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

Abstract

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.

Transfer and Expression of the Catabolic Plasmid pBRC60 in Wild Bacterial Recipients in a Freshwater Ecosystem

“…FL medium composition was based on the nitrogen and dissolved inorganic carbon levels found in Lake Opinicon and on inorganic ion concentrations typical for hardwater lakes (29). It contained 1.25 mM CaCO3, 650 ,uM MgCl2, 300 ,IM MgSO4,450 ,uM NaHCO3, 10 ,uM NaNO3, 5 ,uM (NH4)2SO4, 1 ,uM…”

“…The rate of uptake of 3Cba and the subsequent production of CO2 by pBRC60 hosts were determined by using L-[U-'4C]3Cba (specific activity, 20.3 mCi/ mmol; Sigma Radiochemicals). Measurements were carried out on batch cultures of FL medium amended with 50 FM 3Cba or on samples taken from 10 ,uM 3Cba; FL medium continuous cultures were grown at a dilution rate of 0.01 h-1. All continuous cultures were inoculated with a batch culture of the appropriate species grown in 3Cba or 3Cba/ye and left to equilibrate for at least three volume changes (12 days) before uptake and carbon dioxide production measurements were made.…”

Distribution of the catabolic transposon Tn5271 in a groundwater bioremediation system

“…Very little is known concerning the distribution of this catabolic genotype in contaminated sediments or freshwater. A preliminary study employing colony hybridizations with a pBRC60 probe indicated that elevated numbers of probe-positive colonies could be recovered from samples from Bloody Run Creek relative to samples from an uncontaminated control creek (5). However, because a whole-plasmid probe was used, the results could be interpreted as an increased incidence of IncP,B plasmids similar to pBRC60 in the samples.…”

“…strain DON2 (30), Alcaligenes sp. strain H850 (General Electric Co., Schenectady, N.Y.), and Comamonas acidovorans NR4 (5). In addition, cultures representing four pBRC60 recipient groups isolated following the transfer of pBRC60 from Alcaligenes sp.…”

“…7H20, 0.22 mM 3-chlorobenzoate, 75 ,uM EDTA, 25 ,uM FeSO4. 7H20, 25 ,uM toluene, 17 ,uM CUSO4 5H20, 6 puM Pb (CH3COO) 3H20, 6 ,uM 2,4-dichlorophenol, 5 ,uM MnSO4* 4H20, 1 ,uM NaMoO4 2H20, and 0.05% yeast extract, pH 7.2. The phenols and toluene were added to the sterile medium after autoclaving.…”