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April 2011 an international team led by researchers at the European Mo- Ilecular Laboratory in Hei- delberg, Germany announced in Nature that the mind-boggling mix of mi- crobes in the human gut could be neatly grouped into categories called enterotypes (1). Hailed as a finding that might some- day help researchers address the long-in- tractable problem of antibiotic resistance, the discovery of gut microbial signatures in people raised the possibility that in- dividuals might have a defined enterotype, like a blood type, regardless of age, sex, or ethnicity (1). The study, which garnered attention in scientific and journalistic circles, follows a long-running initiative funded by the Fig. 1. (A) Carl Woese examining film on which ribosomal signatures are displayed (2003). (Photo by Jason Lindley; used with permission of the College of Liberal Arts and Sciences, University of Illinois at National Institutes of Health called The – B Human Project, whose goal is Urbana Champaign.) ( ) George Fox (1999). (Used with permission of the Department of Biology and at the University of Houston.) to catalog the genetic diversity of the tril- lions-strong microbial communities that inhabit our bodies. The hope is to deter- organization on microbial diversity,” says In the next 5 years, Woese documented mine how changes in the microbiome—the University of Colorado, Boulder molecu- the formation of parts of the bacterial genetic endowment of our microbial lar biologist Norman Pace, a self-avowed protein-synthesizing machinery—the selves—might influence health. follower of Woese. ribosome—as the slumbering The microbiome project turns on emerged from the spores, and studied researchers’ ability to compare - Biology, by Way of how radiation could be used to inactivate arily conserved gene sequences in human- A child of the 1930s Depression era, the spores (3). At the end of his post- associated microbes. Such a comparison Woese was born in Syracuse, New York, doctoral stint, in the fall of 1960, Woese might yield signatures that can help fore- where he was raised under straitened set up his own laboratory at General tell how our bodies might respond to diets, circumstances. Ever in search of com- Electric’s Knowles Laboratory in Sche- diseases, and drugs. “With the recognition forting, objective truths, he was drawn to nectady, New York, where he continued that the human body is an ecosystem that the reassuring consistency of ’ to explore the molecular biology of spore is host to ten times as many microbial cells often-categorical laws. That is partly why germination. While waiting for his labo- as human cells, the prospects of the pro- Woese graduated with a bachelor’s de- ratory equipment to arrive, Woese read ject for personalized medicine become gree in physics from , voraciously on a challenge that in- clear,” says Nigel Goldenfeld, a professor in 1950. There, he was in- creasingly preoccupied the decade’s of physics at the University of Illinois at spired to pursue as a career by leading molecular biologists in the wake Urbana–Champaign who has worked on physicist William Fairbank. Later, Woese of the discovery of DNA structure: the evolution of biological complexity. began doctoral studies in un- cracking the . Recent advances in DNA sequencing der the guidance of On the eve of the molecular biology technology have no doubt accelerated the physicist Ernest Pollard, whose con- revolution, the question of how cells effort, but the microbiome project, like tributions to the use of radar in World made proteins began to intrigue many others aimed at documenting biological War II earned him a permanent place in researchers. Of particular interest was the diversity, has its roots in a once-contro- the history of radiation physics. “Pollard process by which the assemblage of the four versial discovery now enshrined in the came from a respectable lineage of nucleotide bases that make up DNA was annals of evolutionary biology. physicists,” Woese says, referring to an interpreted by the cell into the sequence of Memorialized in a 1977 PNAS article by academic pedigree replete with physics amino acids that make up proteins. Al- biologists Carl Woese and George Fox heavyweights like J. J. Thomson, Ernest though RNA’s role as a messenger in the (pictured in Fig. 1), the discovery Rutherford, and James Chadwick. For his protein-making process was suspected at helped reclassify cellular life into three dis- doctoral thesis, Woese studied how radi- the time, the mechanics of protein syn- tinct domains, upending conventional views ation and heat could inactivate thesis was a mystery. Before long, the ex- on biological classification and offering like Newcastle disease , which af- istence of transfer RNA, a molecule that deep insights into the origin of life on Earth. flicts poultry. In 1953, a standout year bridged messenger RNA and amino acids, To this day, Phylogenetic structure of the in molecular biology’s history that was came to light, and the molecule was prokaryotic : The primary kingdoms, marked by the discovery of the double thought to act as an adaptor in accordance which courted controversy and challenged helical structure of DNA, Woese gradu- with the hypothesis advanced by Francis the reigning dogma of its day, remains a ated from Yale. After an inspired but breakthrough—one that emphatically re- unsuccessful foray into medicine that traced the branches on the to lasted 2 years, he returned to post- fl ’ better re ect its evolutionary roots (2). doctoral research in Pollard s laboratory, See Classic Article “Phylogenetic structure of the prokary- “Without Woese’s 1977 report, today’s focusing on the molecular changes un- otic domain: The primary kingdoms” on page 5088 in issue microbial sequencing efforts would not be derlying the germination of dormant 11 of volume 74. meaningful. Woese put a framework of spores of the bacterium Bacillus subtilis. See Classic Perspective on page 1011.

