Download The Archaea: an Invitation to Evolution

The Archaea: an Invitation to Evolution
by
Carl R. Woese
Department of Microbiology
University of Illinois
Carl R. Woese
Urbana, Illinois 61801
In memory of Wolfram Zillig: A founder of the archaeal revolution.
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The Archaea: an Invitation to Evolution
Beginnings
The discovery of the archaebacteria was serendipitous, but not
unexpected. In the late 1960s I had begun assembling the program for
inferring (organismal) genealogical relationships through rRNA
sequence comparisons (42). It was time to determine the structure of
the universal phylogenetic tree (whatever it be). Molecular evolution
had been on the scene for the better part of a decade, and a universal
framework within which to study evolution from the molecules on up
was needed. Our initial emphasis was necessarily on the microbial
world (bacteria in particular), for almost nothing was known about
microbial phylogenies.
My objective in establishing the phylogenetic program, however, was
not to refine bacterial taxonomy per se, but to restore an evolutionary
perspective/spirit to biology. But this time the focus would be on the
evolution of the cell itself; in particular, the evolution of its
translation mechanism (and the information processing systems in
general) (38; 41). A reductionist discipline of molecular biology had
deliberately ignored evolution ─ asserting that a fundamental
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Carl R. Woese
understanding of life could be got without considering its evolution!
This was unacceptable to me, anathema: a Biology that is not
fundamentally evolutionary is not true Biology.
The cell's translation mechanism (and the other cellular information
processing systems) are far too complex to have arisen in anything
approaching their modern "fully evolved" state (38; 41). Therefore,
each must have come from far more primitive beginnings and, so,
have undergone profound and telling evolutionary changes. To
reconstruct even the final stages in this evolutionary progression was
to come face to face with biological organization. [Biological
organization cannot be understood apart from its evolution. All
biological form is ultimately complex dynamic process ─ process
involuted beyond our current capacity to understand it. In an
important sense biological form is four dimensional].
How exactly to go about determining a universal phylogenetic tree
was, then, the question. Since the 1950s the idea that an enormous
phylogenetic record lay buried in the sequences of macromolecules
had been there ─ a record that could be read through comparative
sequence analysis (6; 49). At that point the approach had been used,
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The Archaea: an Invitation to Evolution
however, solely with proteins ─ in part because nucleic acid
sequencing had yet to be developed; in part because most biologists
had convinced themselves that proteins were the only way in which
this could be done. The enterprise had even been labeled "protein
taxonomy" initially (6). Having worked with ribosomes, I thought
the approach might work with the ribosomal RNAs if only we had the
proper method for characterizing them.
Fortunately, that method had come along in 1965 ─ Sanger's
oligonucleotide cataloging approach to RNA sequencing (25). The
Sanger method and ribosomal RNA seemed the perfect combination:
the method was powerful (yielded far more sequence information
than anything that had preceded it), and rRNAs were ubiquitous,
relatively easy to isolate, and their sequences quite highly conserved
(9). The universal phylogenetic (genealogical) tree was only the first
step in a journey, however: a world map, if you will, that helps to
locate relics of the evolutionary past. I guess I didn't realize how
long it was going to take just to find that map!
Several years were spent in tuning the Sanger method to suit our
needs. By the early '70s we were finally up and running, albeit very
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Carl R. Woese
slowly. Initially we groped our way tentatively into the darkness of
bacterial phylogeny, starting with garden variety microorganisms ─
E. coli (here we had only to improve on what others had already
done) (33); Aerobacter aerogenes (42); Bacillus (half a dozen or so
species) (43); and various others. Almost immediately it became
apparent that we were going to need help in order to cover the full
range of the bacteria ─ at least in any reasonable time frame. Many
organisms would just be too difficult for us to grow. Enlisting the aid
of the experts, that is, those of them who were willing to grow their
organisms in the proper radioactive medium, was essential. Such
people were hard to find. But, once the right constellation of experts
started to form, we were on our way in earnest ─ characterizing
rRNAs from mycoplasmas, other pathogens, a host of clostridia,
many lactobacilli, bacteroides, a representative collection of
photosynthetic bacteria, and, of course, key species of what would
become the archaebacteria, and so on.
