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| Project Identification - PMIS 86662 | |
| Project Title: Determine Cave Microbial Diversity | |
| Park/Unit: Timpanogos Cave National Monument | Region: Intermountain |
| States: UT | Congressional District: 03 |
| Old Package Number: | Reference Number: |
| Project Type: Undefined | Financial System Package Number: TICA 086662 |
| Contact Person: Mike Gosse | Contact Phone: 801-756-5239 |
| Project Status - PMIS 86662 | |
| Date Created: 03/11/02 | Review Status: Region-Reviewed on 04/29/2002 |
| Date of Last Update: 03/14/02 | Updated By: Tica (Tica) |
| Project Narratives - PMIS 86662 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Description Abstract A comprehensive survey of microbial species in the Timpanogos Cave system is needed to understand their role in the cave�s ecology. The Biotechnology Center at Western Kentucky University has developed a "DNA fingerprinting" technique that allows many different microorganisms to be surveyed in a single sample. The growing database of DNA sequence and phylogenetic information along with fragment sizes from the cave clone database provides a means for recognizing and monitoring bacterial species in cave sediments, without the need to isolate and culture the organisms. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Justifications Problem Statement A comprehensive survey of microbial species in the Timpanogos Cave system is needed to understand their role in the cave�s ecology. It is known that microbes play a vital role in acid production, which breaks down limestone caves over time. Microbes are also a critical indicator species for the presence of human waste, sewage, and petroleum in caves. The time consuming practice of culturing organisms from the environment has had limited success for only a few species, and most organisms cannot be grown in a lab. The Biotechnology Center at Western Kentucky University has developed a "DNA fingerprinting" technique that allows many different microorganisms to be surveyed in a single sample. The technique extracts DNA directly from cave sediments and amplifies bacterial 16S rDNA using the polymerase chain reaction (PCR) with specific primers. Genetic libraries of bacterial 16S rDNA have been generated, and cloned 16S rDNA sequences from cave bacteria have been analyzed by DNA sequencing and fragment analysis. Species are being identified or taxonomically classified by phylogenetic sequence analysis and comparison to electronic nucleic acid databases, and characteristic fluorescent restriction fragment lengths have been tabulated for cloned or cultured cave bacterial 16S rDNA and standards. The 16S rDNA sequence and fragment database constitutes a reference to which DNA profiles of cave sediment bacterial communities can be compared. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Measurable
Results Methods Genetic identification of environmental strains. Using modern DNA technology, bacteria can be identified and classified according to the sequences of their genes encoding 16S ribosomal RNA (16S rDNA). Different species of bacteria possess characteristic 16S rDNA sequences. Bacterial 16S rDNA sequences may be selectively amplified from the mixture of DNA fragments extracted from the environment to create many copies for more detailed studies. With this technique, bacterial species can be identified and their genetic relationships can be determined without the need to culture individual strains in the laboratory. Furthermore, environmental bacteria that cannot be grown in the laboratory can still be detected by the presence of 16S rDNA (Siering, 1998; Angert et al. 1998; Holmes et al. 2001). Sampling and DNA extraction. Sediment from Timpanogos Cave is scooped wearing latex gloves into sterile centrifuge tubes and kept on ice shipped to the BioTechnology Lab at Western Kentucky University. The DNA is extracted from 1 gram of cave sediment using a simplified procedure, and the mixed environmental nucleic acids are visualized by agarose gel electrophoresis. Cave sediment contains many microorganisms, including bacteria, fungi, protozoans, and even larger cave invertebrates with small particles of dead plant and animal material. All of these things contribute to the mixture of DNA fragments that can be extracted directly from cave sediment. Amplification of 16S rDNA. To study the DNA of cave bacteria among all the DNA fragments present, specific DNA sequences are