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The Archaea: A Personal Overview of the Formative Years

“—a new scientific truth does not triumph by convincing its opponents and making them
see the light, but rather because its opponents eventually die, and a new generation grows
up that is familiar with it.” Max Planck
The year 2002 marks the 25th anniversary of the discovery of the Archaea. Data
to support this discovery were presented in the Proceedings of the National Academy of
Science of October 1977 (Fox et al., 1977). However, most people learned about
methanogens, “a third form of life,” from the front pages of their newspapers on Thursday,
November 3, 1977, the day the PNAS issue was available. The National Science
Foundation (NSF) and the National Aeronautics and Space Administration (NASA)
supported the research of Carl Woese and were pleased to sponsor and receive publicity
for the press release he presented. When discussing the importance of the discovery with
reporters prior to the press release, Woese had difficulty communicating in scientific
terms that they could understand, until he hit upon the phrase, “a third form of life.” They
could relate to this phrase, and it became prominent in their articles. Methanogens, though
prokaryotes, were only distantly related to other prokaryotes.
The immediate response of the scientific community to the press release was
negative with disbelief and much hostility, especially among microbiologists. Scientists
were suspicious of scientific publication in newspapers, and only a very few were familiar
with the use of 16S rRNA oligonucleotides to define relationships among
organisms.Among the phone calls that I received the morning of November 3, the one by
S. E. Luria was the most civil and free of four-letter words. Luria was a Professor of
Microbiology, when I joined the Department at Illinois in 1953 and had later moved to
MIT. Luria: “Ralph, you must dissociate yourself from this nonsense, or you’re going to
ruin your career!” “But, Lu, the data are solid and support the conclusions: they are in the
current issue of PNAS.” Luria: “Oh yes, my issue just arrived.” “If you would like to
discuss the paper after you have had a chance to look at it, give me a ring.” He did not
call again.
I wanted to crawl under something and hide. Fortunately I was able to escape the
hostility and left graduate students to cope, because my wife and I were leaving for
Philadelphia to help celebrate her father’s 90th birthday. We collected a few newspapers
in airports. Carl was on the front page of the New York Times with his Adidas-clad feet
prominently displayed on his desk. Much later he would say, “You know, Adidas never
offered me a contract.” In Philadelphia, I explained in dismay to my father-in-law what
my colleague had done, and his response was: “You know, in my long life I have observed
something, if you don’t overstate your case, no one will listen.” I felt better. However, in
hindsight, the press release polarized the scientific community, and the majority refused
to read the literature, delaying acceptance of the Archaebacteria for perhaps a decade. The
discovery of the Archaea resulted from the intersection of two independent lines of
research; the culmination of the informational-macromolecule line was the research of
Carl Woese, whereas the biochemistry of methanogenesis was pursued by my research
group.
Informational Macromolecules
This line began with the publications of Sanger and coworkers (Sanger and Tuppy,
1951; Sanger and Thompson, 1953), who showed each amino acid in the two chains of
insulin occupies a precise position in the protein molecule.This discovery had enormous
implications for genetics and for the emerging area of molecular biology. By the 1960s,
insulin molecules from various animal species had been sequenced, and it was apparent
that insulin from different species possessed variations in the sequence of certain amino
acids. Zukerkandl and Pauling (1965) pointed out that these differences could be used to
determine the relationship among the molecules, and hence, the organisms from which
they were obtained; macromolecules which showed only minor sequence changes were
closely related, whereas those with larger differences were more distantly related. The
next macromolecules to be sequenced were other small proteins, for example,
cytochromes, but the limitations of protein molecules to study relatedness among
organisms soon became apparent.