Of all the numerous suggestions we had gotten for organisms to
study, the one I solicited from my colleague at the University of
Illinois Department of Microbiology, Ralph Wolfe, turned out to be
the most important. Ralph was in the process of working out the
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The Archaea: an Invitation to Evolution
biochemistry of methanogenesis, which made it natural for him to
suggest we characterize the methanogens. It was not the organisms
per se, but the evolutionary conundrum they posed, that intrigued
me. If the methanogens were taxonomically grouped on the basis of
common methanogenic biochemistry, then the resulting taxon
contained a wide variety of morphologies (including Gram stain
differences) and growth conditions. The alternative morphologicallybased grouping would cast methanogenic biochemistry to the
taxonomic winds. This last is how the 7th edition of Bergey's
Manual had done it (2); but in the 8th edition (methanogenic)
biochemistry had been used as the taxonomic decideratum (22). [A
similar conundrum was posed by bacterial photosynthesis: in which
case molecular analysis would resolve the issue differently ─ in favor
of a reticulated biochemistry spread across the bacterial organismal
phylogenetic tree]. We had the technology here and now to settle the
phylogenetic issue methanogenesis raised. Methanogens went to the
top of the to-do list.
The only difficulty was that, at the time ─ which at latest was spring
1974 (the semester before George Fox joined the lab as my post-doc)
─ a technology that would allow growing methanogens readily and
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Carl R. Woese
safely in radioactive medium had yet to be developed. But, it would
soon be (1). William (Bill) Balch, a graduate student in Wolfe's lab,
was about to develop such a method for other reasons: he would grow
methanogens in pressurized serum bottles, a technique that was not
only efficient and adaptable, but, what was important to us, safe for
working with radioactively labeled cultures. By 1976 it had become
possible for the two labs to collaborate.
When the first methanogen 16S rRNA oligonucleotide catalog rolled
off the production line (June, 1976), I was stunned by what we had;
and thereafter our entire focus was to be on methanogens and the new
group of organisms, the archaebacteria, that they would come to
represent. We had discovered a "third form of life", a new
"urkingdom"! Methanogens (and their yet undiscovered relatives)
stood genealogically completely apart from the other bacteria we had
so far characterized (which we came to call "eubacteria") and from
the (small but representative) cluster of eucaryotes whose rRNA
catalogs we had done (44). We were in for the ride of our scientific
lives!
Hitching up the Team
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The Archaea: an Invitation to Evolution
Like everyone else in those days, I had tacitly accepted all bacteria to
be "procaryotes". That is, they were all of a kind; all had stemmed
from some common procaryotic ancestor (29; 31; 32). Suddenly
confronted with a procaryote that didn't seem to be a procaryote, I
had to ask myself why I had believed all bacteria to be procaryotes in
the first place? No good reason, it turned out! When analyzed
scientifically the "procaryote" didn't wash; there was no hard
evidence, not even sound scientific reasoning to back it up (31; 32).
We had hard evidence in hand and were in the process of gathering a
lot more.
Within six months we had the rRNA catalogs of another five or so
methanogens, with others in production: and all of them possessed
the same anomalous type of rRNA (10; 11). That was it! We had
shown methanogenesis to be monophyletically distributed. The
exceptional variability in the group's morphologies and habitats were
what needed explaining.
I had shared my "eureka moment" (upon assembling the first
methanogen rRNA catalog) with my then post-doc, George Fox, who,
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Carl R. Woese
along with Bill Balch, had gotten the project actually up and running.
George and I became the first to see the phylogenetic "light", to
realize that there were actually three primary lines of descent
(urkingdoms) on this planet, not two!
Convincing ourselves was not the problem. Convincing others was.
It would be a hard sell. For reasons I could not understand at the
time, literally all biologists believed in "the procaryote". And it was
not your typical scientific belief ─ always open to question. This was
dogma, unshakable doctrine! Some biologists would take years to
overcome their procaryote prejudice. Some have yet to do so. The
majority of those who came to accept the three urkingdom notion
were in fact not even genuine "converts": while they accepted
eubacteria
and
archaebacteria
to
be
genealogically
distinct
(representing two separate origins of bacteria), they held firm to the
notion that both of them still had essentially the same lcellular
organization (31; 32; 41).
So many times I have heard that archaea are "still just bacteria
(procaryotes)" or words to that effect ─ they have the same cellular
organization! What does this mean? In a purely scientific sense, it
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The Archaea: an Invitation to Evolution
means nothing! The resemblances between the two are in essence
negative ─ features not found in eucaryotic cells (viewed under a
light microscope).
It means nothing that archaebacteria and
eubacteria both show simple rod, coccoid, or spiral shapes; it means
nothing that both are typically much smaller than eucaryotic cells,
that neither has any microscopically visible intracellular inclusions.
All that these facts tell us is that neither type possesses certain
features characteristic of eucaryotes. Nothing has been (or could be)
said about the "common organization" the two supposedly share!
