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Woese had noticed something about Mitch Sogin during their early interactions: the kid was not just smart but also handy around equipment. Some combination of talents—dexterity, mechanical aptitude, precision, patience, a bit of the plumber, a bit of the electrician—made him good not just at experimental work but also at creating the tools for such work. Sol Spiegelman had ordered and paid for a collection of apparatus to be used for RNA sequencing by the Sanger method; but now Spiegelman was off to Columbia, leaving behind the tools.
“So Carl inherited that equipment. But he had no one that knew how to use it.” No one, that is, until Sogin joined his lab. “I was essentially responsible for importing all the technology”—importing it from Spiegelman’s lab, and other sources, into the Woese operation. Sogin learned as much as possible from Bishop about Fred Sanger’s techniques before Bishop decamped to New York, and then Sogin became Woese’s handyman as well as his doctoral student, assembling and maintaining an array of hardware to enable the sequencing of ribosomal RNA.
Woese himself was not an experimentalist. He was a theorist, a thinker, like Francis Crick. “He never used any of the equipment in his own lab,” Sogin said. None of it—unless you count the light boxes for reading films. Sogin himself had built these fluorescent light boxes, on which the film images of RNA fragments, cast by radioactive phosphorus onto large X-ray negatives, could be examined. He had converted an entire wall of bookshelves, using translucent plastic sheeting and more fluorescent bulbs, into a single big, vertical light box, like a bulletin board. They called it the light board. Viewed over a box or taped up on the light board, every new film would show a pattern of dark ovals, like a herd of giant amoebae racing across a bright plain. This was the fingerprint of an RNA molecule. Recollections from his lab members at the time, as well as a few old photographs, portray Carl Woese gazing intently at those fingerprints, hour upon hour.
“It was routine work, boring, but demanding (#litres_trial_promo) full concentration,” Woese himself recalled later. Each spot represented a small string of bases, usually at least three letters but no more than about twenty. Each film, each fingerprint, represented ribosomal RNA from a different creature. The sum of the patterns, taking form in Carl Woese’s brain, represented a new draft of the tree of life.
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The mechanics of this effort in Woese’s lab, during Mitch Sogin’s time and for much of the next decade, were intricate, laborious, and a little spooky. They involved explosive liquids, high voltages, radioactive phosphorus, at least one form of pathogenic bacteria, and a loosely improvised set of safety procedures. Every boy’s dream. Courageous young grad students, postdocs, and technical assistants, under a driven leader, were pushing their science toward points where no one, not even Fred Sanger or Linus Pauling, had gone before. The US Occupational Safety and Health Administration (OSHA), though recently founded, was none the wiser.
The fundamental goal was to sequence variants of a molecule from the deepest core of all cellular life, compare those variants, and deduce the history of evolutionary relationships since the beginning. Woese had already settled on that one universal element of cellular anatomy, the ribosome, the machine that turns genetic information into proteins, but there remained a crucial decision: Which ribosomal molecule should he study? Ribosomes comprise two subunits, as I’ve mentioned—a small one snuggled beside a larger one, like an auricle and a ventricle of the heart, each constructed of both RNA and proteins. The RNA fractions include several distinct molecules of different lengths. At first, Woese targeted a short RNA molecule from the large subunit, known as 5S (“five-S”) for obscure reasons that I don’t ask you to contemplate. Just remember 5, a smallish number. That molecule proved unsatisfactory because its very shortness limited the amount of information it contained. The alphabet of nucleotides composing RNA is slightly different from that of DNA—it’s A, C, G, and U (for uracil) in place of T (for thymine)—and there was just not enough of the A-C-G-U alphabet in any little 5S sequence to distinguish different creatures from one another. So he switched to a longer molecule in the small subunit, and at the risk of causing your eyes to roll back in your head, I’m going to tell you its name. Why? Because it’s important, and once you’ve got it, you own it: 16S rRNA. There. Not so bad?
In English we say: “sixteen-S ribosomal RNA.” It’s a structural component of every bacterium on Earth, and bacteria were what Woese studied initially.
There’s a close variant, 18S rRNA, in the ribosomes of more complex creatures, such as animals and plants and fungi. This 16S molecule and its 18S variant, therefore, could serve as the reference standard, the great clue, for deducing divergence and relatedness among all cellular organisms. It was, arguably, the single most reliable piece of evidence, molecular or otherwise, for drawing a tree of life. And that recognition, though it never made the front page of the New York Times, was Carl Woese’s single greatest contribution to biology in the twentieth and twenty-first centuries.
The immediate goal for Woese, back in the early 1970s, was to extract ribosomal RNA from different organisms, to learn as much as possible about the genetic sequence of the chosen rRNA molecule from each organism, and to make comparisons from which he could gauge degrees of relatedness. He started with bacteria, because many kinds of bacteria are easy to grow in a lab, and their collective history is very ancient. Looking at bacteria from numerous different families allowed him the prospect of seeing contrasts, even in such a slowly evolving molecule as 16S rRNA. He and his team proceeded by extracting ribosomal RNA from the bacterial cells, purifying samples of the 16S molecules in each, and cutting those molecules into variously sized fragments with enzymes. Then they separated the fragments by electrophoresis, using an electrical field and a racetrack of soaked paper or gel.
In electrophoresis, a solution of mixed fragments is added to the racetrack, the power is turned on, and the electrical force pulls small fragments along faster than large ones, causing them to separate as distinct bands or ovals along the track. In Woese’s effort, each fragment comprised just a few of those A, C, G, U bases—maybe three, maybe five, maybe eight, maybe as many as twenty, but always a minuscule fraction of the full molecule. Those small fragments could then be pulled again, this time in a sideways direction, and their exact sequence would begin to come clear, based on the chemical and electrical differences among A, C, G, and U. Small fragments were easier to sequence by this method than one mammoth chain. AAG was easier to discern, as you might imagine, than AAUUUUUCAUUCG.
There were several stages of work. The primary run began the process of separating the fragments from one another. The secondary run, in a sideways dimension, revealed more about each fragment, which grew discretely recognizable as it raced not just down the racetrack but also now across. Those fragments, because of their radioactive content, showed as ovals burned onto the X-ray films. The oval-marked films would let an expert interpreter such as Woese infer the sequences—that is, to sort the As, Cs, Gs, and Us from one another and determine their order in each fragment. Once illuminated that way, a fragment became more like a word than like a shadowy amoeba. It had its own spelling. What was the spelling of this little word, this fragment, or that one? Was it CAAG? Or was it CAUG? Was it something a little longer and quite different—maybe CUAUGG? The answers were important because from those words, added up into paragraphs, Woese would deduce the degree of relatedness of the creatures from which they had come.
If the sequences were still ambiguous after a secondary run, as they often were, at least for longer fragments, then those were cut further, using other enzymes, and a third run was made. Rarely there might be a fourth run, but that was usually impracticable (as well as unnecessary) because the short half-life of the radioactive phosphorus that had been fed into these bacteria meant that its radiation faded quickly, and, after two weeks, the bits wouldn’t burn their images onto film. With experience, Woese developed a good sense of how to cut the fragments and get it all done in three runs at most.