www.pnas.org/cgi/doi/10.1073/pnas.1120749109 PNAS | January 24, 2012 | vol. 109 | no. 4 | 1019–1021 Downloaded by guest on September 27, 2021 Sogin and William Balch, technician Linda Magrum, and others, Woese painstakingly assembled a database of differences in 16S rRNA among a laundry list of microbes that included both eu- karyotes, a group of organisms defined by the presence of a membrane-enclosed nucleus, and , a group defined solely on the basis of its differences from . Meanwhile, the scientific community’s attention was consumed by other developments in molecular biol- ogy’s early days, and Woese’s labors went largely unnoticed. Not for long. By 1976, Woese’s team had developed genetic signatures for dozens of different microbes, including methane producers that thrived in oxygen-starved environments like sewage and cow intestines. “It was a heroic enterprise to develop RNA catalogs for representatives of the different forms of life as we knew it then,” says Fox, now at the University of Houston, Texas. As a picture emerged, Woese realized that the methane producers were not bacteria. In fact, their 16S rRNA signatures suggested that they were fundamentally different from life forms then known as prokaryotes or eukar- yotes. Years later, Woese named the group of , which included heat- and salt-loving microbes that occupied extreme Fig. 2. (A) Grand Prismatic Hot Spring, Yellowstone National Park. The red, orange, and green pigments niches like deep sea vents and thermal around the spring are microbial mats fueled by photosynthesis and geochemicals from the hot spring. (B) springs, (see Fig. 2). Other bio- preparing to sample a hot spring in upper Hayden Valley, Yellowstone National Park. chemical signatures of archaea unearthed (Photo courtesy of Norman Pace.) by researchers in Germany and elsewhere lent support to the group’suniqueness. Crick, codiscoverer with of Ribosomal RNA: Life’s Timekeeper Through calculations of similarities DNA’s double helical structure. By then, Woese had accepted a faculty between the 16S rRNA sequences of Then, molecular biologists banded to- position in at the University bacteria, eukaryotes, and methane pro- gether to decipher how the 20 amino acids of Illinois at Urbana–Champaign on the ducers, Woese and Fox proposed in the found in cells corresponded to triplet invitation of molecular biologist Sol 1977 PNAS report that the methanogens codes of bases in the messenger RNA. A Spiegelman, whom he had met during represent a separate of life, “ mechanistic understanding of the genetic a sabbatical at the in suggesting that [the living world] is not ’ code became one of molecular biology s . Soon, Woese set out to catalog ri- structured in a bipartite way along the bedeviling challenges, and sure enough, bosomal RNA sequences in a range of lines of the organizationally dissimilar the ensuing years saw a spate of discover- — and . Rather, it is microbes. One form of rRNA named ” ies that unraveled the molecular mecha- 16S for the rate at which it sediments in (at least) tripartite. nism of protein synthesis. Yet despite the laboratory experiments—turned out to be The report unleashed a minor contro- attention lavished on the code, Woese la- versy among microbiologists even as The the yardstick of choice for evolutionary ments, its evolutionary origin was largely New York Times announced in November comparison, largely because the molecule ignored. “Evolution was dismissed as the same year, “Scientists discover a form a historical accident that didn’tneedto forms a part of the protein-making ma- of life that predates higher organisms” (4). be invoked to explain the code, which chinery at the heart of all cells. So, the A few notable researchers denounced the was seen merely as a series of chemical reasoning went, unlike other adaptive proposal of a phylogenetic classification of interactions between molecules,” embellishments in cells, it was likely to be life into three kingdoms as a misguided Woese recalls. conserved over evolutionary time and move to impose a new order, citing mi- To truly crack the code, Woese held, large enough for meaningful genetic crobiologists’ long unsuccessful attempts at the question of how codon assignments comparisons among organisms. classifying prokaryotes, an endeavor writ- evolved or why, for example, the triplet “The conserved nature of ribosomal ten off as Sisyphean. nucleotide code CCC encodes the amino RNA makes it an ideal molecule to trace Yet the reigning bipartite division of life acid , had to be addressed from an a vertical line of descent,” Pace says. was largely utilitarian, relying on differ- evolutionary perspective–aprofoundbut Further, the molecule’s universality ences of little evolutionary significance. As radical view that might shed light on the meant that it was unlikely to be shuttled evidence of archaea’s uniqueness moun- evolution of the cell itself. To that end, he laterally among organisms, unlike other ted, the group’s ranks swelled, and its adapted sequencing techniques, developed genes that were likely swapped freely in evolutionary differences from bacteria by molecular biologist Frederick Sanger, a still-evolving evolutionary soup. became established, pointing to a three- to compare the sequences of RNA in the Together with then-postdoctoral fellow pronged tree of life with a still-mysterious ribosomes of a range of microbes. George Fox, graduate students Mitchell but common root. “Archaea could not

