The Students: Jessica Byrd, Ben Davidson, April Haskins, Donna Henderson, Katey Irwin, Roya Ougouag, Elizabeth Shotton, Joseph Shotton, Aaron Stanton,
The Teacher: Clinton A. Kennedy
Thermus Aquaticus is an aerobic heterotrophic eubacterium found in hot springs, an ideal environment for an organism whose optimal growth temperature is between 50° and 80°C and whose ideal pH lies between 7.5 and 8. T. Aquaticus was first discovered in Yellowstone National Park (U.S.A.) in 1965 and has since been isolated in thermal environments worldwide. T. Aquaticus often lives near cyanobacteria, obtaining its energy for growth from the photosynthesis of these organisms, but it can also be found in temperatures too high for photosynthesis, and in these cases, thrives on the organic matter present in the water. The unique biochemistry of T. Aquaticus has become essential to the polymerase chain reaction (PCR) method, which is used to produce millions or even billions of copies of DNA, as the DNA polymerase (Taq polymerase) is heat stable, unlike the previously used enzyme which denatured at high temperatures. Taq polymerase is widely used in both medicinal and forensic fields.
The particular gene that distinguished T. Aquaticus from other thermophiles is the 16s rRNA gene. The sequences of the 16s rRNA genes of 20 T. Aquaticus were determined to a high fidelity by using automated DNA sequencing and fluorescent-dye-labelled primers. The strains tested included members of the three validly named T. Aquaticus and representatives of major taxonomic clusters defined previously for this genus. The parsimony method was used to reconstruct the phylogeny of the strains from the aligned sequences, and a bootstrap analysis revealed a number of well-supported clades. Our results are not consistent with groupings inferred from numerical taxonomy data but support the conjecture that the genus Thermus contains more species than the three currently recognized species. (Saul DJ, Rodrigo AG, Reeves RA, Williams LC, Borges KM, Morgan HW, Bergquist PL; 1993)
Media Preparation. T. Aquaticus was cultured in Castenholz TYE media (see Figure 1). After autoclaving, the media was mixed together in a ratio of 5 parts Castenholz Salts to 1 part TYE to 4 parts DI water under a hood using aseptic techniques . All flasks, pipettes, and containers were flamed before and after use, while all surfaces were disinfected with bleach or ethanol prior to and following each use. Once the ingredients had been mixed thoroughly, the medias pH was brought up to 7.6 to support ideal growth of T. Aquaticus. The pH was measured from a sample of the media rather than directly from the media bottle to avoid contamination from the pH meter, and sterile .1N NaOH was used to bring the pH up.
Sampling. Aseptic techniques were employed all around; forceps were flamed and sterile collection jars (32 ounce Wide Mouth Mason Jars) and ------- collection jars with 25 mL of Castenholz-TYE media were used. The Wide Mouth Mason Collection Jars were approximately 1/4 full with the sample water, and small stones, matting samples, and sticks were added at each of the eight sampling sites. Water samples (approximately 1.5 mL) and matting samples from five of the eight sites were inoculated directly into Castenholz - TYE media in the ------- media jars using sterile pipettes and forceps. The temperature (49 - 89°C), pH (7 - 9), and distance from the source of each site were measured and recorded, and the collection jars were labeled appropriately, and the appearance of each site was documented with a digital camera. Algae mats ranged in color, with the white and green matting found in temperatures ranging from 80 - 89°C, the orange and red matting found in 60 - 84°C temperatures, and the black matting found in temperatures of 49 - 63°C. Samples were then transported to a local laboratory, where all of the water samples in media and half of the water samples in the Mason Jars were stored in an oven at 65 - 70°C, while the other half of the Mason Jar water samples were stored in a refrigerator at approximately 4°C. Once back at the laboratory at the Idaho National Engineering and Environmental Laboratories (INEEL), the water samples in media were transferred directly to a 70°C water bath, while the Mason Jar water samples were inoculated into media.