Mitch Sogin and his successors did the culturing of microbes, the extraction of RNA, the cutting, and the electrophoresis. They added improvements to the methodology—different enzymes for cutting, modifications of the electrophoresis—and by 1973, the Woese lab had become the foremost user of Sanger-type RNA-sequencing technology in the world. While the grad students and technicians produced fingerprints, Woese spent his time staring at the spots. Was this effort tedious in practice as well as profound in its potential results? Yes. “There were days,” he wrote later, “when I would walk home (#litres_trial_promo) from work saying to myself, ‘Woese, you have destroyed your mind again today.’” The years between 1968 and 1977 were lonely and long. Today sequencing is a snap, but Woese was ahead of his time, gathering data like a man crawling across desert gravel on his hands and knees. He couldn’t have done it without a strong sense of purpose.
Being his assistant or his student called on a certain gravelly fortitude too. Mitch Sogin described the deliveries of radioactive phosphorus (an isotope designated as P-32, with a half-life of fourteen days), which by 1972 amounted to a sizable quantity arriving every other Monday. The P-32 came as liquid within a lead “pig,” a shipping container designed to protect the shipper, though not whoever opened it. Sogin would draw out a measured amount of the liquid and add it to whatever bacterial culture he intended to process next. “I was growing stuff with P-32. It was crazy,” he said, tossing that off as a casual memory. “I don’t know why I’m alive today.” Because the bacteria were cultured in growth media lacking other phosphorus, a vital nutrient, they would avidly seize the P-32 and incorporate it into their own molecules. Sogin would then extract and purify the ribosomal RNA, “all the while not contaminating the laboratory.” That was the hope, anyway. For separating 16S from the other ribosomal fractions, he used “home-built electrophoresis units,” cylinders of acrylamide gel through which the different molecular fragments would migrate at different speeds. (Acrylamide is a water-soluble thickener, sometimes used in industry as well as in science.) Then he would freeze the gel and attempt to slice it, like bologna, with a very precise knife. The slicing was difficult: slices would fall off when they shouldn’t, he had to work the material at just the right temperature, and “this was pretty radioactive stuff.” Sogin then cut the 16S molecules into fragments with an enzyme, and those fragments would run a race of their own, not through cylinders of gel but along a racetrack of special, absorbent paper.
One end of the long paper strip went into a receptacle known as a Sanger tank (as developed by Fred Sanger), containing a liquid buffer. The strip passed over a rack, beyond which its far end dropped into another Sanger tank, and both tanks were wired to an apparatus that provided the electrical pull. At the bottom of the tanks were high-voltage platinum electrodes, covered by three inches of liquid buffer and then at least fifteen inches of Varsol, a solvent not unlike paint thinner, intended to cool the paper strip. “Varsol is both volatile and explosive,” Sogin said. The power source delivered around 3,500 volts and plenty of amps, he recalled—“certainly enough to kill you.” Also enough, with an errant spark into the Varsol, to blow you up.
This whole panoply of dangerous, intricate machinery dwelt within a shielding hood that could be closed behind large sliding doors, floor to ceiling, in a nook off the main lab known as the electrophoresis room. Set up the system, close the doors, turn on the juice, hope for the best. “I was too stupid to be afraid of anything,” Sogin told me. “Too naïve. Too young. Immortal.” He was also lucky. Nobody got hurt.
Around the time Sogin finished his doctorate and prepared to leave, Woese hired a young woman named Linda Bonen, a walk-in from a different building, to take on some of the technical work. Raised in rural Ontario, she had come down to the University of Illinois and gotten a master’s degree in biophysics. Woese trained her for this new lab work himself—how to chop the RNA into fragments, how to run the electrophoresis in two dimensions, how to prepare the films, even a bit about how to interpret them, deducing which spot on a film represented which fragment, which little blurt of letters. Was it UCUCG, or was it UUUCG? Tricky to tell. But here’s GAAGU, obviously different. Woese coached her patiently on the tasks and their meaning.
“He was very good about bringing me along,” Bonen recalled four decades later, when I visited her at the University of Ottawa, where she was by then a biology professor herself, gray haired, deeply expert in molecular genetics, gentle mannered as a schoolteacher. “The end product would be a ‘catalog’ for microbe X,” she said, meaning simply a list of the different fragments found within the 16S rRNA molecules of that creature. A catalog. If the fragments resembled words, these catalogs were the paragraphs. Comparing one catalog with another revealed the degree of similarity between any two organisms, by a very precise standard, and more dissimilarity could be taken to reflect more distance in evolutionary time. Where had the great limbs diverged from the trunk, the big branches from the limbs—and why there, and why then, and leading to what creatures? Beyond the mind-numbing methodology of data collection, those were the questions Woese hoped to answer.
What was he like, I asked Bonen, as a boss and a teacher?
“Well, he never came across as a boss,” she said. “He was very soft-spoken and quiet, reserved. I’m sure you’ve …” She hesitated. “Did you know him yourself? Did you ever meet him?”
Never. I didn’t explain to her, but the reason was simple: Woese died, in late 2012, an old man taken down hard and fast by pancreatic cancer, just before I picked up the trail.
“To everybody, he was Carl,” she said. “He was not a boss.”
Bonen showed me a photograph, a memento from her personal files: the youngish Carl Woese in his lab, bathed by yellow-green light, jaw set firmly, gazing up at a pattern of dark spots. Short brown hair, striped sport shirt, handsome and jaunty enough to have stood onstage amid the Beach Boys. Almost apologetically, she said: “That’s the only good picture I have.” This was all different from what I had expected. My mental image was of the later man: the shy, crotchety, and august Dr. Carl Woese.
He was shy, yes, Bonen said. But “august,” no, that was wrong, not a word she would ever … and here again her voice fell away. Then she added: “I only knew him in a short period of time.”
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Ken Luehrsen, soon after Linda Bonen’s short period, had a different sort of experience in the Woese lab. He was an undergraduate at Illinois when he first encountered Woese as one of the instructors for a seminar in developmental biology, well outside Woese’s field of expertise. The logic behind this mismatch, according to Luehrsen, was that “other professors just liked to hear Carl’s take on things so they might incorporate some of his ideas into their own research.” Woese was notoriously brilliant, full of ideas, but jealous of his expended effort. “Undoubtedly, Carl found an opportunity to get a teaching credit where he didn’t have to do too much work.” In a seminar, students would be assigned to make presentations explicating this journal paper or that one, and Woese could easily moderate the discussion. He hated classroom teaching of the more arduous sort—preparing and delivering lectures, God forbid—because “he felt it took him away from his real love (#litres_trial_promo): understanding the origin and evolution of life.”