In the early 1960s, Carl Woese’s study of the ribosome convinced him that this
structure was highly conserved. He reasoned that because the ribosome is of ancient
origin, is universally distributed, and is functionally equivalent in all living cells, it would
be the ideal structure to use for study of evolution. In addition, the ribosome has only one
function, translation of the genetic code into the amino acid sequence of a protein, and
may be somewhat “insulated” from the variables of phenotypic encounters. Then, Sanger
et al. (1965) published a method for a twodimensional (2-D) fractionation of radioactive
nucleotides, which could be used to sequence RNA. Woese’s seminal insight was to
recognize that ribosomal RNA was the ideal molecule to follow evolution to very ancient
events. He modified the Sanger RNA sequencing technique and explored the use of
ribosomal 5S, 16S, and 23S rRNA. He chose 16S rRNA as a “statistical ensemble” of
1540 monomers that would allow investigations of organisms to reach the very root of
the tree of life. Carl Woese was alone in developing the use of 32P-labelled
oligonucleotides from T1 endonuclease digests of 16S rRNA to generate a similarity
coefficient that could be used to determine the relatedness of organisms (Sogin et al.,
1972; Pechman and Woese, 1972).
I was fascinated by this new approach, for as a graduate student I had been
prejudiced against “bug sorting” and its tenuous results by my professor, and I resolved
never to get involved in taxonomy with its constant reshuffling and renaming of species.
But here was something I could believe in, for it had a sense of permanence. I was
especially impressed by results of Woese’s initial study of the genus, Bacillus (Woese et
al., 1976). I became a believer. Today with greatly improved technology, data can be
generated in one day that took Woese months to do in 1969. By 1976, Woese had obtained
the 16S rRNA similarity coefficients of 60 microbes, representing a wide diversity.
Biochemistry of Methanogenesis
This line had an early beginning with the experiments of Alessandro Volta in 1776
which showed that the gas produced by decaying vegetable residues in sediments was
combustible; its identity as methane came a century later, and definitive experiments with
methanogenic bacteria came with the isolation of Methanobacillus omelianskii (Barker,
1936; Barker, 1940). For over 20 years after 1936, Barker’s laboratory was the leader in
studying methanogens, and many observations led him to conclude that these organisms,
though morphologically diverse, had a common physiology. In 1961, I was attracted to
and began to investigate the unexplored biochemistry of methanogenesis. An enzymically
active cell extract had not been prepared. Because of the formidable challenges in
cultivating these organisms, knowledge of their biochemistry lagged decades behind
other organisms Barker’s culture of M. omelianskii was mass cultured, and the first cell-
free formation of methane was obtained (Wolin et al., 1963). In collaboration with Bryant
and Wolin, Barker’s culture of M. omelianskii was found to be a symbiotic association
of two organisms, i.e., interspecies hydrogen transfer had been discovered (Bryant et al.,
1967). The methanogenic organism from the mixed culture oxidized hydrogen and
reduced carbon dioxide to methane. My laboratory developed a technology for mass
culture of the organism on hydrogen and carbon dioxide (Bryant et al., 1968), which was
scaled up to the 200-liter level. With kilogram quantities of cells available, the first unique
coenzyme of methanogenesis was discovered and named “coenzyme M” (McBride and
Wolfe, 1971). Its structure was determined, and its methylated active form was
synthesized (Taylor and Wolfe, 1974a). The unusual coenzyme was found to be a
“vitamin” required by a methanogen from the cow’s rumen, Methanobacterium
ruminantium (Taylor et al., 1974b).To assay this vitamin, this organism was grown via a
new procedure, the Balch modification of the Hungate technique, involving use of a
pressurized atmosphere of hydrogen and carbon dioxide (Balch and Wolfe, 1976). After
exhaustive analyses, CoM was found nowhere else in nature. A second unique coenzyme
(Eirich et al., 1978), the blue-green fluorescent deazaflavin, F420, was found to be the
coenzyme for formic dehydrogenase (Tzeng et al., 1975). By 1976, evidence for other
unique coenzymes was in hand in my laboratory.
