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Home , Thermus aquaticus

Copyright 0 1997 by the Genetics Society of America Perspectives Anecdotal, Historical And Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

The Value of Basic Research: Discovery of 117termus aquaticus and Other Extreme

Thomas D. Brock

E. B. Fred Professor of Natural Sciences Emeritus, Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706

OLYMERASE chain reaction (PCR), the revolution- Year” award. Taq from T. aquaticus was des- P ary technique for DNA research, depends on Taq ignated the first awardee. polymerase, an from Themnus aquaticus, an or- Discovery of T. aquatim I began my scientific ca- ganism that I first isolated from a hot spring in Yel- reer in 1951 as a microbial physiologist and did exten- lowstone National Park. The PCR technique is so im- sive work in and portant that Hoffmann-LaRoche, the giant Swiss phar- through the mid-1960s. Although I found this work sat- maceutical company, paid more than $300 million to isfjmg, I had always been interested in the outdoors. acquire world rights for this procedure. Essentially, In 1963 my professional circumstances were such that Hoffmann-LaRoche was acquiring the rights to two pa- I could branch out into new areas of research. It was tents, those of GELFAND et al. (1989) on at this time that I initiated what was to become a long- and of MULLISet al. (1990) on the PCR technique. term research program in microbial ecology.‘ My eco- The importance of Taq polymerase was recognized logical work focused on the study of by the White House in 1991. D. hLAN BROMLN, the directly in their natural environments. I worked on a Director of the Office ofScience and Technology Policy variety of habitats, including marine intertidal pools, and the President’s chief science advisor, testified be- freshwater lakes, and soils, but for 10 years I concen- fore the House of Representatives: trated my research on hot springs and , which I viewed as model systems for basic research in microbial Different kinds of research and development tend to have different kinds of returns. With basic research- ecology. the majority of which is done by individual scientists and My discovery of T. aquaticus and other high-tempera- small groups of scientists at universities-it is very diffi- ture could not have been made without studies cult to predict when, where, and to whom the returns directly in the natural environment in Yellowstone Na- will eventually accrue. Yet even work that can seem highly tional Park. At the time I began this work, thermophilic abstract canhave surprisingly immediate impacts. To take just one example, in 1968 Thomas Brock, a microbiolo- bacteria were thought to have temperature optima of gist at the University of Wisconsin, discovered a form of only 55”, and the upper temperature for life was stated bacteriain the thermal vents of Yellowstone that can as73” (KEMPNER 1963). However, I was not initially survive at very high temperature. Fromthese bacteria interested in defining the upper temperature limits of an enzyme was extracted that is stable at near-boiling life. My first Yellowstone work involved a study of the temperatures. Nearly two decades later this enzyme proved to be vital in theprocess known as the polymerase distribution of photosynthetic microorganisms (primar- chain reaction,which is used to duplicate specific pieces ily ) along the thermal gradients created of DNA. Today, PCR is the basis of a multimillion dollar by the outflow channels of hot springs. The research business with applications ranging from the rapiddiagno- was part of a broader study on the thermal control sis of disease to forensic medicine.’ of and primary productivity in natural In 1989 Science magazine (December 22, 1989 issue) environments (BROCKand BROCK1967,1968). The hot established a new award called “The Molecule of the springs wereviewed as “steady state” ecosystems or what I came to call “experiments in nature” where Address fm correspondence: Thomas D. Brock, 1227 Dartmouth Rd., critical ecological research could be carried out. High- Madison, WI 53705. temperature bacteria were an unexpected discovery. ’ Testimony of D. ALLANBROMLEY, Director, Office of Science and I did my first research in Yellowstone National Park Technology Policy, before the Committee on Science, Space, and Technology, House of Representatives, February20, 1991. BROMLEY’S statement should be corrected. The discoverywasmade in 1966 (pub For background on my career, and how I came to work on thermc- lished in 1969),and I was then at Indiana University in Bloomington. philic bacteria, see BROCK(1995a).