After the seminar acquaintance, Luehrsen went to this formidable figure and asked to do an honors project under his guidance. Woese not only accepted him but also, to Luehrsen’s surprise, “he plopped me down in his office (#litres_trial_promo),” a very small room containing two desks, both covered with chaotic stacks of papers, and said (either seriously or as a tease) that it was so he “could keep an eye on me.” Luehrsen was befuddled. Should he really be there? Should he scram whenever the phone rang and give Woese his privacy? His discomfort eased when he saw that Woese himself spent little time in that office and most of his time in the lab, “reading 16S rRNA fingerprints at his light board.”
After Woese’s death, Ken Luehrsen wrote a short memoir describing the man’s work, his temperament, and their interactions so long ago, for publication with other Woese tributes in a scientific journal. He brought it all to mind again when I tracked him down in San Carlos, California, on the edge of Silicon Valley, where he was now a senior scientist and biotech inventor in the late afternoon of his career, consulting for a small company lodged behind glass doors in an office park. By that time, he held many patents in biotechnology, for methodologies to create antibodies and other molecular products, and lived comfortably in an old counterculture enclave across the peninsula, a place known as Half Moon Bay, from which he could commute to the action. He worked when he felt like it. At this firm, he was the grizzled elder, surrounded by smart young colleagues seated in carrels, for whom “Woese” was at most a dimly recognizable name, like “Darwin” or “Fibonacci.” Tall and thin, with a goatee, relaxed and a little sardonic, Luehrsen suggested we escape downtown for sushi—after which we talked for most of the afternoon.
“I may have been a junior at the time,” he said about his first acquaintance with Woese. “I didn’t know anything.” Despite Luehrsen’s ignorance, the great man invested some effort in him; a private tutorial was less abhorrent to Woese than lecturing at banks of indifferent faces. “He explained to me what he was doing. I maybe understood a quarter of it.” But the youngster paid close attention and caught on fast. “I think he saw somebody who was interested, and I was a pretty hard worker.”
It was 1974 when Luehrsen joined the Woese lab as an undergraduate assistant, paired with a graduate student and assigned the unenviable job of extracting radioactive rRNA from bacterial cultures. They would dump ten millicuries (a large dose) of P-32 into this culture or that and, after overnight incubation to let the bacteria suck it up, spin the mixture in a centrifuge to gather the hot bacteria into a little pellet. After dissolving the pellet in a buffer, they would squash that brew through the laboratory version of a French press, not too unlike the one you might use for coffee. This served to rip open the bacterial cells and set their innards adrift. Luehrsen and his partner would then pull out the ribosomal RNA by chemical extraction, after which the different fractions—the 16S molecules versus the others, including that shorter one, known as 5S—were separated using Mitch Sogin’s home-built cylinders of acrylamide gel. In addition to acrylamide (today recognized as a probable carcinogen), they were working with phenol, chloroform, ethanol, and the radioactive phosphorus. “What a mess that often was! (#litres_trial_promo) The Geiger counter was always screaming,” Luehrsen wrote in his memoir.
One of the bacteria he cultured and squashed was Clostridium perfringens, the microbe responsible for gas gangrene, an ugly form of necrosis that takes hold in muscle tissue made vulnerable by wounds, especially the sort that lay open among injured soldiers on battlefields. When he realized this, Luehrsen complained, but Woese “just chuckled and said not to worry (#litres_trial_promo)” in the absence of an open wound. He had been to medical school for “two years and two days,” Woese said, and he could assure Luehrsen that Clostridium perfringens was unlikely to give him gangrene. Luehrsen took the episode as a lesson—not a lesson to trust Woese but to rely on his own perspicacity more—and never probed the matter of why Woese had quit medical school two days into his third-year rotation in pediatrics.
After graduating from Illinois in 1975, Ken Luehrsen stayed to work toward a PhD under Woese’s supervision, just as Woese shifted the lab’s focus, slightly but critically, in a way that would lead toward his most startling discovery. So far, they had targeted their molecular analyses on common bacteria and a few other single-celled organisms such as yeast—easy to obtain, easy to grow in the lab. But that was just a preliminary effort as they refined their methods. “One of the things he wanted to do was to look at unusual bacteria,” Luehrsen told me. Woese hoped this might give a view “deep into evolution,” where he could see “deep divergences” between one big branch of life and another. So he struck up a collaboration with a colleague in the Microbiology Department, Ralph Wolfe, one of the world’s leading experts in culturing a group known as the methanogens.
Methanogens: their name derives from an odd aspect of their biochemistry, producing methane as a byproduct while metabolizing hydrogen and carbon dioxide in environments lacking oxygen. To say it more plainly, these bugs generate swamp gas in muddy wetlands, from which it bubbles up, and similar gas in the bellies of cows, whence it emerges by belch and fart. Certain methanogens also thrive beneath the Greenland ice cap, deep in the oceans, and in other extreme environments, such as hot desert soils. Despite these shared metabolic traits, Ralph Wolfe advised Woese, there was an odd discontinuity among the assemblage of methanogens—discontinuity in terms of their shapes. Some were cocci (spherical), some were bacilli (rod shaped). Since the cocci and the bacilli were considered two distinct kinds of bacteria, microbiologists had been puzzled about how to classify the methanogens—together by metabolism or separately by shape. That conundrum captured Woese’s interest.
Having told me this much, and more, Ken Luehrsen finished our conversation and sent me away with some gifts. One was a black-and-white print of a photo he took in the mid-1970s, a snapshot, showing Woese at his light board, engrossed before a pattern of dark spots, with a handful of felt-tip pens for color coding what he saw, a pencil for data registry behind his right ear. Luehrsen’s other gift was a single yellowing sheet—not a copy, the original—from his own notebook of the time. It was a catalog of fragments from an organism, more of those telling blurts of the four coding letters, neatly recorded in two columns. UCUCG. CAAG. GGGAAU, and dozens more. At the top, also hand lettered, an abbreviation indicated the name of the organism as it was known at the time: Methanobacterium ruminantium. Later, I realized that, notwithstanding the name, this was no bacterium. Luehrsen had given me the genetic rap sheet on a separate form of life.
Annotating RNA fragments on a “fingerprint” film.
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How do you classify the methanogens? Where do they fit on the tree of life? To what other little bugs are they most closely related? Those questions, which Woese and his colleagues were asking themselves in the mid-1970s, fell within the scope of an important discipline with a dry name: bacterial taxonomy. That’s the enterprise of sorting bacteria into nested groups: species, genera, families, etcetera. You name something Methanobacterium ruminantium, and then where do you put it?
This may sound like an exercise in arcana, a marginal activity of risible triviality beside which stamp collecting looks like an adventure sport. Bacteria are tiny, relatively simple, invisible. But if being invisible made things unimportant, gravity and microwaves would be unimportant too. It’s useful to recall that most life-forms on Earth are microbial, that they determine the conditions of existence for the rest of us, and that even the human body contains at least as many microbial cells (those tiny passengers that live in your gut, on your skin, in the follicles of your eyelashes, and elsewhere) as human cells. Your environment is highly microbial too. Your food. The air you breathe. Microbes run the world, and a very large portion of those microbes are bacteria. Some of them serve as helpful partners of humanity. Some are benign. Some are rapacious, ready to poison your blood, fill your lungs, kill you. So it’s no small matter, telling one bacterium from another.