Culture. Before culturing was initiated, microscope slides of the T. Aquaticus sample provided by Dr. Robertos lab were prepared to get an idea of what T. Aquaticus looked like. When the cultures were started, 25 mL of media was added to an autoclaved 125mL flask capped with a foam plug. The samples were then inoculated with 0.5mL of the T. Aquaticus sample provided by Dr. Robertos lab, and all of the cultures were properly labeled. After inoculation, the cultures were incubated in a 70°C water bath. Once the original cultures started to grow, they were subcultured every two days into a freshly autoclaved 125 mL flask, adding 0.5 mL of the old sample and then transferring the original to a 4°C refrigerator to ensure that there was a stock of back-up cultures in the event that the cultures in the water bath failed to grow.
Cell Lysis/DNA Extraction. Alkaline chemical lysis methods (see Figure 2) similar to those utilized for lysis of bacteria such as E. Coli, were initially employed. Proteins were precipitated using a Potassium Acetate solution, cells and proteins were pelleted using an ultra micro centrifuge, and a 100% ethanol was implemented to precipitate the DNA, which was then dissolved in TE. Electrophoresis in 0.8% agarose and TBE Buffer was used to confirm the presence of DNA, indicating that the cells had been correctly lysed. A Hind III Lambda DNA Molecular Weight Marker was added to lane one as a positive control to confirm correct electrophoresis protocol, and E. Coli was used as a positive control to confirm a correct lysis protocol. The gels were stained with 0.5% Ethidium Bromide for 15 - 20 minutes and were then viewed with a shortwave ultraviolet transilluminator. A combination of mechanical and chemical lysis was next employed (see Figure 3). Because T. Aquaticus is fairly resilient to chemical lysis and DNA extraction, a commonly employed method involves the physical breaking of the cell wall. The cell walls were first softened with buffer, lysozymes, SDS, and heat incubation. Acid washed beads were then added and loaded into a bead beater, which knocked the cells and beads together, eventually breaking the cell wall. Phenol chloroform was used to extract the DNA while isopropanol precipitated the nucleic acids. The supernatant was decanted, leaving the pellet intact for purification with ammonium acetate. The pure DNA was then transferred to a new tube where it was precipitated with isopropanol. The sample was then centrifuged and the supernatant disposed of. The pellet was rinsed with ethanol and air dried before it was resuspended in TE buffer and electrophoresed. Once it was determined that the cell had been correctly lysed, the PCR protocol for MasterAmp Tfl DNA Polymerase (see Figure 4) was implemented, using aseptic techniques. The Reaction Mix was assembled (see Figure 5) under a hood, using sterile aerosol PCR tips to transfer the reagents into the solution. The final reaction volume was the required 50 µl. The reagents were thoroughly mixed and transferred to PCR tubes and were immediately put on ice. A thermocycling program was then implemented (see Figure 6). 10µl of the DNA sample was added to a new 1.5mL tube along with 1µl of loading dye for electrophoresis. Two markers, Lambda and 1kb, were also run to determine the size of the sample and to serve as a positive control for electrophoresis.
Diagnostics. The presence of DNA from Thermus Aquaticus was confirmed using PCR on the 16s rRNA gene. The primers used were 1492 (E. Coli Numbering System) 5-GGT TAC CTT GTT ACG ACT -3 as a reverse primer and 4FA (E. Coli Numbering System) 5-TCC GGT TGA TCC TGC CRG-3 as a forward primer. The DNA was cloned using TOPO-TA techniques and then sequenced using the dideoxy DNTP (Sanger Method).
Culturing, Media, and Sampling. The cultures of T. Aquaticus provided by Mark Delwiche of Dr. Robertos lab were inoculated into Castenholz-TYE media and grown in a shaking hot water bath. The cultures soon developed a deep yellowish color and several long, slimy filaments, which, upon microscopic examination, were confirmed to be T. Aquaticus. The subculturing of these samples was performed every two to three days, depending on the density of the bacteria in the media, as indicated by depth of color. The bacterial cultures were multiplying at an extremely high rate, until the shaking water bath blew a fuse and stopped shaking. Attempts to remedy the malfunction were futile, and although the T. Aquaticus continued to grow, the growth rate decreased dramatically because the flasks containing the culture were not being properly aerated.
