Research Product

Plasmid pSI30 was constructed to increase the sensitivity of detection of a genetically engineered microorganism (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. In filter-sterilized water, numbers of viable P. putida cells declined by less than one log in 64 days. In natural freshwater, numbers of P. putida cells fell rapidly over the first 5 days and then slowly declined to cell densities of approximately 100 per ml. 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. The addition of 3-chlorobenzoate to natural freshwater containing as few as 3 RC-4 (pSI30) cells per ml resulted in an increase in their numbers. In flow-through microcosms RC-4 (pSI30), undetectable as freeliving 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.