amplified out of the mixture using the polymerase chain reaction (PCR) with specific taxonomic primers. For the test study that focuses on the bacterial community, primers 27f and 1492r are used(Lane, 1991; Layton et al. 1994). Cloning and Sequencing. The amplified 16S rDNA are spliced into a cloning and sequencing vector plasmid DNA. The circular recombinant plasmid molecules thus produced are used to transform E. coli for studies of individual copies of the environmental genes. A cave clone library of E. coli host cells carrying cave DNA sequences was created that can be compared to other cave samples. Other clones on file in the library are related to Gram positive species, Planctomycetes, and various uncharacterized bacteria commonly found soils. Some of the library clone matches can raise ecological red flags by indicating the presence of bacteria that derive energy through biodegradation of petroleum, creosote, heavy metals, or sewage. Fragment Analysis. Rather than commit to cloning and sequencing from every cave sample examined, a snapshot of bacterial diversity can be generated easily and quickly for a larger number of samples by terminal restriction fragment length polymorphism (TRFLP) analysis. Snapshots from environmental samples depict multiple types of bacteria within the community in a given sediment sample, and the profile generated is a �fingerprint� with information about the microorganisms present and their relative abundance. Budget
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Rating Criteria
Significance of the Resource or Issue to the
Park: The Timpanogos Cave system is the monument's primary resource and subject of enabling legislation. The caves were set aside by presidential proclamation because of "their scientific interests and importance". This project initiates gaining a better understanding to the role that microbes play in Timpanogos Cave system. (Score Value=5) (Weight=1) Total 5 x 1 = 5 Severity & Urgency of Resource Threat, Problem, or
Need: This project will measure the health of the cave ecosystem. Currently, the extent of the cave watershed extends beyond the boundary of the monument. Grazing, camping, ATV and off-road vehicle use, and wildfire activity all occurs within the known cave watershed. The scientific study of microbes is making great discoveries that are impacting the whole world. New genera of bacteria capable of expressing genes with medical and practical applications have been discovered and are now the focus of many cave microbial studies (Angert et al.1998; Holmes et al. 2001; Northtup et al. 2000). This project may also lead to a vital sign indicator to characterize the health of the cave ecosystem at its most basic level. Any delay may lead to the extinction of some unknown cave species that contains DNA for unique medical properties or research. (Score Value=5) (Weight=1) Total 5 x 1 = 5 Problem Resolution: This project will provide a snapshot of the microbial species present in the Timpanogos Cave system. If these species indicate the presence of E. coli from human waste or sewage, petroleum products, or large carbon particles then specific management actions will be prescribed to restrict activities occuring within the cave watershed, and protect the fragile non-renewable cave resources. Future research on the caves microbes will also produce a larger DNA sequence and phylogenetic database that can be described in even greater detail. (Score Value=5) (Weight=1) Total 5 x 1 = 5 Scientific Resource Management: References Amann, R.I., Stromely, J., Devereux, R., Key, R. & Stahl D.A. (1992). Molecular and Microscopic Identification of Sulfate-Reducing Bacteria in Multispecies Biofilms. Appl. Env. Microbiol. 58(2): 614-623. Angert, E.R., Northrup, D.E., Resenbach, A-L., Peek, A.S., Goebel, B.M. & Pace, N.R. (1998). Molecular analysis of a bacterial community in Sulphur River, Parker Cave, Kentucky. American Mineralogist 83: 1583-1592. Elliott, L., Wright, S., Coakley, T. & Groves, C. (2000). Microbial Ecology of Conduit Stream Sediment Interstitial Fluids of the South Central Kentucky Karst Aquifer: Impacts on Aquifer Development, in Proceedings of Mammoth Cave National Park�s Eighth Science Conference, Mammoth Cave, Kentucky, pp 57-60. Holmes, A.J., Tujula, N.A., Holley, M., Contos, A., James, J.M., Rogers, P. & Gillings, M.R. (2001). Phylogenetic structure of unusual aquatic microbial formations in Nullarbor caves, Australia. Environmental Microbiology 3(4): 256-264. Hugenholtz, P., Pitulle, C., Hershberger, K.L. & Pace, N.R. (1998). Novel Division level bacterial diversity in a Yellowstone hot spring. J. Bacteriology 180(2): 366-376. Lane, D.J. (1991). 