Scientists once believed it might be possible to do this from visual evidence obtained through a microscope. They even presumed that the concept of species, as understood for animals and plants and fungi, could be applied to bacteria. These were useful simplifications in their era—like the simplifications of Newtonian physics, before correction by Einstein—but that era was a long time ago.
The early hero in the field was a man named Ferdinand Julius Cohn, a botanist and microbiologist at the University of Breslau (now Wrocław, Poland) during the late nineteenth century. Cohn is an appealing figure, and only partly because his important contributions have been overshadowed by those of better-remembered contemporaries whose accomplishments were more practical and dramatic: Louis Pasteur, Robert Koch, Joseph Lister. They worked on disease, agriculture, and wine. Cohn worked mainly on describing and classifying microscopic organisms. No one makes Hollywood movies about bacterial taxonomists.
Cohn wasn’t the first researcher to classify bacteria, making distinctions between kinds, trying to place the whole group in its proper position on the tree of life. But his effort was more hardheaded and percipient than the others, and he did much to bring bacteriology out of a fog of confusions that had lingered for more than a century, ever since startled observers such as Leeuwenhoek had noticed these little creatures through simple microscopes. Several insights and adjustments of method helped him make progress. Microscopy improved, with better lenses and precision instruments in which they were mounted. Cohn’s lab started culturing bacteria on solid media such as slices of cooked potato, not in liquid nutrient, the old way. That allowed Cohn to choose, cultivate, and consider different strains separately. Also, he recognized that physiological and behavioral characteristics as well as structural ones could be useful for distinguishing bacterial species: How do they grow in different media? How do they move? By this time, too, Cohn had embraced Darwin’s theory of evolution, and so it made sense to him that bacterial strains might change and adapt over time. This was incremental change, very different from the sort of utter transformation—one bacterial form suddenly morphing into another—that some scientists imagined to occur. Cohn didn’t buy transformation. He saw bacteria as fundamentally stable in their identities. Finally, he published his system, dividing them into four tribes: spherical, rod shaped, filamentous, and spiral, each of which got an imposing Latinate name. Within the tribes, he drew finer distinctions, separating them into genera and species.
Not everyone in the field accepted Cohn’s classification of bacterial species or his conviction about their stable identities, and the idea of shape-shifting bacteria lingered for more than a decade. The longer judgment of science historians was good to him, as a man and a scientist, noting his “reserve” against self-promotion, his modesty, his eloquent lecturing, and his success in “disentangling almost everything that was correct (#litres_trial_promo) and important out of a mass of confused statements on what at that time was a most difficult subject to study.” Besides arguing for the reality of bacterial species and sketching a way to classify them, Cohn did much, along with Pasteur, to kill the resilient delusion that new life-forms arise by spontaneous generation. They don’t, he showed. When bacteria seem to appear out of nowhere, it’s because they have arrived from somewhere: contamination, floating through the air, reawakening spores. Cohn’s work was “entirely modern in its character and expression (#litres_trial_promo),” according to an authoritative chronicler of the field, writing in 1938, “and its perusal makes one feel like passing from ancient history to modern times.” But what looked modern in 1938, of course, doesn’t look modern now.
Even the devoutly empirical Ferdinand Cohn made mistakes. For one: after all his research, he still believed, as many of his colleagues did, that bacteria belong to the kingdom of plants. So his tree of life, by later standards, was badly wrong. For another: the premise of radical transformation, one bacterial form to another, turns out to be vastly more complicated than he could imagine.
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Chaos” was the name of the group (#litres_trial_promo) into which Linnaeus, the great systematizer, in the 1774 edition of his Systema Naturae, had lumped Leeuwenhoek’s bacteria and other little creatures. That was a durable judgment. Even well into the twentieth century, decades after Ferdinand Cohn, experts were still arguing about whether bacterial taxonomy was a meaningful enterprise or hopelessly chaotic.
Beginning in 1923, the standard source for identifying bacteria was a thick compendium, Bergey’s Manual of Determinative Bacteriology, edited by the bacteriologist David Hendricks Bergey. But as microbiology progressed, it became clear that the Bergey’s system was vague, inconsistent, and, on some fundamentals, inaccurate. It didn’t offer a tree of bacterial life. It was only a glorified field guide. Still, other researchers who critiqued Bergey’s Manual, and then tried to improve on it, found the critiquing much easier than the improving. The task of bacterial classification was just so difficult. There was almost no fossil record of bacterial ancestors. There weren’t enough differences of external shape and internal anatomy, even as seen through powerful microscopes, to support fine distinctions. Physiological characters could also be misleading, if they reflected parallel adaptations rather than shared ancestry. What did that leave for a classifier to use? (Hint: Carl Woese would offer an answer, but not until 1977.) This conundrum came to a head in 1962, when two of the world’s leading microbiologists, C. B. van Niel and Roger Stanier, essentially threw up their hands in despair.
Van Niel was a Dutchman, educated in Delft, who in 1928 decamped to California, where he taught at a marine biological station that was part of Stanford University. His particular interests were bacterial physiology and taxonomy. Roger Stanier was a younger Canadian who became van Niel’s student, then his special protégé, then his collaborator. In 1941, when Stanier was still just twenty-five years old, he and van Niel coauthored an influential paper on bacterial classification.
That paper stood as definitive for a generation—until both authors renounced it. Stanier himself later admitted some embarrassment about it, all the more so because he had arm-twisted van Niel to sign on as coauthor—student and teacher together, although the work was mainly Stanier’s. What the paper contained, besides a pointed critique of Bergey’s Manual, was a shiny new proposal for classifying bacteria—not just a checklist or a field guide but a “natural” system reflecting their evolutionary relationships. That system divided the familiar bacteria into four major groups (as Ferdinand Cohn had done) and placed them in a kingdom of simple creatures along with just one other group: the blue-green algae.