Discovery of the Archaea
The simple method of growing methanogenic cells in an anaerobic pressurized
atmosphere of hydrogen and carbon dioxide was pivotal to the discovery of the Archaea,
for hydrogen and carbon dioxide could be replenished with safe containment of high
levels of 32P and without exposure of cells to oxygen or contamination. After analysis of
the 2-D chromatographic pattern of the T1 endonuclease digestion fragments from the
16S RNA of the first methanogen, I asked about the results. Woese was puzzled, for the
pattern was unlike any he had seen; he could only conclude that: “Somehow we must
have isolated the wrong RNA.” So the experiment was repeated with special care, and
this time, his response was: “Wolfe, these methanogens are not bacteria.” “Of course they
are Carl; they look like bacteria.” “They are not related to any bacteria I’ve seen.” Because
Woese had spent nearly 10 years alone developing the method and analyzing the 2-D
chromatographic patterns of T1 endonuclease digestion patterns of 32P-labelled 16S
rRNA from 60 different bacteria, he was easily able to discern that methanogens were
different! But what should the group be called?
In November 1977, Woese and Fox proposed that ribosomal RNA sequence
characterization could be used to define three “aboriginal lines of descent” (Woese and
Fox, 1977). One line, the typical bacteria, was designated “eubacteria.” “Archaebacteria”
was proposed as a name for the methanogen line, and the term “urkaryotes” was proposed
for the cytoplasmic component of eukaryotic cells. So the methanogens were
Archaebacteria. The name Archaebacteria was suggested by David Nanney. In a way, the
term was unfortunate because the case was being made that the methanogens were “old”
(i.e., represented a very ancient divergence in evolution), so that they were no more
related to bacteria than to eukaryotes. On the other hand, they were bacteria. (Over a
decade later, Woese, Kandler, and Wheelis would propose the name “Archaea” for the
Archaebacteria [Woese et al., 1990]. By that time, much evidence had accumulated
showing the Archaea clearly belonged on the eukaryotic line of descent, and it would be
less confusing if the word “bacteria” in Archaebacteria was deleted.)
All of the available methanogens in pure culture were obtained and subjected to
16S rRNA analysis. The results were startling (Fox et al., 1977); none of the species was
related to typical bacteria, but they fell into natural groups. Additionally, the unusual
biochemistry of methanogens now assumed importance. Methanogens are the only
organisms that produce methane as their metabolic product, and they do so by a
biochemistry that employs unique enzymes and coenzymes. So, the initial concept that
methanogens were different rested on 16S rRNA analysis and was supported by unique
biochemistry. While these studies were underway, it occurred to me that, if methanogens
were so different in these aspects, they should show differences in other areas. So, I wrote
to Otto Kandler in November 1976 explaining that Carl Woese had determined
methanogens to be only distantly related to bacteria and asked him whether he would be
interested in determining the structure of the cell walls of methanogens, which we could
send to him. He was enthusiastic about the project for his laboratory had been studying
the cell walls of diverse bacteria including Halococcus morrhuae (Schleifer and Kandler,
1972; Steber and Schleifer, 1975) and Methanosarcina (Kandler and Hippe, 1977) which
were shown to lack peptidoglycan. This letter would have an effect far beyond its simple
question, for Kandler found a gold mine and became convinced that the Archaebacterial
concept represented a major turning point for prokaryotic phylogeny. In 1977, he made a
trip to Urbana, Illinois, to obtain the most direct information. He became convinced that
German microbiologists should begin serious work on the Archaebacteria. His laboratory
soon showed that species of Methanobacterium lacked typical peptidoglycan (murein)
cell walls, known to be a characteristic of all bacteria. Other species of methanogens had
no peptidoglycan derivatives at all (Kandler and König, 1978), and when the project was
complete, Kandler’s laboratory had shown that there were as many variations in the cell
walls of methanogens as in all bacteria combined! This was a breakthrough—a third pillar
to support the concept that the methanogens were different.
The year 1978 was eventful in fine tuning the concept that methanogenic bacteria
(10 species) were so distantly related to typical bacteria that they should be considered as
a distinct phylogenetic group, the Archaebacteria. Tornabene et al. (1978) investigated
the lipids of Methanobacterium thermoautotrophicum and found that phytanyl-glycerol
ethers and squalenes were major lipid components. A fourth pillar to support the
Archaebacterial concept was in place. This unusual lipid composition was similar to that
of Halobacterium cutirubrum reported by Kates et al. (1965) and Tornabene et al. (1969).
