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Use of a novel plasmid to monitor the fate of a genetically engineered Pseudomonas putida strain
Author(s) -
GENTHNER F. J.,
CAMPBELL R. P.,
PRITCHARD P. H.
Publication year - 1992
Publication title -
molecular ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.619
H-Index - 225
eISSN - 1365-294X
pISSN - 0962-1083
DOI - 10.1111/j.1365-294x.1992.tb00169.x
Subject(s) - biology , pseudomonas putida , plasmid , dna , kanamycin , gene , plasmid preparation , pseudomonas , bacteria , recombinant dna , microbiology and biotechnology , escherichia coli , amp resistance , genetics , pbr322
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, freeliving 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.