Algae? Yes, the blue-green algae, as they were then called, had long been an ambiguous group, because they seemed to straddle the line between bacteria and plants. (This was partly what allowed Cohn to believe that all bacteria were plants—the blurry lines around blue-green algae.) Algae was a catchall term for a loose assemblage of creatures that photosynthesize, including these tiny blue-green creatures, but that didn’t mean all algae shared a single common ancestor. Did they? Stanier and van Niel said no. By their new definition of things, blue-green algae were more similar to bacteria than to other algae, and these two groups should be lumped together in a kingdom of their own, apart from everything else. Eventually they labeled such cells procaryotic—meaning “before kernel,” as I’ve mentioned—and set them in contrast to eucaryotic cells, comprising all else. (Their spellings were later corrected, from more accurate transliteration of the Greek roots, to prokaryotic and eukaryotic.) The kernel in question was a cell nucleus. Just as a bacterium doesn’t have one, neither do the creatures that were then known as blue-green algae (and are now classified as cyanobacteria). Advances in microscopy since the end of World War II, including electron microscopy, had given microbiologists a better view of those distinctions and others, making possible a fresh analysis of what a bacterium is—and what it isn’t. Stanier and van Niel offered that fresh analysis along with the prokaryote category in a new paper, published in 1962, titled “The Concept of a Bacterium.” By their lights, the “abiding intellectual scandal of bacteriology” (#litres_trial_promo) was that no such concept had ever been clearly delineated. What was a bacterium? Um, hard to say.
They tried to correct that by placing bacteria and blue-green algae together as prokaryotes, and setting them in contrast to the alternative category, eukaryote, which encompasses all other forms of cellular life. The chief distinguishing features of a prokaryote, according to Stanier and van Niel, were: (1) no cell nucleus, (2) cell division by simple fission, rather than the elaborate process of chromosome pairing known as mitosis, and (3) a cell wall strengthened by a certain sort of latticework molecule with a fancy name, peptidoglycan. I know, it looks like the moniker of a flying reptile from the Jurassic. Forget about it for now, and when peptidoglycan comes back as an important clue toward understanding the deepest structure of the tree of life, and the twig on the branch on the limb from which we humans have sprouted, I’ll remind you.
The dichotomy between prokaryotes and eukaryotes, creatures without cell nuclei and those with, relatively simple beings and relatively complex, became a fundamental organizing principle of biology. Stanier and his two coauthors of a textbook would later say that it “probably represents the greatest single evolutionary discontinuity (#litres_trial_promo) to be found in the present-day living world.” It was also a salubrious reminder to humans of our inescapable linkage to other creatures, including some very humble ones. We are, at the most basic level of classification, eukaryotes. So are amoebae. So are yeasts. So are jellyfish, sea cucumbers, the little parasites that cause malaria, and rhododendrons. To an average person, the gap between an amoeba and a bacterium may seem narrow (partly because most of us have never, or at least not since high school biology, looked through a microscope at either), but the prokaryote-eukaryote distinction reveals it as oceanic. You could think of the living world—and, beginning from Stanier and van Niel’s 1962 paper, biologists did think of the living world—as divided into proks and euks.
Besides putting that idea into play, “The Concept of a Bacterium” is notable for having signaled surrender, by Stanier and van Niel, in the battle of bacterial taxonomy. About this they were candid, confessional, and brusque. Ever since Leeuwenhoek, microbiologists had been seeking the best way to classify bacteria. Ever since Darwin, they had been arguing about how one bacterium was related to another. Enough was enough. “Any good biologist finds it intellectually distressing (#litres_trial_promo) to devote his life to the study of a group that cannot be readily and satisfactorily defined.” C. B. van Niel himself had devoted forty years. He and Stanier now alluded to the “elaborate taxonomic proposal” they had published (#litres_trial_promo) back in 1941, “which neither of us cares any longer to defend.” Never mind that. They admitted having “become sceptical about the value” of any such formal systems, or the effort spent to develop them, although they still affirmed the importance of figuring out just what the devil bacteria are.
This skepticism, this taxonomist’s despair, had been wiggling up inside van Niel for a long time. Two decades earlier, even as he was signing onto that first elaborate proposal, he had confessed his gloom to Stanier in a letter: “Many, many years ago I often went around with a sense of futility of all our (my) efforts. It made me sick to go around in the laboratory (this was in Delft) and talk and think about names and relations of microorganisms.” Was any of it real? Was there any value to putting bacteria into labeled boxes? “During those periods (#litres_trial_promo) I would go home after a day at the lab, and wish that I might be employed somewhere as a high-school teacher.” Not that he would enjoy such teaching, he realized, but at least “it would give me some assurances that what I was doing was considered worth-while.” Nowadays we might see that as a signal of bipolar disorder, but it’s just as likely that van Niel simply viewed bacterial taxonomy with great clarity.
Under their revised spellings, prokaryote and eukaryote, those two became enshrined for a generation as the most fundamental categories of life. Eukaryotes had cell nuclei. Prokaryotes did not. That dichotomy seemed to represent, as Stanier and his coauthors had written, the greatest single evolutionary divide in the living world. There were two basic kinds of creature, the proks and the euks, and there was nothing between.
What makes this worth knowing is that Carl Woese proved it wrong.
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As of early 1976, with Ken Luehrsen and others still helping, Woese had done his unique form of catalog analysis on samples from roughly thirty species, using differences in ribosomal RNA molecules to measure their relatedness. Most were prokaryotes, but he also looked at a few eukaryotes (which carried that slightly different molecule in their ribosomes, 18S rRNA instead of 16S), including yeast, for purposes of gross comparison. He could tell a prok from a euk just by inspecting the spots on a sheet of film. And he was eager to see those “unusual bacteria,” the methanogens, about which Ralph Wolfe had alerted him.
The tricky thing about methanogens was that, since oxygen poisoned them, they were hard to grow in a laboratory. But Wolfe’s lab team included an ingenious doctoral student, Bill Balch, who had solved that problem by devising a way to culture methanogens in pressurized aluminum tubes with black rubber stoppers, and using syringes to move things in and out. Balch gave the methanogens an atmosphere of hydrogen and carbon dioxide instead of oxygen, plus a liquid growth medium, and they thrived. Woese sent his own postdoc, a rangy young man named George Fox, trained in chemical engineering, to work with Balch on growing some of these methanogens and tagging them with radioactive phosphorus. Fox, Ken Luehrsen, and other members of the Woese lab then combined their efforts on the rest of the process: extracting the radioactive RNA, purifying it to get concentrations of 16S and 5S molecules, chopping those molecules into pieces, running the electrophoresis to separate the fragments, and printing the spots onto films. Their first methanogen carried a formal name so long (Methanobacterium thermoautotrophicum) that even Woese himself dismissed that as “a fourteen-syllable monstrosity (#litres_trial_promo)” and preferred using a shorter label, denoting the particular laboratory strain: delta H. Examining its primary fingerprint on his light board, Woese noticed something odd.
He was practiced enough by now at reading such fingerprints that he could immediately recognize a certain pair of small fragments, common to all bacteria, that “screamed out” their membership in the prokaryotes (#litres_trial_promo). He looked for them on the primary film from delta H. They were missing. Intrigued but patient, he waited for the secondary fingerprint, with the fragments pulled sideways to reveal more detail. He got that from his technician several days later. On June 11, 1976, he taped the primary film up on his light board again, with the secondary now in front of him on the light table, and began trying to interpret what he saw. He intended, as usual in this stage of the process, to use the secondary film as a guide for inferring the base sequences of the fragments in the primary pattern. Apart from his board and his table, the room was dark. His face, we can imagine, reflected an eerie glow. Quickly he noticed more oddities.
