DNA. The clc genes encode enzymes that convert 3-chlorocatechol to maleylacetic acid permitting the host, Pseudomonas putida RC-4, to grow on 3-chlorobenzoate. This catabolic phenotype was exploited using enrichment procedures to detect RC-4(pSI30) cells, free-living in the water column or when irreversibly bound to surfaces. The eukaryotic DNA sequence provided a unique target allowing positive identification by DNA:DNA hybridization. Using the eukaryotic DNA sequence as a probe, no transfer of the plasmid to indigenous bacteria was detected. Persistence of RC-4(pSI30) and its ability to multiply upon addition of 3-chlorobenzoate were demonstrated 78 days after its addition to natural freshwater. In flow-through microcosms RC-4(pSI30), undetectable as free-living cells, was found by enrichment as irreversibly bound sessile forms. These experiments revealed the stability of pSI30 and its utility in a 'combination' detection system for tracking the survival of a GEM and its DNA in environmental samples.
Genthner
F J
FJ
US Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL 32561.
Campbell
R P
RP
Pritchard
P H
PH
D001673:Biodegradation, Environmental; D002723:Chlorobenzoates / Q000378:metabolism; D015342:DNA Probes; D004274:DNA, Recombinant / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D017753:Ecosystem; D005618:Fresh Water; D005818:Genetic Engineering / Q000009:adverse effects; D010957:Plasmids / Q000235:genetics*; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D014871:Water Microbiology
D002723:Chlorobenzoates; D015342:DNA Probes; D004274:DNA, Recombinant; C036427:3-chlorobenzoic acid