Consider an analogous situation on the metazoan level. Three kinds
of eyes exist among animals. Their similarity is defined only
functionally. Structurally the three are worlds apart. Might not the
same situation exist with regard to archaebacterial and eubacterial
organizations (41)? After all, there have never been any facts to
refute the idea, as there has never been a reasonable body of fact to
support a monophyletic taxon “prokaryote”; thus the extremely gross
traits (cited above) that all "procaryotes" were supposed to share
could easily be rationalized in general terms having to do with
ancestral life-style more than anything else. Its organization is the
most complex attribute the cell has. No biologist would accept that a
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Carl R. Woese
trait this complex could have evolved more than once. Nor would any
biologist accept that two independently evolved "procaryotic" cell
types would be so organizationally similar that their differences
would be trivial, not be worthy of elucidation! The above metazoan
example makes perfectly clear that if evolution builds two complex
traits that resemble one another in some overall function sense, it
does not (cannot) build them so alike on the underlying (complex)
structural level that they will fail to show major (non-homologic)
differences.
In sum; the notion that eubacteria and archaebacteria could have
evolved independently yet have arrived at so similar a cellular
organization as not to be of interest to the biologist, is absurd; it
contradicts all that we know about evolution! Microbiologists were
simply having their cake and eating it too. More has to be said about
the "procaryote" simplification and will be below, for it is pivotal to
microbiology's development (or non-development) in the 20th
century.
Mapping out the Territory
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The Archaea: an Invitation to Evolution
Having discovered what we thought to be a third primary line of
descent (represented at the time solely by methanogens), we needed
to explore its evolutionary implications. In the process we would per
force put the doctrine of genealogical descent with modification to
perhaps its greatest test to date. Evolution as we know it demanded
that the new "urkingdom" display two fundamental characteristics:
1- comprise a number of major organismal groups very different from
one another in their overall phenotypes. [My colleague Norman Pace
used to refer to this as "kingdom level" diversity].
2- that there be phenotypic features integral to the warp and woof of
the basic archaeal cell design that strongly distinguish it from the
eubacterial (and vice-versa) ─ features at least as striking as those
that distinguish plants from animals—and more deeply significant in
that they involve the organization of the cell itself.
Fortunately, a small cadre of scientists had quickly come round to the
three urkingdom perspective (in addition to our collaborators Wolfe
and Balch). Particular mention should be made of the Germans Otto
Kandler, Wolfram Zillig and Karl Stetter. Kandler, whose studies at
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Carl R. Woese
the time centered on (bacterial) cell walls, had visited the University
of Illinois in early 1977 (prior to the publication of our work). He was
one of the first outsiders whom we told about the three kingdom
concept, and the only one to understand and accept it upon first
hearing! Wolfram Zillig, a group leader at the Martinsried Max
Planck Institute, was an expert in DNA-dependent RNApolymerases. Both he and Kandler had encountered certain anomalies
in their own research that could be neatly explained on the basis of a
three urkingdom hypothesis. Karl Stetter, who had trained with both
Kandler and then Zillig, also appreciated the significance of the
archaebacteria ─ and proceeded to fashion a career as the greatest of
the "swash-buckling" archaea hunters of the 20th century; I wouldn't
be surprised if the majority of isolated archaeal species today still
come from his laboratory. These three individuals were the most
effective of all initially in promoting the study of the archaebacteria,
especially among Europeans. They were a strong second front, as it
were, in part because Europeans had been less affected by the
procaryote propaganda that so dominated (micro)biology in North
America.
Where are the cousins?
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The Archaea: an Invitation to Evolution
Methanogens offered no clues as to their cousins. The unusual coenzymes that methanogenic biochemistry utilizes seemed all to occur
almost nowhere else. However, with Kandler's visit to Urbana there
came a break. Kandler already knew (as did others working in the
cell wall field) that the walls of extremely halophilic bacteria did not
contain peptidoglycan, which at the time had been taken as a defining
characteristic of the "procaryote" (31). Moreover, he had just
determined that the wall of one methanogenic species was also
aberrant (though not of a halobacterial type) (15).
However, the walls of the would-be archaebacteria were not the most
important clue, for it would turn out that cell wall compositions are
variable among the archaeabacteria. But the walls were pointing us in
the right direction. The critical clue was to be atypical lipids
possessed by the halophiles ─ ether-linked and branched chain (not
ester-linked and straight chain, as the lipids of bacteria and
eucaryotes are). These strange lipids would ultimately be found in all
the archaebacteria characterized. They were the first of the universal
phenotypic characteristics found exclusively in the archaebacteria!
