Development of a Technique for the Rapid Screening of Microbial Diversity in Environmental Samples
Background and Objectives
Microbial Ecology is the study of the interaction of microbes with the environment and with each other. Before the advent of highly sensitive analytical techniques for the detection of cellular components such as nucleic acids (DNA and/or RNA) and lipids, studies of microbial ecology were primarily accomplished by isolating organisms from the environment and studying their characteristics. Now, the extraction, amplification, and separation of the aforementioned cellular components is routine. However, the identification of even 100 microbes in a single sample of soil is a time-intensive process. Many of the revolutionary studies in microbial ecology have involved a single sample or a handful of samples from a small spatial area. While it is true that the distribution of microbes can vary on millimeter scales, microbes also influence their environment on larger scales. It is our proposition that the study of microbial diversity at the field scale will yield useful information about the synergistic relationship between microorganisms and their geochemical environment. This kind of information is particularly needed for studies of the mobility of potentially toxic trace metals in environments affected by mining activities. Studies of microbial ecology at the field scale will necessitate repeated sampling of several sites in a geographic area in order to monitor changes in the microbial populations from site to site and at a single site as a function of time. In order to perform these studies, techniques that lessen the processing time of a single sample are needed. The main objective of this task is to develop new high-throughput methods of screening samples for microbial diversity based on the chromatographic separation of amplified fragments of environmental DNA based on size and/or sequence. The instrument employed for these analyses is a denaturing gradient high-performance liquid chromatography (DHPLC) unit recently acquired by the USGS, and located in the Menlo Park laboratory.
Highlights and Key Findings
Several different types of methods under development, are being used in two internally-funded and one externally-funded projects. One method to be developed is the chromatographic separation of amplified DNA fragments on the basis of fragment size [Diagram below]. The length-heterogeneity PCR technique, currently specific only to eubacteria (i.e., will not detect archaea or eukaryotes) will supplant the current time-consuming method of agarose gel-based analysis of enzymatically digested DNA fragments. Although analysis of the LH-PCR fragments by DHPLC has advantages over analysis on a DNA sequencer, both in terms of costs (the capital outlay for a sequencer is at least twice that of a DHPLC) and in terms of time: because the amplified DNA fragments must be fluorescently labeled for analysis by sequencer, the analysis must be repeated without the label for use in sequencing. In the case of DHPLC, the fragments can be collected directly, concentrated, and sent for sequencing. Other methods being developed rely on the chromatographic separation of same-sized amplified DNA fragments on the basis of fragment sequence. One method under development produces fragments from all types of eubacteria, and another method is selective for sulfate-reducing bacteria. The methods under development in this subtask are being applied in studies linked to scientific activities in other USGS-funded projects. Several of these methods will allow the analysis of over 30 samples from sulfide-oxidizing microbial communities in Alaska (Modeling and Mineralized Systems Project, Task 5.4). The method for evaluating the composition of sulfate-reducing microbial communities is being applied to studies of mercury methylation at sites impacted by historic gold (Au) dredge and hydraulic mining in Northern California. Sulfate-reducing bacteria are currently recognized as the primary agents of mercury methylation (Pathways Project, Tasks 2 and 4). Methods developed under this task will also be applied in a study of arsenic-accumulating microbial mats from a lode-gold mining area in the Sierra Foothills, California (IAG between EPA and USGS).
Comparison of Techniques and Methods for Analysis of Community DNA: an Example of a 3-Species Mixture. [Return to text above]
Abstracts, Presentations, and Posters for Professional Technical Meetings
Ashley R.P., Rytuba, J.J., and Foster, A.L., 2005, Minimizing mercury release and methylation in river restoration projects in a watershed impacted by historic placer gold dredge and hydraulic mining: CALFED Annual Science Meeting, Sacramento, California, December, 2005. (Poster)
Foster, A.L., Munk, L., Koski R.A., Shanks, W. C., and Stillings, L.L., 2005, Microbial communities in mine-impacted environments of Prince William Sound, Alaska [abs.]: Geological Society of America 2005 Annual Meeting, Salt Lake City, Utah, October 16-19, 2005, Abstracts with Programs, v. 37, no. 7, p. 49. (Abstract and presentation) Available online at http://gsa.confex.com/gsa/2005AM/finalprogram/abstract_93255.htm.
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