The two missing fragments were still missing, but it wasn’t just that. Woese turned to a different part of the pattern, expecting to see another familiar fragment—a “signature” sequence in all prokaryotes (#litres_trial_promo). Not there. Instead, he found a strange fragment, a longish sequence that shouldn’t have been present at all. “What was going on? (#litres_trial_promo)” he later recalled wondering. This methanogen rRNA just “was not feeling” prokaryotic. And the more fragments he sequenced, the less prokaryotic it felt. By this time, he knew the sequences of ribosomal RNA in bacteria so intimately that his “feel” for the molecule was a persuasive standard of normality. And something in this particular creature, delta H, was abnormal. Some bacterial fragments were appearing where expected, as expected, yes. But some others looked eukaryotic, suggesting a completely distinct form of life: a yeast, a protozoan, what? And still others were just weird. What was this RNA? he wondered, and what manner of organism did it represent? It couldn’t be from a prokaryote. It wasn’t eukaryotic. It wasn’t from Mars, because it contained too many familiar stretches of RNA code. “Then it dawned on me (#litres_trial_promo),” he wrote. There was “something out there”—out there in the teeming ecosystems of planet Earth, he meant—other than prokaryotes and eukaryotes. A third form of life, separate.
Woese called this, whimsically, his “out-of-biology experience.” (#litres_trial_promo) It would be the watershed moment of his scientific life.
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After his death in December 2012, Woese’s files of scientific correspondence, manuscripts, journal articles, and other materials went to the University of Illinois Archives to be indexed, curated, and preserved. The archives are held in several different locations, one of which is the Archives Research Center, a sort of annex, housed in an old, barnlike building of red brick on Orchard Street near the south edge of the campus. A sign in front identifies this, confusingly but historically, as the Horticulture Field Laboratory; a bank of yew bushes and a riot of hostas guard the entrance. Inside, filed neatly in thirty-four boxes that can be accessed by request, are the Carl Woese Papers. I was working there at a table one hot July afternoon, reading through letters, looking for clues about the human side of this peculiar man, when John Franch arrived, wearing a dark T-shirt and a ball cap. Franch is the assistant archivist who was sent to clean out Woese’s lab after the funeral, and who knows the material found there better than anyone else. He had heard about my interest and wanted to show me something.
He led me toward the back of the building, where the roof arches high, and unlocked a door. This was one of the “vaults,” he told me, that formerly served for storing fruit—apples in particular—from the horticultural research orchards from which Orchard Street got its name. At one point, there were 125 varieties of apple grown just behind the building, and they came in by the basket and the crate to be stored here or pressed for cider and vinegar. Beyond the door, we entered an air-conditioned room, empty of apples now but lined along its left side with tall metal shelves, along its right side with tables. The shelves held hundreds of large, flat yellow boxes—the original packaging of Kodak medical X-ray films—representing the library of Woese’s RNA sequencing fingerprints. Each box was labeled along its edge with a date and the organism whose fragments were depicted.
Across the room, some films lay on the tables, where Franch had been working over them. He showed me three large sheets, carefully taped together, forming a triptych of images. I stared at the patterns of dark spots: amoebae galloping on a plain. To me, they made no particular sense. But to Woese, they had spoken eloquently of identity, relationship, evolution. If something was odd, he would have seen it.
This is delta H, Franch said.
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Immediately after his epiphany, Woese shared it with George Fox, the postdoc he had assigned to work with Bill Balch on growing the methanogens. As recalled later by Fox, Woese “burst into my room in the adjoining lab (#litres_trial_promo)” with the announcement that they had something unique. From there he proceeded throughout the lab, among his young students and assistants, “proclaiming that we had found a new form of life (#litres_trial_promo). He then pointed out,” by Fox’s memory, tart and amused, “that this was of course contingent on my having not screwed up the 16S rRNA isolation.” Being cautious, they repeated the whole process with delta H and got the same result. So no, Fox hadn’t screwed up.
“George was always skeptical,” Woese himself wrote (#litres_trial_promo) later about their reactions to the discovery, adding that he valued such skepticism as good scientific instinct. Fox’s doctorate in chemical engineering suited him well to offset epiphanic leaps, even by the boss, with empirical caution. In fact, their shared instinct for skepticism about such a startling result helps explain why these two men worked so well together. But the anomalies in the fingerprints persuaded Fox too. By his account, they seemed to “jump off the page (#litres_trial_promo),” and he agreed that those differences suggested a third, very distinct form of life.
Still, Woese and Fox both knew that convincing other scientists of such an epochal discovery would be difficult. More data were needed. So the Woese lab went back to work, with Balch’s methodology and help, on culturing and fingerprinting still another methanogen. Woese and his colleagues worked quietly, for the time being. By the end of 1976, they had five additional genetic catalogs from five more methanogenic microbes, all quite different from one another but sharing signs of a much greater, much deeper, and shared difference from anything else known to exist.
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Bacteria are versatile and diverse. That’s an understatement. Widespread—another understatement. They are hard to categorize, hard to identify, hard to sort into related groups, as even Stanier and van Niel finally admitted. They are nearly ubiquitous across most parts of Earth’s expanse, in both natural and human-made environments, floating through the air, coating surfaces everywhere, awash in the oceans, even present in rocks deep underground. Your skin as I’ve said is covered with them. Your gut is teeming. Your human cells may be outnumbered by them at a three-to-one ratio in your body. Bacteria live also in mudholes and hot springs and puddles and deserts, atop mountains, deep in mines and caves, on the tabletops at your favorite restaurant, and in the mouths of you and your dog.
A species called Bacillus infernus has been cultured from core samples of Triassic siltstone, buried strata at least 140 million years old, drilled up from almost two miles beneath eastern Virginia. Under the Pacific Ocean, 35,755 feet deep in the Mariana Trench, lie sediments that have also yielded living bacteria. In Antarctica, a body of water known as Subglacial Lake Whillans, lidded by half a mile’s thickness of ice and supercooled to just below zero, harbors a robust community of bacteria. They thrive there in the darkness and cold, eating sulphur and iron compounds from crushed rock.
Then again, some like it hot. Those are called thermophiles. Among the most famous of thermophilic bacteria is Thermus aquaticus, first cultured from a sample collected in Yellowstone National Park by the microbiologist Thomas Brock and a student, Hudson Freeze, in 1966. Brock and Freeze had found it in a steaming, multicolored pool called Mushroom Spring, in Yellowstone’s Norris Geyser Basin, at a temperature of about 156 degrees Fahrenheit. Functioning in such heat, Thermus aquaticus contains a specialized enzyme for copying its DNA, one that performs well at high temperatures, which became a key element in the polymerase chain reaction technique for amplifying DNA. That technique, widely useful in many aspects of genetic research and biotech engineering, earned its chief developer (but not Thomas Brock) a Nobel Prize.