16S/23S rRNA Sequencing, in Nucleic Acid Techniques in Bacterial Systematics, E. Stackenbrandt and M. Goodfellow (eds), John Wiley and Sons, Inc. New York, pp115-148. Layton, A.C., Lajoie, C.A., Easter, J.P., Jernigan, R., Sansaverino, J.& Sayler, G.S. (1994). Molecular Diagnostics and Chemical Analysis for Assessing Biodegradation of Polychlorinated Biphenyls in Contaminated Soils, J. Indust. Microbiol. 13: 392-401. Minz, D., Fishbain, S., Green, S.J., Muyzer, G., Cohen Y., Rittman, B. & Stahl, D.A. (1999). Unexpected Poplulation Distribution in a Microbial Mat Community: Sulfate-Reducing Bacteria Localized to the Highly Oxic Chemocline in Contrast to a Eukaryotic Preference for Anoxia, Appl. Env. Microbiol. 65(10): 4659-4665. Moyer, C.L., Dobbs, F.C. & Karl, D.M. (1994). Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active hydrothermal vent system, Loihi Seamount, Hawaii, Appl. Env. Microbiol. 60(3): 871-879. Northrup, D.E., Dahm, C.N., Melim, L.A., Spilde, M.N., Crossey, L.J., Lavoie K.H., Mallory, L.M., Boston, P.J., Cunningham, K.I. & Barns, S.M. (2000). Evidence for geomicrobiological interactions in Guadalupe caves. Journal of Cave and Karst Studies 62(2): 80-90. Rusterholtz, K.J. & Mallory, L.M. (1994). Density, Activity, and Diversity of Bacteria Indigenous to a Karstic Aquifer. Microbial Ecology 28:79-99. Siering, P.L. (1998). The double helix meets the crystal lattice: The power and pitfalls of nucleic acid approaches for biomineralogical investigations. American Mineralogist 83: 1593-1607 (The following text was truncated from the measurable results block in November 2002, when the measurable results field was shortened in the database): style='width:.75in;border:solid windowtext 1.0pt; border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt: solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt'> Cost | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
DNA tests for 25
samples -
$85/sample (DNA extraction,
TRFLP restriction digest, interpretation) |
$
2,125 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Water chemistry
analysis for 6 samples per quarter @
$580/sample ($300
Anion/cation, $50 carbon analysis, $200 pescides, $30 Fecal/E.Coli
count) |
$
3,480 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Personnel
Support � RM Specialist GS-07 for 4 pp @
$1365/pp (Sample
collection, additional interpretation, and report
writing) |
$
4,095 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Supplies (Centrifuge
tubes, cooler, shipping) |
$
240 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Total |
$
9,940 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Rating Criteria
Significance of the Resource or Issue to the Park:
How
important is the resource or issue to the park involved, relative to its
other resources and issues?
The Timpanogos Cave system is the monument's primary resource and subject of enabling legislation. The caves were set aside by presidential proclamation because of "their scientific interests and importance". This project initiates gaining a better understanding to the role that microbes play in Timpanogos Cave system. (Score Value=5) (Weight=1) Total 5 x 1 = 5
Severity & Urgency of Resource Threat, Problem, or Need:
This project will measure the health of the cave ecosystem. Currently, the extent of the cave watershed extends beyond the boundary of the monument. Grazing, camping, ATV and off-road vehicle use, and wildfire activity all occurs within the known cave watershed. The scientific study of microbes is making great discoveries that are impacting the whole world. New genera of bacteria capable of expressing genes with medical and practical applications have been discovered and are now the focus of many cave microbial studies (Angert et al.1998; Holmes et al. 2001; Northtup et al. 2000). This project may also lead to a vital sign indicator to characterize the health of the cave ecosystem at its most basic level. Any delay may lead to the extinction of some unknown cave species that contains DNA for unique medical properties or research. (Score Value=5) (Weight=1) Total 5 x 1 = 5
Problem Resolution:
Will the proposed use of funds
contribute directly to decisions or actions that, when implemented, will
meaningfully resolve a management issue?
