how we discovered a third domain of life

The Mechanism

*Carl Richard Woese* (born Syracuse, New York, *15 July 1928*; died Urbana, Illinois, *30 December 2012*, aged 84) was, in the 1970s, a professor of microbiology at the *University of Illinois at Urbana-Champaign* who had set himself a problem most of his colleagues regarded as unsolvable. The problem was the *deep phylogeny of microbial life*: bacteria are too morphologically simple, and reproduce too asexually, for the comparative-anatomy and cross-breeding methods that work on plants and animals to be applied. There was no agreed way to construct an evolutionary tree of bacteria — and therefore no agreed way to determine which prokaryotes were close relatives, which were distant, or what the relationships were between bacteria and the eukaryotes that contained them. Woese's bet was that the sequence of the *16S ribosomal RNA* — the structural RNA component of the small subunit of the bacterial ribosome — could be used as a *molecular clock* for the deep tree. The 16S rRNA is *universal* (every cellular organism has it), *essential* (its sequence is constrained by the protein-synthesis machinery), and *slowly varying* (most mutations are quickly eliminated by purifying selection). Comparing 16S sequences between organisms would, Woese argued, reveal phylogenetic distance at the deepest scales. The technique Woese used to capture sequence information — a *T₁ ribonuclease oligonucleotide cataloguing method* developed largely in his own laboratory through the late 1960s and early 1970s — was *brutally laborious*: rRNA was extracted from a culture, digested with *RNase T₁* (which cuts after every G residue), the resulting oligonucleotides were separated by *two-dimensional paper electrophoresis*, and each spot on the resulting fingerprint was hand-identified and hand-sequenced. A single 16S catalogue required *months of work per species*. Through the early-to-mid 1970s Woese's group catalogued the 16S of dozens of bacterial and eukaryotic species and produced the first molecular phylogenies of microbial life. In *June 1976*, Woese's group received from collaborator *Ralph S. Wolfe* — a fellow Illinois microbiologist who had been culturing methane-producing organisms for years — a sample of *Methanobacterium thermoautotrophicum*, a strict-anaerobe methanogen, for 16S cataloguing. The oligonucleotide fingerprint that came off the paper electrophoresis plate in mid-June was *unlike anything in their bacterial catalogues*. The characteristic *signature oligonucleotides* found in every previously-catalogued bacterium — short sequences universally conserved across *E. coli*, *Bacillus*, *Streptococcus*, *Mycoplasma*, and dozens of other eubacterial groups — were *missing*. In their place were oligonucleotides that did not match the bacterial reference set, *and* did not match the eukaryotic reference set. The methanogen, by 16S signature, was as different from a typical bacterium as it was from a yeast. Woese, recalling the moment later, said he spent days walking the corridors of the microbiology building before he was willing to commit the conclusion to paper: the tree of life was not bipartite but *tripartite*. The methanogens, and a small but growing collection of other extremophile prokaryotes that he and Wolfe and *George E. Fox* (then a postdoctoral researcher in Woese's lab) began re-cataloguing through 1976-77 — the *extreme halophiles* (organisms thriving in saturated salt), and selected *thermoacidophiles* (organisms thriving in hot acid) — all shared the same anomalous 16S signature, and were as distant from typical bacteria as bacteria were from eukaryotes. The paper, *"Phylogenetic structure of the prokaryotic domain: the primary kingdoms,"* by Woese, C.R. and Fox, G.E., appeared in *Proceedings of the National Academy of Sciences*, volume 74, pages 5088-5090, in *November 1977*. Three pages long, it proposed that the living world consisted not of two primary lineages but of *three*: the *Eubacteria* (typical bacteria), the *Archaebacteria* (the methanogens-plus-halophiles-plus-thermoacidophiles), and the *Urkaryotes* (the cytoplasmic ancestors of eukaryotes). The proposal was received, in late 1977 and through 1978, with *near-universal scepticism*. The dean of microbiology at Illinois, on first reading, asked Woese privately whether he was certain enough to be staking his career on it. *Salvador Luria*, who had won the Nobel Prize ten years earlier for the discovery of bacterial mutation, telephoned Wolfe and urged him to dissociate himself from Woese's project for the sake of his own reputation. *Ernst Mayr*, the dean of twentieth-century evolutionary systematics, argued in print for years that prokaryotes could not possibly contain a phylogenetic division as deep as the prokaryote-eukaryote one. The skepticism slowly eroded through the 1980s as additional 16S catalogues, then full 16S sequencing, then complete genome sequencing of methanogens, halophiles, and thermophiles confirmed the result at progressively higher resolution. By the time Woese, Otto Kandler, and Mark Wheelis renamed the three domains as *Bacteria, Archaea, and Eukarya* in 1990 (*Proceedings of the National Academy of Sciences* 87:4576-4579), the tripartite tree had become the consensus framework. Subsequent genomic sequencing has shown that the Archaea share most of their genetic-information-processing machinery — DNA replication, transcription, translation — with eukaryotes rather than with bacteria, suggesting that *eukaryotes are the descendants of an archaeal lineage that engulfed a bacterial endosymbiont* (the Lokiarchaeota hypothesis, 2015). Carl Woese died in Urbana on 30 December 2012; the 16S rRNA cataloguing method, now generalised to metagenomic sequencing of environmental DNA, is the foundation of modern microbial ecology and is used to identify the unculturable majority of life on Earth — most species of which were undescribed before Woese's method. He received the *Crafoord Prize in Biosciences* in 2003 (the Royal Swedish Academy's parallel honour to the Nobel) but was never awarded a Nobel Prize.