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Carl R. Woese
Ether-linked lipids had been discovered by Morris Kates in the early
1960s (16; 20). Kates took them, as did everyone else at the time, to
be adaptive, arising independently in certain organisms that grow in
extreme environments. Shortly thereafter, the microbiologist Thomas
Brock isolated two strange new types of bacteria that inhabited
thermophilic niches, Sulfolobus and Thermoplasma (the former
discovered also in Italy, and named Caldariella) (3; 7; 8). Another
Tom, Thomas Langworthy, went on to show that the lipids of Brock's
new isolates were of the ether-linked type, but somewhat different
than the extreme halophile lipids: Thermoplasma and Sulfolobus had
"tetra-ether" lipids, which were back-to-back covalently linked
versions of the halophile (di-ether) lipids (20; 21).
The pieces of the archaebacterial puzzle now began to fall into place.
Not only were the phenotypically diverged cousins of the
methanogens beginning to show up, but so were the traits common to
all
archaebacteria.
The
rRNA
sequences
that
defined
the
archaebacteria, though phylogenetically very telling, should not be
considered traits in the true (informative) phenotypic, organismal
sense. These sequences (in effect gene sequences) tell you nothing
about the overall phenotype, either of the group as a whole, or of the
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The Archaea: an Invitation to Evolution
subordinate sub-groupings therein. A tree based upon rRNA
sequence comparisons is basically just an abstract genealogical tree.
Look at it as only a road map interconnecting towns. The contours of
the map begin to appear, the towns bustle with people, only when the
phenotypically-informative traits become known. Those features
common (and unique) to all archaebacteria then reveal the overall
landscape of the archaebacterial world. The first of them to appear
were:
1- the unusual ether-linked, branched chain lipids.
2- the characteristic subunit structures of their DNA-dependent RNA
polymerases (more similar to their eucaryotic than their (eu)bacterial
counterparts) (48).
3- an unusual and characteristic modified version of the so-called
common arm sequence in tRNAs, the archaeal version of which
uniquely had the sequence Ψ-Ψ-C-G, rather than the customary
eubacterial and eucaryotic T-Ψ-C-G (12). Also, the so-called "Dloop" of archaebacterial tRNAs contains no "D", dihydrouridine (12).
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Carl R. Woese
4- cell walls that lack peptidoglycan; a negative trait, equivalent, say,
to our metaphorical landscape's having no lakes (15).
5- a largely unique spectrum of antibiotic sensitivities vis a vis the
eubacteria (4).
Now the rRNA analyses began to tell us even more: the new
archaebacterial urkingdom was larger than we first thought. While
the halobacteria and Thermoplasma lay within the phylogenetic
confines defined by the methanogens (46), the rRNAs of the
Sulfolobales showed these organisms to be only a sister group to the
others. [The in-group species would be called the Euryarchaeota, the
Sulfolobales and relatives, in turn the Crenarchaeota (45)]. And, a
notably deep phylogenetic divide (still not really understood)
separated the two groups (39; 46).
Reactionary science at its best
To this point our archaebacterial adventure had felt like one does
emerging from a dark forest (ignorance) and seeing before him a
beautiful panorama, with the clear sky of a bright future above you ─
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The Archaea: an Invitation to Evolution
a sky clear except for one small cloud on the horizon (a small cloud
called the "procaryote"). As it came nearer, however, that small
cloud would darken and broaden into a violent storm (as we shall
now see).
Things had gone well with the “archaes”, as we fondly called them,
to begin with ─ before their formal presentation to the scientific
community and the public at large. By the fall of 1977 the time for a
formal debut had come; now the world would see the new
urkingdom, the hitherto unknown "third form of life". I had alerted
both the NSF and NASA, my funding agencies, that what we were
about to publish might be of particular interest to them; might make a
little splash ─ the storm had yet to materialize. The two agencies
were indeed interested and decided to make a joint statement to the
press on the day the first of our two articles appeared in print. That
would be the 3rd of November, 1977. I realized something bigger
than anticipated was afoot when one or two days before the formal
press release the phone started ringing and a reporter from The New
York Times showed up in my office.
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Carl R. Woese
November 3rd. There they were! The archaebacteria! Right there on
the front page of the New York Times (and a lot of other newspapers
around the globe).
But unbeknownst to anyone at the time, a telling coincidence had
occurred, one that set the stage for an epic drama, a struggle for the
soul of biology ─ a drama in which we are today all actors.