However, the discovery of these unusual lipids in Halobacterium had not created wide
interest at the time of publication, for their possession was largely perceived to be a
consequence of adaptation to growth in saturated salt environments. Similarly, organisms
previously isolated from thermoacidic aquatic environments, Sulfolobus and
Thermoplasma summarized by Brock (1978), had been shown to contain only negligible
amounts of ester-linked lipids (Langworthy et al., 1972; DeRosa et al., 1975; DeRosa et
al., 1976). Cell walls of Sulfolobus had been shown by Brock et al. (1972) to be deficient
in peptidoglycan, and of course, species of mycoplasma were known to have only a
protein covering. Again it was presumed that perhaps the lipids and lack of peptidoglycan
were related (somehow) to growth in hot acidic environments. Then Woese, Magrum,
and Fox brought it all together in an article entitled “Archaebacteria” (Woese et al., 1978),
and expanded the concept to include the methanogens, extreme halophiles, and
thermoacidophiles, the common factors being: 1) the possession of characteristic
ribosomal RNAs and tRNAs; 2) the absence of murein cell walls; and 3) the presence of
etherlinked lipids in phytanyl chains.
In 1978, Zillig and coworkers began to report results of experiments on the
remarkably similar component patterns of the DNA-dependent RNA polymerases of
Archaebacteria and eukaryotes (Zillig et al., 1978; Zillig et al., 1979; Stetter et al., 1980;
Sturm et al., 1980). In addition, the Archaebacterial polymerases showed characteristic
eukaryotic resistance against the inhibitors rifampicin, streptolydigin, and α-amanitin.
Because the DNA-dependent RNA polymerases of eubacteria have the standard four-
component composition β’βασ, it was assumed that all prokaryotes would be similar.
However, the Archaebacterial RNA polymerases contained 7 to 12 different components,
showing a pattern similar to Saccharomyces cerevisae. The results summarized by Zillig
et al. (1985) were so astounding that many viewed them as conclusive evidence, alone,
that Archaebacteria indeed represented a third domain of life. Another pillar to support
the Archaebacteria was in place.
In 1979, an interesting observation was made in the laboratory of Friedrick Klink
by M. Kessel. He found that in vitro protein synthesis by halobacterial preparations was
inhibited by diphtheria toxin (Kessel and Klink, 1980). These studies were extended to
18 Archaebacteria (Kessel and Klink, 1982). The state of the field was summarized by
Klink (1985). The Archaebacteria have an elongation apparatus in the ribosome that is
distinct from those of typical bacteria and eukaryotes. All Archaebacteria contain a
structural domain in elongation factor-2 (EF-2) that renders it a substrate for diphtheria
toxin. Archaebacterial EF-2 is highly specific for Archaebacterial ribosomes and does not
work with ribosomes from bacteria or eukaryotes. These experiments attracted wide
interest because they exploded the perceived dogma that all prokaryotes are insensitive
to the diphtheria toxin; so another pillar to support the Archaebacterial concept was in
place.
However, the concept of the Archaebacteria, outside of a very few believers, was
not embraced by the microbiological community. So in 1978, I suggested to William
Balch, a graduate student in my laboratory, that we gather together in a review article all
of the evidence in support of methanogens as Archaebacteria. He did a magnificent job
dedicating his considerable talents to the task and working closely with Woese. With the
dedicated help of Marvin Bryant, a new classification of methanogens based on 16S
rRNA was included in the article. The review displayed eloquently the power of 16S
rRNA to show the relatedness of organisms, i.e., a poorly understood group of
methanogens as diverse as all bacteria combined was finally organized into a phylogeny
that made sense. Marv Bryant, the world authority on methanogens, pronounced the
results “beautiful, beautiful.” The four types of cell walls, the C20 diphytanyl glycerol
diethers, the C40 dibiphytanyl diglycerol tetraethers, and C30H30 squalene of the neutral
lipid fraction were Archaebacterial. The structure and function of coenzyme M and
coenzyme F420 had been determined (Taylor and Wolfe, 1974a; Taylor et al., 1974b;
Eirich, L. D., et al., 1978; Tzeng et al., 1975) and were clearly unique to the biochemistry
of methanogenesis. Although initially the uniqueness of these coenzymes supported the
Archaebacterial concept, it soon appeared that they were not found at the time in known
species of extreme halophiles or thermoacidophiles and so were a general characteristic
of methanogens only. In addition to ribosomal RNA, transfer RNAs of methanogens were
found to be unique; they do not contain the universal common arm sequence. The review,
“Methanogens: Reevaluation of a unique biological group,” appeared in the June 1979
issue of Microbiological Reviews (Balch et al., 1979) and was received with respect by
the microbiological community. The work attracted the interest of a number of new
investigators to the Archaebacteria.