Other heat-loving bacteria can be found around hydrothermal vents on the sea bottom, where they help anchor the food chains, producing their own organic material from dissolved sulfur compounds vented out with the hot water, and being fed upon by little crustaceans and other animals. A giant tube worm, one of those gaudy red creatures that waggle around such vents, with no mouth, no digestive tract, gets its nutrition from bacteria growing within its tissues.
By one estimate, the total mass of bacteria exceeds the total mass of all plants and animals on Earth. They have been around, in one form or another, for at least three and a half billion years, strongly affecting the biochemical conditions in which most other living creatures have evolved. That we don’t see bacteria is simply because our eyes are not calibrated to the appropriate scale. There may be more than a billion bacterial cells in an average ounce of soil, and five million in a teaspoon of fresh water, but we can’t hear their crackle or their fizz. A single kind of marine bacteria known as Prochlorococcus marinus, which drifts free in the world’s tropical oceans and photosynthesizes like a plant, may be the most abundant creature on Earth. One source places its standing population at three octillion individuals, a number that looks like this: 3,000,000,000,000,000,000,000,000,000.
They vary in shape and in size—interestingly in shape, drastically in size. A bacterial cell, on average, is about one-tenth as big as an animal cell. At the upper end of the range is Thiomargarita namibiensis, an odd thing discovered on the sea floor near Namibia, its cells ballooning up to three-quarters of a millimeter in diameter, stuffed with pearly globules of sulfur. At the lower end of the range is Mycoplasma hominis, a tiny bacterium with a tiny genome and no cell wall, which manages nonetheless to invade human cells and cause urogenital infections.
Bacterial shapes, as I’ve mentioned, range through rods, spheres, filaments, and spirals, with variations that in some cases represent adaptations for movement or penetration. It turns out that their geometries, notwithstanding the efforts and convictions of Ferdinand Cohn, are unreliable guides to their phylogeny. Shape can be adaptive, but adaptations can be convergent as well as ancestral. Roundness may be good as a hedge against desiccation. Elongation as a rod or filament seems to help with swimming, and a flagella definitely does. Filamentous bacteria that are star shaped in cross section, recently discovered in a wonderfully named substance called “mine-slime,” (#litres_trial_promo) deep in a South African platinum mine, may profit from all their surface area by way of enhanced absorption in nutrient-poor environments. The twisting motion of spirochetes, such as the ones that cause syphilis and Lyme disease, evidently allows them to wiggle through obstacles that other bacteria can’t easily cross, such as human organ linings, mucous membranes, and the barrier between our circulatory system and our central nervous system—a fateful degree of access. Even the less dynamic shapes, the short rods known as bacilli, the spheres known as cocci, and the rods slightly curved like commas, serve well enough the bacteria responsible for a long list of diseases: anthrax, pneumonia, cholera, dysentery, hemoglobinuria, blepharitis, strep throat, scarlet fever, and acne, among others.
Although many bacteria live as solitary cells, taking their chances and meeting their needs independently, others aggregate into pairs, clusters, little scrums, chains, and colonies. The coccoid cells of Neisseria gonorrhoeae, which cause gonorrhea, lump together by twos, forming bilobed units resembling coffee beans. The genus Staphylococcus gets its name from Greek words for “granule” (kókkos, the spheroid aspect) and “a bunch of grapes” (staphylè), because staph cells tend to bunch. Most of the forty staph species are harmless, but Staphylococcus aureus can inflict skin infections, sinus infections, wound infections, blood infections, meningitis, toxic shock syndrome, plus other nasty conditions, and if you’re so unlucky as to pick up a dose of those little grapes in one of their antibiotic-resistant forms, such as MRSA, a monstrous product of horizontal gene transfer (as I’ve mentioned, and to which I’ll return), you could be in a world of hurt. Cells of Streptococcus species, including those that cause impetigo and rheumatic fever, stick together like beads on a chain.
Bacteria can also form stubborn, complex films on certain surfaces—the rocks of a sea floor, the glass wall of an aquarium, the metal ball of your new artificial hip—where they may cooperate together in exuding a slimy extracellular substance that helps nurture them collectively, maintain the stability of their little environment, serve as a sort of communications matrix among them, and even protect them from antibiotics. These living slicks, known as biofilms, can be thinner than tissue paper or as thick as a good dump of snow, and may incorporate multiple species. The little rods of Acinetobacter baumannii are infamous for their ability to lay down persistent biofilms on dry, seemingly clean surfaces in hospitals.
Cyanobacteria, including that monumentally abundant Prochlorococcus, convert light to energy and deliver, as byproduct, a large share of Earth’s free atmospheric oxygen. Purple bacteria photosynthesize too, but do it by drawing upon sulfur or hydrogen instead of water as fuel for the process, and they don’t produce oxygen. Lithotrophic bacteria, the rock eaters, deriving their energy from iron, sulfur, and other inorganic compounds, exist in more ingenious variants than you care to know. Japanese researchers have recently discovered a new bacterium, Ideonella sakaiensis, that digests plastic. Certain enterprising ocean bacteria, such as Marinobacter salarius, have risen to the challenge of degrading hydrocarbons from the Deepwater Horizon oil spill. Other bacteria are quite capable, in the presence of oxygen or without it, of feasting on garbage, sewage, various inorganic compounds, plants, fungi, and animal tissue, including human flesh. Lactic acid bacteria, which may be rod shaped or spherical, turn up in milk products, busy at their task of carbohydrate fermentation and resistant to the acid they create. Many of them also like beer.
Not all such particulars were known to Carl Woese in 1977 as he examined the fingerprints from his first few methanogens. But the vast scope, ubiquity, and multifariousness of bacteria certainly were. The terrain of bacteriology was known even better to Ralph Wolfe, who had trained in the classic fundamentals under van Niel and others. Woese’s reaction to his own preliminary results must have seemed all the more radical, then, all the more shocking, as he shared it not just with George Fox and members of his own lab but also with Wolfe, just after they repeated the rRNA analysis of delta H, the first methanogen. “Carl’s voice was full of disbelief (#litres_trial_promo),” Wolfe wrote in a memoir, “when he said, ‘Wolfe, these things aren’t even bacteria.’”
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Ralph Wolfe told me the same story, with some elaboration, thirty-nine years later when I called on him in Urbana. By then, he was an emeritus professor of microbiology, ninety-three years old, a frail and slender gentleman with a quick smile, still maintaining his office and coming to it, as though retirement were not an entirely satisfying option. On the wall behind his desk hung a replica of Alessandro Volta’s pistola, a gunlike device invented by Volta in the late 1770s for testing the flammability of swamp gases, including methane. On the desk itself were papers and books and a computer.
Woese’s lab back in the day had been in Morrill Hall, on South Goodwin Avenue, and Wolfe’s was in an adjacent building, connected by a walkway. Woese would occasionally trundle over on various business. “He came down the hall and happened to see me,” Wolfe recalled, “and says, ‘Wolfe, these things aren’t even bacteria!’” Wolfe laughed gently and, for my benefit, continued reenacting the scene.