The samples collected from eight different sites, at different temperatures, and varying pH levels from Vulcan Hot Springs were also grown in Castenholz-TYE media. Mattings of various colors and pure water samples were collected in autoclaved sample jars, and selected samples were inoculated directly into media at the site. The growth rate of T. Aquaticus did not seem to be affected by whether the samples were inoculated immediately into media or inoculated later in the laboratory. The samples that had the highest growth rate were taken from Sites 4, 5, and 7 (see Figure 7), as was indicated by the deep yellow color of the media and the later microscopic examination of the cultures contents. Site 4 had a temperature of 82°C and a pH of 8.4, and the inoculation consisted of stick with white organic material. Site 5 was inoculated from a brownish mat and had a temperature of 71°C and a pH of a 8.5. Site 7 had a temperature of 60°C with a pH of 8.8 and was inoculated from orange organic material. The other cultures had minuscule amounts of T. Aquaticus growing.
Four and a half weeks into the project, the Castenholz-TYE media became severely contaminated. The source of contamination was traced to contamination of the TYE yeast extract. All of the inoculated samples in the water bath except one were found to be contaminated with microorganisms, and new subcultures and media were prepared. Samples that had been stored in the refrigerator were chosen because they had not been contaminated and had demonstrated viability when previously cultured were inoculated into freshly mixed media.
Lysis and Extraction. Thermophilic bacteria, including T. Aquaticus, are very resilient organisms designed to live in conditions that are typically fatal to other organisms. Because T. Aquaticus has developed many survival mechanisms, lysis is often more difficult with it than most other bacteria. Therefore, results indicating T. Aquaticus had been lysed were not expected to be obtained when a chemical alkaline lysis protocol was run. An E. Coli sample was included in this procedure to serve as a positive control to ensure proper lysis protocol, and electrophoresis of an agarose gel was used to confirm the successful lysis of cells. A Hind III Lambda Molecular Weight marker was run in lane one to confirm correct protocol on electrophoresis and it showed good separation when stained with 0.5% Ethidium Bromide for 15-20 minutes and viewed with an Ultraviolet Transilluminator (see Figure 8). The results of this experiment showed that the E. Coli had been successfully lysed but that T. Aquaticus had not been broken apart. Lanes 2 and 3 of the agarose gel were the first sample of T. Aquaticus, lanes 4 and 5 were samples of E. Coli, and lanes 6 and 7 were the second T. Aquaticus sample. E. Coli showed distinct DNA bands in both lanes, while the bands of T. Aquaticus were absent, which led to the conclusion that alkaline lysis was not an efficient means of lysing T. Aquaticus. Several other protocols were researched, but the ultimate decision was to pursue a mechanical disruption procedure involving a Bead Beater in conjunction with chemical lysis/extraction procedures. Upon gel electrophoresis after the completion of this new procedure, the presence of DNA from T. Aquaticus was confirmed (see Figure 9), as is evident when a band of DNA appeared along the lane that was loaded with the sample from Vulcan Hot Springs.
PCR. Following the amplification of the 16s rRNA gene using PCR, electrophoresis confirmed that there was T. Aquaticus DNA present in the Vulcan Hot Springs PCR sample. However, there were several extra bands that appeared on the Vulcan Hot Springs lane that did not line up with any of the other samples run in comparison (see Figure 10).
Sequencing. After completing the miniprep and sequencing methods, the product was loaded into the automated sequencing machine and left to run overnight. The resulting sequence of the T. Aquaticus sample from Vulcan Hot Springs was entered onto the Basic Local Alignment Search Tool to search for possible matches, but the results were inconclusive.
