Isolation and Characterization of Mobile Genetic Elements from Microbial Assemblages Obtained from the Field Research Center Site
Considerable knowledge has been gained from the intensive study of a relatively limited group of bacterial plasmids. Recent efforts have begun to focus on the characterization of, at the molecular level, plasmid populations and associated mobile genetic elements (e.g., transposons, integrons) occurring in a wider range of aquatic and terrestrial habitats. Surprisingly, however, little information is available regarding the incidence and distribution of mobile genetic elements extant in contaminated subsurface environments. Such studies will provide greater knowledge on the ecology of plasmids and their contributions to the genetic plasticity (and adaptation) of naturally occurring subsurface microbial communities. We requested soil cores from the DOE NABIR Field Research Center (FRC) located on the Oak Ridge Reservation. The cores, received in February 2003, were sampled from four areas on the Oak Ridge Site: Area 1, Area 2, Area 3 (representing contaminated subsurface locales) and the background reference sites. The average core length (24 in) was subdivided into three profiles and soil pH and moisture content were determined. Uranium concentration was also determined in bulk samples. Replicate aliquots were fixed for total cell counts and for bacterial isolation. Four different isolation media were used to culture aerobic and facultative microbes from these four study areas. Colony forming units ranged from a minimum of 100 per gram soil to a maximum of 10,000 irrespective of media composition used. The vast majority of cultured subsurface isolates were gram-positive isolates and plasmid characterization was conducted per methods routinely used in the Sobecky laboratory. The percentage of plasmid incidence ranged from 10% to 60% of all isolates tested. This frequency appears to be somewhat higher than the incidence of plasmids we have observed in other habitats and we are increasing the number of isolates screened to confirm this observation. We are also characterizing the plasmid populations at the molecular level. Isolates cultured from the background control site exhibited the lowest occurrence of plasmids (10%). Aliquots of samples were also used in enrichment assays to isolate metal resistant subsurface isolates. Samples were subjected to three different metals (chromium, mercury and cadmium) at two different concentrations and incubated following a conditioning period in which samples were amended with a carbon, nitrogen and phosphorus source. Isolates were plated on metal selection, purified to single isolates and plasmid content determined.
- Research Organization:
- Georgia Institute of Technology, Atlanta, GA; Rutgers University, New Brunswick, NJ
- Sponsoring Organization:
- USDOE Office of Science (SC)
- OSTI ID:
- 895496
- Report Number(s):
- CONF-NABIR2004-45; TRN: US200702%%886
- Resource Relation:
- Conference: Annual NABIR PI Meeting, March 15-17, 2004, Warrenton, VA
- Country of Publication:
- United States
- Language:
- English
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