“‘Of course they are, Carl.’” They look like bacteria in the microscope, Wolfe had told him. But Woese wasn’t using a microscope. He never did. He was using ribosomal RNA fingerprints.
“‘Well, they’re not related to anything I’ve seen.’” Coming back to the present, Wolfe said: “That was the pivotal statement that changed everything.”
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We went into fast-forward mode (#litres_trial_promo),” Woese recalled in his account of these events. By the end of 1976, his team had done fingerprints and catalogs on five additional methanogens, with more in the pipeline. And sure enough, he wrote, none of the new catalogs was prokaryotic, not in the prevailing sense of that word, which meant bacteria and only bacteria. None of the organisms was eukaryotic, either. But “they were all of a kind!”—a third kind, something else, something anomalous, something hitherto unsuspected to exist. Woese started thinking that he would need to declare a new kingdom of life—create a new name, invent a huge new category—to recognize their uniqueness and contain them. It wasn’t really a new kingdom, of course. It was a newly discovered natural grouping of life-forms, which had existed apart for a long time, unrecognized, and which might be called a “kingdom” or an “urkingdom” or a “domain,” according to preferred human convention.
Woese believed that this discovery, still unannounced, offered “a rare opportunity to put the theory of evolution (#litres_trial_promo) to serious predictive test.” He meant Darwin’s theory of evolution, as opposed to any others—the one that recognized hereditary continuity plus a degree of random variation over long stretches of time, and explained the shaping of that variation, to yield adaptation and diversity, mainly by way of natural selection. If Woese’s preliminary findings were correct, he noted, those findings should serve as a guide for predicting roughly what further data and discoveries would appear. From the premise that 16S rRNA represented a very slow-ticking molecular clock, with a minimum of selected variation, he deduced that his newly found kingdom must represent a very old division. Very old—having originated near the beginning of cellular life, maybe three and a half billion years ago. Now he would try to sketch its boundaries and its characteristics. As he and his team added more microbes to its membership—more methanogens and maybe other creatures too, each known by its catalog of RNA fragments—Woese expected two things: that this unnamed kingdom would remain dramatically distinct from the rest of the living world and that it would nonetheless encompass great diversity. “Testing these two main evolutionary predictions (#litres_trial_promo),” he wrote, “drove our work from that point on.”
Three domains and (within the eukaryotes) four kingdoms, four types of cell.
In August the team published a carefully limited paper, just a hint of what was coming, in the Journal of Molecular Evolution, the same journal at which Emile Zuckerkandl continued to serve as editor. It was a logical match of subject and outlet because Zuckerkandl, back in his days as Linus Pauling’s sidekick, had helped articulate the very premise that Carl Woese was now putting dramatically to use: that the branching of lineages “should in principle be definable (#litres_trial_promo) in terms of molecular information alone.” The molecular information at issue in this case consisted of ribosomal RNA sequences from the first two methanogens Woese’s team had characterized. One of those methanogens was a strain of M. ruminantium, isolated from rumen fluid (from the paunch of a cow) donated by a friendly contact in the university’s Department of Dairy Science. The other was delta H, the conveniently nicknamed strain of the fourteen-syllable monstrosity, M. thermoautotrophicum, known to live at high temperatures and metabolize hydrogen. I asked Ralph Wolfe where they had gotten their starter sample of that exotic beast, delta H.
“It was isolated here from the sewage.” More specifically, from a sewage sludge digester.
“In Urbana?”
“Yeah.”
The first author on this discreet paper was Bill Balch, Wolfe’s graduate student, who had earned his authorship priority by developing the sealed-tube technique of growing and labeling methanogens. “It was because of that technique,” Wolfe told me, “that we could now do these experiments with Carl. Because everything was sealed, and you could now inject the P-32 into the culture.” P-32, remember, was the radioactive phosphorus. “Whereas the previous techniques, you had to keep opening the stopper and flushing it out, and it would have been a radioactive nightmare to do it that way.” Balch’s system allowed for injecting the P-32 by syringe through the black rubber stopper. Balch grew the microbes, George Fox extracted the RNA, and Woese’s trusted lab technician at the time, a young woman named Linda Magrum (she had replaced the earlier Linda in that role, Linda Bonen), prepared the fingerprint films for Woese to analyze. All three of them, plus Ralph Wolfe himself, appeared as coauthors, with Woese’s name last, reflecting his role as senior author. Besides describing the methodology, this paper noted drily that the two methanogens didn’t look much like “typical” bacteria (#litres_trial_promo). It mentioned that the divergence might represent “the most ancient phylogenetic event (#litres_trial_promo) yet detected”—a big claim, vague enough as stated to pass almost unnoticed.
In October the team published a second paper, in a more far-reaching journal, the Proceedings of the National Academy of Sciences (known as PNAS). This time George Fox was first author, and the data covered ten species of methanogen, each one assessed for similarity to the other nine and to three species of what the authors still cautiously called “typical bacteria.” Fox had created a simple measurement system by which the catalog of one microbe could be compared with the catalog of another, yielding a decimal number—a coefficient—representing degree of similarity. Comparing each of these thirteen microbes with all the others gave an overall picture of which were how closely similar to which others. The data could be arranged in a rectangular table, names down the left margin, names again across the top, numbers at each cross point, as in a chart showing the various mileage distances between all pairs in a list of cities. Instead of mileage: a similarity coefficient. From those numbers, and the premise that similarity reflected relatedness, Fox generated a dendrogram, a branching figure, showing nodes of divergence between major lineages and a branch for each organism. Although they printed this dendrogram sideways—like a bracket for the NCAA basketball tournament—rather than vertically, it was, in fact, a tree: the first of the new trees of life in the era of Carl Woese. There would be many more.
This one showed the “typical bacteria” occupying one major limb. The ten methanogens all branched from a second major limb. “These organisms,” said the paper, “appear to be only distantly related (#litres_trial_promo) to typical bacteria.” Again the five authors were saying less than what they believed. The phrase “typical bacteria” was an interim delicacy that would soon disappear.
A third paper, the most bold and dramatic, appeared in PNAS a month later under the authorship of Woese and Fox alone. Its title hinted only obliquely at its intent: to reorganize “the primary kingdoms” of life. Again using Fox’s similarity coefficients, it compared methanogens against one another and against “typical bacteria,” and each of those also against several eukaryotic organisms, including a plant and a fungus. Its conclusion was radical: there are three major limbs on the tree of life, not two. The prokaryote-eukaryote dichotomy, as proposed by Stanier and van Niel, as generally accepted throughout biology, is invalid. “There exists a third kingdom (#litres_trial_promo),” Woese and Fox wrote, and it includes—but may not be limited to—the methanogens. It isn’t the bacteria, and it isn’t the eukaryotes, they explained. It’s a separate form of life.
