Bacterial Adaptation to Chlorobenzoate Contamination in the Niagara Region Investigated by DNA:DNA Colony Hybridization

Chloroaromatic biodegradation was studied in samples of water and the sediment-water interface of Bloody Run Creek, a tributary of the Niagara River with a source adjacent to the Hyde Park Industrial Landfill in New York State. Alcaligenes spp. were found which metabolized chlorobenzoates by virtue of an 85 kilobase plasmid, pBR60. These isolates were obtained following continuous culture enrichment of populations from the sediment-water interface, selecting isolates for their metabolism of 3-chlorobenzoate as a carbon and energy source. To determine whether plasmid deoxyribonucleic acid (DNA) sequences were present in a significant number of organisms in the water and surface sediments of the creek, samples were diluted and spread on nitrocellulose niters overlying solid media containing chlorobenzoates and traces of yeast extract. A control creek with a similar superficial geology but lacking contaminants was also sampled. Colony hybridization with phosphorus-32 (³²P)-labeled pBR60 revealed differences between samples from the two creeks in both the number of positive signals and the signal strength. Isolates, obtained from the creek water and surface sediments, which were capable of growth on 3-chlorobenzoate were screened by dot-blot hybridization using a specific probe, derived from pBR60, which detected sequences involved in chlorobenzoate catabolism. Evidence for the mobility of the plasmid between Alcaligenes and Pseudomonas species was also obtained. Technical problems with this approach, alternative methods, and applications are discussed.