As is the case in any scientific experiment, many obstacles were encountered, particularly when it came to culturing the T. Aquaticus samples. It was determined that the samples multiplied at a much higher rate when the water bath was shaking and aerating the flasks than when it stopped shaking. However, even once the water bath stopped shaking, it was still more effective than an oven because less evaporation occurred when the samples were in the water bath. Foam plugs on the flasks worked better than plastic caps because the plugs allowed air to circulate throughout the flasks, though this aeration may have been a source of contamination.
The media seemed to be very effective in supporting the growth of T. Aquaticus until contamination in the form of other microorganisms was discovered, reinforcing the concept that strict adherence to proper aseptic techniques is of the utmost importance when media is being mixed and the samples are being subcultured. It is entirely possible that the media was contaminated when a non-sterile pH probe was accidentally placed directly into the media.
The samples gathered from organic mats at Vulcan Hot Springs at temperatures ranging between 60 and 80°C and a pH of approximately 8.5 produced more T. Aquaticus in a shorter period of time than did the other samples. Furthermore, pure water samples with no organic matter yielded no visible strains of T. Aquaticus even after five weeks of attempted culturing. It would be interesting to continue to gather samples from other local hot springs to determine whether or not any T. Aquaticus is present in the water, and, if so, to compare the DNA of those samples to each other to determine the relatedness. Another interesting occurrence to note is the observation of diatoms found in the 70°C Vulcan Hot Springs sample. It would be interesting to determine whether or not these diatoms actually live in a silica rich granitic hot springs environment and if they can, in fact, be cultured.
The electrophoresis gels seemed to work well as the only potential problems we ran into involved ensuring the proper gel thickness and the proper loading of the samples.
After much trial and error with lysis, it was concluded that Alkaline SDS lysis is not efficient in and of itself. However, when mechanical means were used in conjunction with chemical lysis, the results were impressive. A reference in a Thermus Aquaticus polymerase article suggested the implementation of ultrasound to break apart the T. Aquaticus cells was very effective, and it would be interesting to try this in the future, perhaps using a hospitals ultrasound.
Due to time constraints, the sample was not purified completely before sequencing, and therefore contaminants overshadowed the actual insert when. However, there were traces of a thermophile in the sequence, leading us to believe that running another experiment would be worthwhile if the time was taken to purify the sample.
Despite the lack of clarity in the DNA bands on the gel after PCR, the procedure seems to have worked well as it has served our purposes nicely, leading us to consider our project an overall success because T. Aquaticus was isolated and confirmed by a PCR of the 16S rRNA gene. Sequencing is still in progress to determine whether or not the T. Aquaticus isolated from Vulcan Hot Springs is identical to the standard T. Aquaticus or if it is, rather, a sub-species that is genetically unique. As we take our project back to our home lab with us, we hope to continue our work with isolates from other area hot springs and would love to continue our collaboration with Dr. Robertos laboratory.
We would like to extend the sincerest thanks to Dr. Frank Roberto, our mentor at the INEEL, for all the training, support, time, and assistance he provided. Without the opportunity to come to the INEEL and work in conjunction with his lab, none of this research would have been possible. We also extend our heartfelt appreciation to Dr. Robertos team who graciously donated their valuable time to train and assist us. We especially appreciate the sampling and culturing help and technical advice that Mark Delwiche provided us. He took us under his wing and led us down the path to success. Likewise, Heather Silverman provided us with the biochemical expertise necessary to complete our project. Her patience and thoughtfulness were especially appreciated considering the technical skill level we began with. We feel as though with her help, our skill and knowledge levels have improved exponentially. Megan Whitmore, Jamie Snyder, and Marilyn Tsang were also particularly helpful in the assistance they provided individual team members in many of the actual hands-on tasks necessary to complete our project. Finally, we would like to extend our appreciation to Julene Messick of the INEEL Institute for her major role in getting the funding to bring us here and providing us with the opportunity to work here for the past 8 weeks. Julene made this possible, and we are forever grateful to her for the opportunity she provided us.
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