Genetics 146 1207-1210 (August, 1997) 1208 T. D. Brock in June 1965 when I was Professor of Microbiology at By the fall of 1968, I had obtained a large number Indiana University. I studied a large group of thermal of cultures of T. aquaticus and had characterized them. pools, many of which had good effluents that provided Most of the thermophilic bacteria that had been de- thermal gradients from boiling down to ambient tem- scribed by earlier researchers were members of a group perature. I was able to show that there was a definite called the spore-forming bacteria (because they pro- upper temperature for photosynthetic life, at around duced heat-resistant spores). The new bacterium was 70-73", but higher temperatures were not devoid of definitely not a spore-former, and it was clear that it life. I observed that in the effluents of certain springs was a member of a new genus of organisms. there were masses offilamentous bacteria living at tem- In preparing a paper for publication, I needed to peratures much hotter than those at which photosyn- create a name for this new genus. I did an extensive thetic organisms were present. One particular spring, survey of the literature of thermophilic bacteria, listing Octopus Spring, hadlarge amounts of pink filamentous all the names that had been used over the years. The bacteria at temperatures of 82-88". Here were organ- new name I first selected was Caldobacter trichogenes, re- isms living at temperatures above the reputed "upper flecting the fact that the organism lived in hot water temperature for life." (caldo is Italian for hot), and under some conditions Attempts to cultivate these pink bacteria were initially forms filaments (tm'cho derives from the Greek for fila- unsuccessful, but during the next severalyears, as I ment or thread). However, after the first draft of the continuedbroader work on the ecology of the Yel- manuscript was written, I decided that this name was lowstone springs, I continued to make occasional obser- too fanciful. I chose instead thename T. balnearius vations on these bacteria. At that time, I was doing a ( referring to heat and balnearius to a spa or large amount of work on the photosynthetic microor- thermal bath). Later, for reasons that I no longer re- ganisms of Mushroom Spring, a large spring in the member, I replaced balnearius with aquaticus (aquaticus Lower Basin (BROCK1967a). The source pool referring to water, where the bacterium grows). This of this spring had a temperature of73", just at the last name, T. aquaticus, became official when the paper known upper temperature limit for photosynthetic life. describing this organism was published (BROCKand Because of the difficulty I had cultivating bacteria at FREEZE 1969). temperatures above go", I decided to study the bacteria The name for Taq polymerase is often abbreviated in Mushroom Spring that lived at 70-73". Beginning "Taq" in common usage. Is a name really a trivial mat- in the fall of 1966, with undergraduate student HUDSON ter? The word Taq rolls readily off the tongue and also FREEZE,I began the work that was to lead to the discov- fits well into molecular biology jargon and advertising ery and culture of T. aquaticus. It was from a sample copy. I suppose if I had kept my original name, Cakdo- collected from Mushroom Spring on September 5,1966 bacter trichogenes, the enzyme might be called "Cat!" that culture YT-1 of T. aquaticus was isolated. This is the At the time that the paper on 7'. aquaticus was being culture that is used today as the source of Taq polymer- written, I also deposited representative cultures of the ase for PCR and is the culture specified in the Taq organism in the American Type Culture Collection in polymerase patent (GELFANDet al. 1989). Washington, D.C. Among these cultures was IT-1, the HUDSONFREEZE and I isolated T. aquaticus culture culture later to be used in PCR. Culture YI"1 became YT-1 in October 1966. During various travels to study ATCC 25104. When DAVIDGELFAND of Cetus conceived thermal areas in other parts of the world, I isolated a of the idea of using a as a source of DNA number of other cultures of T. aquaticus. An interesting polymerase for PCR, he tested all of the available cul- culture was isolated from the hot watersystem of a tures of T. aquaticus from the American Type Culture buildingon the Indiana University campus. Subse- Collection. Strain IT-1 turned out to have the enzyme quently, I was able to show that T. aquaticus was wide- with the best properties for PCR (D. GELFAND,personal spread in artificial hot-water environments, and other communication). workers have isolated it from hot tap water in other Long before GELFAND'Swork on Taq polymerase, bio- parts of the world. chemists hadstarted work on thermostable All of the cultures had an optimum temperature for from T. aquaticus. Even before my first papers on 7'. growth at around70" and were able to grow at tempera- aquaticus, I had been sending out cultures to biochem- tures up to 79". FREEZEalso showed that enzymes from ists who had seen a feature article I had written in T. aquaticus were able to tolerate temperatures higher Science (BROCK196713). A variety of thermostable en- than the maximum growth temperature, even surviving zymes were discovered. One enzyme that worked with boiling water (FREEZEand BROCK1970). Another stu- DNAwas Taq I restriction endonuclease (SATA ut ul. dent in my laboratory, J. GREGORYZEIKUS, studied as- 1977). Also, JOHN TRELAat theUniversity of Cincinnati pects of the protein-synthesizing system of T. aquaticus described a thermostable DNA polymerase from 7: and showed that ribosomes and -activating aquaticus (EDGARet al. 1975).Although there have been enzymes were also active at high temperature (ZEIKUS some fairly expensive legal arguments about whether rt al. 1970; ZEIKLJSand BKOCK1971). TRELA'Senzyme is the same Taq polymerase that GEL- Perspectives 1209