Because Rolf Thauer had close contact with the German Forschungsgemeinschaft,
Kandler asked him in March 1978 to think about a proposal for funds to initiate a
Schwerpunktprogram for research on methanogens and other Archaebacteria. Thauer
agreed to head the Schwerpunkt, but to avoid controversy, they decided not to use the
term “Archaebacteria” in their grant application, since this term was not well accepted at
the time. The DFGSchwerpunkt-program, “Methanogene Bakterien,” was funded in 1979.
Despite the title, research on other Archaebacteria was encouraged. The initial
investigators included J. Andreesen (Göttingen), A. Bacher (Garching), G. Diekert
(Marburg/Stuttgart), G. Fuchs (Marburg/Ulm), G. Gottschalk (Göttingen), O. Kandler
(München), A. Klein (Marburg), H. König (Regensberg/Ulm), P. Scherer
(Jülich/Weihenstephan), P. Schönheit (Marburg/Berlin), R. Thauer (Marburg), J. Winter
(Regensberg). Kandler was the instigating force in the initiation of this effort, which
stands in stark contrast to the rejection of or ambivalence toward the Archaebacterial
concept by microbiologists in other countries, who were quite content with the entrenched
taxonomy of the time.
The Schwerpunkt paved the way for the first international workshop on
Archaebacteria at München-Martinsried, June 27–July 1, 1981. W. Zillig, O. Kandler,
and K. Stetter were involved in setting up the workshop, which was supported by the
Stiftung Volkswagenwerk and the MaxPlanck-Gesellschaft. The workshop was devoted
to all areas of Archaebacterial research, with emphasis on molecular and biochemical
research as well as geochemical, paleontological, and taxonomic areas. Over 50 workers
attended, and it was the first time that many workers in the field had a chance to meet
personally. Participants were enthusiastic about the success of the workshop. It was clear
that the Schwerpunkt was not only focusing the efforts of German microbiologists on
Archaebacteria but was having an international impact as well. It was also clear that
German microbiologists had assumed a leadership role in the study of the Archaebacteria.
The proceedings of the 1st International Workshop were published in May 1982
(Kandler, 1982). The 17 articles covering cell walls, the ribosome, translation apparatus,
transcription, lipids, membranes, coenzymes, CO2 fixation, anaerobic reduction of
molybdenum, phylogeny, new species, and 5S ribosomal RNA well documented the
beautiful flowering of the Archaebacterial concept that had occurred in just five years!
To celebrate the breakthrough of the Archaebacterial concept after the workshop, Kandler
and his wife Trudy took Woese and Wolfe for a hike in the Bavarian Alps to the top of
“Hohe Hiss” 1850 m, 80 km south of Munich. Woese and especially Wolfe were not in
top physical shape, but with some huffing and puffing, they reached the top via a well-
graded path. The photograph (Fig. 1) was taken by Trudy Kandler at the summit. After
we returned to the car, the Kandlers showed us some beautiful Bavarian churches, and
then, because it was a hot afternoon, we stopped at an open-air beer garden for a glass of
beer under a large linden tree. The beer was delivered to us in large graceful glasses, and
after “Prost” (to your health in English), we each had a sip of the cold beer. Otto Kandler
seemed to have consumed about a third of the beer in his glass, when he pronounced, “Ah,
the first sip is the best!” I have always remembered the volume of a German “sip” on a
hot afternoon.
