The Tangled Tree

by David Quammen

  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 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 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 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 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.

  18

  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” 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. 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. Not there. Instead, he found a strange fragment, a longish sequence that shouldn’t have been present at all. “What was going on?” 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,” he wrote. There was “something out there”—out there in the teeming ecosystems of planet Earth, he meant—other than prokaryotes and eukaryotes. A thir
d form of life, separate.

  Woese called this, whimsically, his “out-of-biology experience.” It would be the watershed moment of his scientific life.

  19

  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.

  20

  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” 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. 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 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,” 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.

  21

  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 infecti
ons.

  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,” 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.