FAND et al. (1989) patented, the fact remains that there peratures could be found. I made visits to Iceland and was a longhistory of research on thermostable enzymes New Zealand, where the boiling springs are at low alti- from T. aquaticus before PCR was discovered. tudes and temperatures range up to 100” or a little It is fair to say, however, that it was PCR that put T. higher in superheatedwaters. I was able to find bacteria aquaticus on the map, and there has been a veritable in virtually every boiling spring of neutral or alkaline “feeding frenzy” amongthe media on this subject. pH in these areas, thus extendingto a somewhat higher Television has particularly liked to discuss Taq polymer- value the upper temperature limit for life. ase in the contextof forensic medicine, because footage Deep seavents For many years my Yellowstone work of Old Faithful erupting can be shown. I have ceased had seemed somewhat “exotic” to many microbiolo- trying to note all references in the popular literature gists, perhaps because of the presumed restricted distri- to T. aquaticus, but a brief listing through mid-1995 can bution on earth of hot springs. This attitude changed be found in BROCK(1995a). after the discovery of the deep sea vents, with their In 1991, when I did aretrospective computer search, very high temperatures and the associated diverse and over 1000 papers on T. aquaticus had been published, flourishing life forms. Deep sea thermal vents are wide- and the numbercontinues to increase. Truly, one does spread in the oceans, usually associated with volcanic not know where a research study will lead! activity and tectonic movements of the earth. After the Bacteria in boiling water: Although I had convinced deep sea vents were discovered in the late 1970s, my myself in 1965 that bacteria were living at much higher Yellowstone worktook on broadersignificance, because temperatures than had beenpreviously suspected, I had it not only made it reasonable to hypothesize that mi- still not realized that they were livingin “boiling”water. crobes might be present in some of these high tempera- My ideas had been based only on observations in out- ture systems, it also provided the essential foundation flow channels where “visible” accumulations were pres- for studies on the microbiology of thermal vents. The ent (such as the pink bacteria at temperatures above techniques and principles thatI had developed for 80” in OctopusSpring). Culture studies on extreme proving that bacteria live and reproduce in boiling wa- thermophiles are technically difficult, and if I hadrelied ter could then be applied to the deep sea habitats. onstandard bacteriological isolation proceduresI The upper temperaturelimit for life has still not been might never have been successful. Here is where the completely resolved, but the chance to study microor- ecologist’s approach, study of organisms directly in na- ganisms living in deep sea vents, where temperatures ture, comes to the fore. range up to greater than 300”, has opened up a whole Near the end of the summer of 1967 I started using new approach. Although it is clear that no organisms a sensitive immersion slide technique to demonstrate exist at such high temperatures (even amino acids are microscopically the presence of living bacteria in boil- not stable under these conditions), there arealso ther- ing water. This immersion slide technique had been mal vents in the sea at temperatures between 100 and widely used by microbial ecologists studying “normal” 150”.The German microbiologist KARL STETTERin par- environments. With this technique, I was able to show ticular has done an excellent job of isolating bacteria that although there were no visible accumulations of capable of growth attemperatures above 100” from microorganisms in the boiling pools themselves, bacte- some of these vents. One , Pyrolobus fu- ria were present in microscopic amounts in virtually marii, has an optimum temperature for growth of 105” every boiling pool I looked in. and can still grow at 113” ( MADIGAN and MARR~1997). In 1967 I initiated a systematic search for the pres- (This organism finds 90” too cold!) ence of bacteria in Yellowstone boiling springs. The Microbialprospecting in Yellowstone: Although it experiment was simple: tie one ortwo microscope slides was clear when I ended my project in 1975 that there to a piece of string, drop in the pool, and tie the other were many interesting organisms with potential practi- end of the string to a log, rock, or nail. Return later, cal applications in hot springs, it was not until the ad- retrieve the slides, and examine under the microscope. vent of PCR that widespread attention really focused on The results were dramatic. Virtually every slide, from thermophiles. Not only has the industry every boiling or superheated pool, had heavy bacterial discovered Yellowstone, but the National Park Service growth, readily visible microscopically. In some cases, itself has finally realized that thereis more of biological the density was so heavy that the slides had afilm visible interest in Yellowstone than grizzly bears and lodgepole to thenaked eye. The following summer (1968) pines (MILSTEIN1994). Dozens of research