1020 | www.pnas.org/cgi/doi/10.1073/pnas.1120749109 Nair Downloaded by guest on September 27, 2021 have been prokaryotic outliers, because Carl’s team was finding the same differ- ences between bacteria and different members of archaea,” says University of Illinois at Urbana–Champaign evolution- ary biologist Gary Olsen, whose research interests were shaped by Woese’s dis- covery. Archaea, it turned out, were more closely related to eukaryotes than bacteria (See Fig. 3). “Woese did not set out to uncover a third domain of life; he was interested in the evolution of the protein synthesis machinery and ribosomal RNA,” says Pace. Archaea, then, were a revelation to Woese as well as the rest of the scientific community. Woese says part of the skeptical stance to the 1977 report could be traced back to classical microbiology’s quiet divorce from the decade’s evolutionary thinking. “There was a disconnect between Dar- winists, who had taken over evolution, and microbiologists, who had no use for Darwinian natural selection,” he says. To make matters worse, molecular biology’s application-oriented approach, he adds, Fig. 3. A molecular tree of life. The diagram compiles the results of many rRNA sequence comparisons. smothered evolutionary considerations of (Reproduced from Microbiol. Mol. Biol. Rev., 2009, vol. 73, 565–576, doi: 10.1128/MMBR.00033-09 with life. “There was a tacit agreement be- permission from American Society for Microbiology.) tween evolutionists and molecular biolo- gists—en entente curieuse—that neither for analysis were key to the revised view debt to Woese’s trailblazing cataloging group would criticize the other.” Add to of life,” says Olsen. technique formalized in 1977. these reasons microbiologists’ reluctance The three-kingdom view of life is now “The 1977 paper is one of the most to abandon the existing classification of widely accepted, save some muffled op- influential in microbiology and arguably, life into eukaryotes and prokaryotes, and position. To boot, 16S rRNA sequencing all of biology. It ranks with the works of Woese’s phylogenetic classification met has become a mainstay in the molecular Watson and Crick and Darwin, providing with widespread resistance. “By then, the biology toolbox, helping researchers clas- an evolutionary framework for the in- concept of prokaryotes had become firmly sify biodiversity in a range of environ- credible diversity of the microbial world,” entrenched. When the phylogenetic clas- ments, including the human body. The says fi si cation was proposed, it was as if field of gained steam in Justin Sonnenburg, who studies the re- ” a crutch had been taken away, he recalls. the early 1980s, when Pace found that lationship between human diet and the technique could be used to identify gut microbes. Enduring Legacy individual microbes in a naturally oc- Edward DeLong, a microbiologist at Despite the initial criticism leveled at curring assemblage without the need to Massachusetts Institute of Technology who fi the 1977 paper, its ndings have stood grow them in the laboratory (6). “The explores biodiversity in oceans, adds that the test of time. Nearly 15 years later, realization that we could get RNA se- the “paper and its technique provided the editors of The Prokaryotes, a touch- quences from ecological samples was a quantitative metric for understanding the stone textbook of microbiology, pro- a singular moment that still makes me phylogenetic relationships among all cel- “ ” claimed, The pioneering work of Carl high, Pace recalls. Of the more than 100 lular life. That’s foundational.” Woese in cataloguing and sequencing phyla of bacteria known today, only about Like the discovery of archaea, the rRNA of prokaryotes has, for the two dozen have been successfully grown Woese’s contribution to molecular fi ’ rst time in the , in the laboratory, a fact that puts Pace s , propelled by the ardent em- fi provided a means of establishing a nding in perspective. brace of an evolutionary view of life, truly phylogenetic system for living or- Today, the Human Microbiome Pro- might be his enduring legacy. “Woese ganisms—a goal previously thought im- ject, like others aimed at identifying ” fi continues to champion the central place possible (5). For his ndings, Woese won microbes in terrestrial, aquatic, and aerial of evolution in biology, and with the bio- a MacArthur Foundation grant and the environments, is making strides thanks to medical establishment now facing chal- Royal Swedish Academy of Sciences’ the advent of rapid and inexpensive ge- lenges, such as the evolution of antibiotic . nome sequencing technology, which can resistance, the field is finally beginning to “Carl’s deep understanding of the uni- help researchers sequence entire microbial heed his words,” says Goldenfeld. versality of ribosomal RNA and the genomes for less than $1,000. Yet the field pragmatic ease of getting abundant RNA of microbial genomics owes an incalculable Prashant Nair, Science Writer

1. Arumugam M, et al. (2011) Enterotypes of the human 3. Woese CR (1958) Comparison of the x-ray sensitivity of 5. Balows AHG, Truper M, Harder DW, Schleifer KH (1992) gut microbiome. Nature 473:174–180. bacterial spores. J Bacteriol 75:5–8. The Prokaryotes (Springer, New York), 2nd Ed, Vol. 1, p vii. 2. Woese CR, Fox GE (1977) Phylogenetic structure of the 4. Lyons RD (November 3, 1977) Scientists discover a form 6. Lane DJ, et al. (1985) Rapid determination of 16S ribo- prokaryotic domain: The primary kingdoms. Proc Natl of life that predates higher organisms. NY Times, Sec- somal RNA sequences for phylogenetic analyses. Proc Acad Sci USA 74:5088–5090. tion A, p 1. Natl Acad Sci USA 82:6955–6959.

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