This project will provide a snapshot of the microbial species present in the Timpanogos Cave system. If these species indicate the presence of E. coli from human waste or sewage, petroleum products, or large carbon particles then specific management actions will be prescribed to restrict activities occuring within the cave watershed, and protect the fragile non-renewable cave resources. Future research on the caves microbes will also produce a larger DNA sequence and phylogenetic database that can be described in even greater detail. (Score Value=5) (Weight=1) Total 5 x 1 = 5
Scientific Resource Management:
Is the proposal
scientifically and technically credible?
This project clearly
demonstrates the techniques involved, information assembled, and
references. Rick Fowler and associates at the Biotechnology Center at
Western Kentucky University have been working sites within Mammoth Cave
NP, and surrounding areas, to test these methods. A DNA sequence and
phylogenetic database to interpret the results has been created. The
Biotechnology Center is looking forward to performing all of the lab work
for this project. They are excited to expand their databases to include
caves from across the country. (Score Value=5) (Weight=1) Total 5 x 1 = 5
References
Amann, R.I., Stromely, J., Devereux, R., Key, R. & Stahl D.A. (1992). Molecular and Microscopic Identification of Sulfate-Reducing Bacteria in Multispecies Biofilms. Appl. Env. Microbiol. 58(2): 614-623.
Angert, E.R., Northrup, D.E., Resenbach, A-L., Peek, A.S., Goebel, B.M. & Pace, N.R. (1998). Molecular analysis of a bacterial community in Sulphur River, Parker Cave, Kentucky. American Mineralogist 83: 1583-1592.
Elliott, L., Wright, S., Coakley, T. & Groves, C. (2000). Microbial Ecology of Conduit Stream Sediment Interstitial Fluids of the South Central Kentucky Karst Aquifer: Impacts on Aquifer Development, in Proceedings of Mammoth Cave National Park�s Eighth Science Conference, Mammoth Cave, Kentucky, pp 57-60.
Holmes, A.J., Tujula, N.A., Holley, M., Contos, A., James, J.M., Rogers, P. & Gillings, M.R. (2001). Phylogenetic structure of unusual aquatic microbial formations in Nullarbor caves, Australia. Environmental Microbiology 3(4): 256-264.
Hugenholtz, P., Pitulle, C., Hershberger, K.L. & Pace, N.R. (1998). Novel Division level bacterial diversity in a Yellowstone hot spring. J. Bacteriology 180(2): 366-376.
Lane, D.J. (1991). 16S/23S rRNA Sequencing, in Nucleic Acid Techniques in Bacterial Systematics, E. Stackenbrandt and M. Goodfellow (eds), John Wiley and Sons, Inc. New York, pp115-148.
Layton, A.C., Lajoie, C.A., Easter, J.P., Jernigan, R., Sansaverino, J.& Sayler, G.S. (1994). Molecular Diagnostics and Chemical Analysis for Assessing Biodegradation of Polychlorinated Biphenyls in Contaminated Soils, J. Indust. Microbiol. 13: 392-401.
Minz, D., Fishbain, S., Green, S.J., Muyzer, G., Cohen Y., Rittman, B. & Stahl, D.A. (1999). Unexpected Poplulation Distribution in a Microbial Mat Community: Sulfate-Reducing Bacteria Localized to the Highly Oxic Chemocline in Contrast to a Eukaryotic Preference for Anoxia, Appl. Env. Microbiol. 65(10): 4659-4665.
Moyer, C.L., Dobbs, F.C. & Karl, D.M. (1994). Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active hydrothermal vent system, Loihi Seamount, Hawaii, Appl. Env. Microbiol. 60(3): 871-879.
Northrup, D.E., Dahm, C.N., Melim, L.A., Spilde, M.N., Crossey, L.J., Lavoie K.H., Mallory, L.M., Boston, P.J., Cunningham, K.I. & Barns, S.M. (2000). Evidence for geomicrobiological interactions in Guadalupe caves. Journal of Cave and Karst Studies 62(2): 80-90.
Rusterholtz, K.J. & Mallory, L.M. (1994). Density, Activity, and Diversity of Bacteria Indigenous to a Karstic Aquifer. Microbial Ecology 28:79-99.
Siering, P.L. (1998). The double helix meets the crystal lattice: The power and pitfalls of nucleic acid approaches for biomineralogical investigations. American Mineralogist 83: 1593-1607.