In 1985, owing to the fast-growing field of Archaebacterial research, international
symposia were again organized in Germany. The second International Workshop on
Biology and Biochemistry of Archaebacteria was organized by O. Kandler and K. O.
Stetter and sponsored by the Deutsche Forschungsgesellschaft. A workshop on Molecular
Genetics of Archaebacteria was organized by W. Zillig and A. Böck and sponsored by
the European Molecular Biology Organization (EMBO) and the Max-PlanckGesellschaft
(München). These workshops were held back-to-back June 23–July 1, 1985, in München.
Proceedings of the workshops were published in a volume entitled Archaebacteria ’85
(Kandler and Zillig, 1986).The book contains 65 reprints of papers and 38 extended poster
abstracts and thoroughly documents the astounding progress made in all aspects of the
Archaebacteria in four years (1981–1985). Again, German microbiologists took a
leadership role and were ahead of the international community of microbiologists in fully
accepting evidence for the three domains of life as indisputable; in contrast,
misunderstandings, doubts, and hesitation prevented many microbiologists in other
countries from embracing the Archaebacterial concept. It would be perhaps another 10
years before the concept was fully accepted.
It should be reemphasized that the road was difficult and experimentally
demanding, especially in the early years, when Carl Woese’s laboratory alone generated
the primary data. To obtain the radioactive nucleotides for 2-D fractionation, the
microbial cells had to be grown in media from which phosphate had been removed and
to which high levels of 32P-phosphate were added. The trick was to get cells to take up
enough radioactive phosphate before they were killed by radiation. The procedure worked
well for organisms with a short generation time but was less suitable for slow-growing
microbes. The results of the T1 endonuclease digestion and subsequent fractionation
procedures to identify the sequence of each oligonucleotide, i.e., 5-mers to 18-mers,
provided data for only about 25% of the 16S rRNA. Yet the whole system for obtaining
the similarity coefficient (SAB) for each organism to construct dendrograms of
relationship among organisms was developed by Woese within these limitations. The
SAB was the method of choice until cloning and sequencing of the 16S rRNA genes
became available. The first to appear was the 16S rRNA gene from Escherichia coli
(Brosius et al., 1978), and the first archaeal 16S rRNA sequence appeared in 1983 (Gupta
et al., 1983). There was a long lag before the complete 16S rRNA sequences became
useful, because for comparative studies, a significant number of sequences needed to be
determined. Thus, the method of choice now is to clone rDNA from an organism and
obtain the complete sequence of the gene encoding 16S rRNA. The increasing number of
complete genome sequences of organisms has made analysis relatively simple compared
to the initial procedures used by Woese to define the tree of life.
The second edition of The Prokaryotes (four volumes published in 1992)
recognized the almost unbelievable impact that the work of Carl Woese had in defining
the Archaea as well as a 16S rRNA phylogenetic basis for the microbial world. In the
1990s, microbiology textbooks in the United States began to present phylogeny based on
16S rRNA. In 1997, twenty years after the discovery, the textbook Brock, Biology of
Microorganisms (Madigan et al., 1977) completely endorsed the Archaea and the
universal phylogenetic tree based on 16S rRNA. However, the vast inertia invested in the
morphological approach to taxonomy and phylogeny, since the time of Linnaeus and later
Darwin by the biological community, makes changing the course of such a huge ship very
difficult. This is illustrated dramatically in textbooks of general biology from the 1990s
that include perhaps a paragraph or so on the Archaebacteria with very little or nothing
on the use of monomer sequences of rRNA to reveal a molecular tree of life. This
approach trickles down from college textbooks to high school textbooks, perpetuating the
teaching of biology based on morphology. However, in the United States, the 6th edition
of the general text Biology by Raven and Johnson (2002) refers to taxonomy and
phylogeny based on morphology as the “traditional approach” and then proceeds with a
well-presented three-page treatment of the domains of life: Eubacteria, Archaea, and
Eukarya. So, all is not lost, just delayed. Remember the quote from Max Planck!