groups now THOMASBOTT did an outstanding job of proving that have permits to collect microbial samples in Yel- these bacteria were really growing, by measuring their lowstone. Although another discovery as valuable Taqas growth rates in situ (BOTT and BROCK1969). polymerase may not come out of this work, isit certainly Because of the altitude, water boils at only 92.5” in possible that some of these groups may discover organ- Yellowstone. Once I knew that bacteria could live in isms of value. Never before has industry profited di- boiling springs here, it was natural to plan field trips rectly from living creatures taken from a national park, to thermal areas at lower altitudes, where higher tem- andthe Yellowstone administratorsare concerned 1210 T. D. Brock about whether the Park itself should participate in the LITERATURE CITED largess. Yellowstone, of course, has no monopoly on BUU, T. L., and T. D. BROCK,1969 Bacterial growth rates above thermophiles, but it provides the most accessible loca- 90” in Yellowstone hot springs. Science 164: 1411-1412. BROCK, T. D., 1967a Relationship between standing crop and pri- tion where a wide variety of thermal habitats are avail- mary productivity along a hot spring thermal gradient. Ecology able. 48: 566-571. BROCK,T. D., l967b Life at high temperatures. Science 158: 1012- Also, thermophilic bacteria are nowjustone example 1019. of “,” microorganisms capable of living BROCK,T. D., 1978 Thennophilac Mirroorgunisms and Lifr at High Tpm- under extremeconditions. Other extreme habitats peratures. Springer-Verlag, New York. BROCK,T. D., 1995a The road toYellowstone-and beyond. Annu. where interesting bacteria live are those with high or Rev. Microbiol. 49: 1-28. low pH, high salt, and low temperature. Extremophiles BROCK,T. D., l995b Photographic supplement to “The road toYel- are suddenly “hot” research topics with the biotechnol- lowstone-and beyond.” Available from T. D. BROCK,Madison, Wl . ogy industry (MADIGANand MAKRs 1997). BROCK,T. D., 1995c Bibliogruphy of Thomas L). Brock, Publacattons 1951-1 995. Available from T. D. Brock, Madison, M‘I. BROCK,T. D., and M. L. BROCK,1967 The nleasurement of chloro- phyll, primary productivity, photophosphorylation, and macro- EPILOGUE molecules in benthic algal mats. Iimnol. Oceanogr. 12: 600- 605. BROCK,T. D., and M. L. BROCK,1968 Measurement of steady-state The work I have discussed here is just a small subset growth rates ofa thermophilic alga directly in nature.,J.Bacteriol. of the various research areas that I have been involved 95: 811-815. BROCK,T. D., and H. FREEZE, I969 Thmmus uquatiru.~gen. 11. and in throughout my career. A more detailed overview is sp. n., a non-sporulating extreme thermophile. J. Bacteriol. 98: given in my memoir for theAnnual Review of Microbiology 289-297. EDGAR, D.,A. CHI^ andJ. TREIA,1975 Purification and character- (BROCK1995a) and in two supplements to this memoir ization of a DNA polymerase froman extremethermophilc, (BROCK1995b,c). However, of all the work I have been Thmnus aquaticus. Abstracts, Annual Meeting, Am. Soc. Micro- involvedwith, the Yellowstone project has beenthe biol. 75: 151. FREELF.,H., andT. D. BROCK,1970 Thermostable aldolase from most exciting and has continued to elicit the most inter- Thmnus uquuticus. J. Bacteriol. 101: 541-550. est from others. For 10 yearsI operated a field research GELFANI), D. H., S. STOFFEL.,F. C. LAWTRand R. K. SAIKI,I989 Puri- fied Thermostable Enzyme, United States patentnumber laboratory in West Yellowstone, Montana, which pro- 4,889,818, December 26, 1989. vided facilities for research by a variety of students and KKMPNER, E., 1963 Uppertemperature limit of life. Science 142: postdoctorates, as well as visitors from across the coun- 1318-1319. MADIGA~,M. T., and B. L. MAKKS, 1997 Extrenlophiles. Sci. Am. try and around theworld. Much of this workis discussed April 1997, 82-87. in a book (BROCK1978) and in over 100 research and Mrl.srF.ln., M., 1994 Yellowstone managers eye profits from hot mi- review papers (see BROCK1995c for listing). crobes. Science 264: 655. M~I.I.Is,K. B., H. A. ERI.ICH,D. H. G~FANL),G. HORN andR. K. SAM, Thermostable enzymes were only a small part of this 1990 Process for amplifying, detecting, and/or cloning nucleic work, since my focus was not on biotechnology but on acid sequences using a thermostable enzyme. United States pa- tent number 4,965,188, October 23, 1990. basic research. However, the new microorganisms that SAIA, S., C.A. HL~TCHISON andJ. I. HARRIS,1977 A thermostabk have been discovered in Yellowstone and elsewhere sequence-specific cndo nuclease from Thm~u.~aquaticus. Proc. around theworld havebeen madeavailable to the scien- Natl. Acad. Sci. USA 74 542-546. ZEIKUS,J. G., and T. D. BRO(:K,1971 Protein synthesis at high tem- tific community. Only one of these organisms led to peratures: aminoacylation of tRNA. Biochim. Biophys. Acta 228: PCR, butothers have provided interesting research 736-745. ZEIK~.S,J. G., M. M’. TAYLORand T. D. BROCK,1970 Thermal stability problems for microbiologists, biochemists, geneticists, of ribosomes and RNA from Thrrmus uquutiru.r. Riochim. Biophys. evolutionists, geologists, and ecologists. Acta 204: 512-520.