November 3rd just so happened to be the date chosen by the then
president of the U.S. National Academy of Sciences, Philip Handler,
to release an official statement heralding the dawn of the cloning era
(signaled by the recent cloning in bacteria of the gene for the growth
hormone somatotropin). As can now be appreciated that fortuitous
coincidence was a foretaste of, the first skirmish in, the ideological
struggle between what would become the biomedical-industrial
complex and evolution resurgent. Our "third form of life", which
touched upon one of the deepest chords in human nature (i.e., where
we came from), completely wiped the press release announcing the
era of "Man-the-medical-miracle" off the front pages of the papers. I
was overjoyed at the public's appreciation of our work (10; 44)!
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The Archaea: an Invitation to Evolution
The celebratory mood, the joy at the public and general scientific
reaction, was short-lived, however. As alluded to above resistance to
the concept had already surfaced to some extent in the
microbiological community. Now the storm hit full force! On the
day the front page of The New York Times announced our discovery
of a "third form of life", my colleague Ralph Wolfe received a
telephone call from his friend, the Nobel Laureate Salvador Luria
(whom he did not initially identify), an upset Salvador Luria.
According to Wolfe, Luria told him in no uncertain terms to publicly
dissociate himself from this scientific fakery or face the ruination of
his career. In a recent recounting of the episode (47) Ralph said he
was so humiliated he "wanted to crawl under something and hide";
but he did tell Luria that supporting evidence for the claim had been
published. When informed that the scientific journal was the
Proceedings of the National Academy of Sciences (a fact that had
been mentioned in the New York Times article), Luria, a bit
befuddled, had blurted out that the October issue had just arrived but
he hadn't looked at it yet. Fortunately, Ralph left for a planned family
gathering out of town the next day (47) and thereby escaped any
further humiliation.
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Carl R. Woese
As you might expect, I saw the episode and its overall significance
differently. How could this Luria fellow have the temerity to
excoriate his friend and my colleague like that? What pulpit was he
preaching from? It appears that he had blustered at Ralph something
to the effect that: "Everybody knows that all bacteria are procaryotes;
there can't be any such thing as a 'third form of life'"! Irony of
ironies! As time (and the diligence of a particular scientific historian)
have shown, the fakery lay not in our work, but in the procaryote
concept itself (26): it is now clear that the "procaryote" was mere
guesswork. [More on this below]. But in the hey-day of the
procaryote, which this was, the true believers were out to pillory us
for our heresy: how dare we slander their Procaryote!
That day in November 1977 had laid bare a fundamental structural
problem that affected all of biology; and the condition of
microbiology was only the most obvious symptom of it. The name of
that problem was molecular biology ─ under whose hegemony 20th
century biology had developed. Molecular biology's world view was
that of classical physics ─ a world view that is inimical to biology.
Without doubt spectacular progress occurred under molecular
biology's hegemony, but that proved a superhighway to a biological
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The Archaea: an Invitation to Evolution
dead end ─ unless one looks at bioengineering as the ultimate goal of
biology! Our encounter with microbiologists over the archaebacteria
was by no means the parochial taxonomic squabble one might think.
The issue was not confined to microbiology, nor even biology as a
whole. The ultimate issue is the relationship between biology and
society ─ their conjoined future. The archaebacteria were telling 20th
century biology (microbiology in particular) that it had forsaken its
roots and had no more chance of becoming mankind's ultimate view
of biology than an oak tree has of growing on a flat rock. Such
advice ran afoul of biology's conventional wisdom, and biology,
microbiology in particular, was reacting accordingly.
As I stated earlier, one of my goals in establishing the program of
phylogenetic analysis in the first place had been to restore a sadly
lacking evolutionary perspective/spirit to biology.
I had long
cautioned that biology was on the verge of being taken over by
"science mongers and technological adventurists". The great fear was
that biology would cease being a basic scientific discipline, becoming
merely an olio of separate subdisciplines, whose common focus was
applied problems ─ the complaints of the society, as it were. This is
acceptable and proper up to a point, of course; but beyond that point
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Carl R. Woese
the framework of basic biology crumbles and the basic science turns
into engineering. And this must not happen! The "procaryote" was
both the symbol and the lynch-pin of biology's structural problem.
[Try to imagine how different 20th century biology might have been
had microbiologists stayed true to their birth-right, i.e., the world of
(micro)organisms ─ rather than turning their discipline into a theme
park for molecular gawking].
The discovery of the archaea had provided one of those special
moments when things are put into grand perspective: it was a wakeup call to microbiology and provided a time for biology as a whole to
reassess its head-long dash into reductionism. Introspective
opportunities such as this are rare, ephemeral, transient. In contrast
the grip of convention is strong, persistent, and compelling. The
discovery of the archaea was a moment that should have been
grasped. Some of us tried hard to bring this about. If only
microbiology would change back to the kind of discipline that
Beijerinck knew and was trying to develop there would be hope. But
there were so many road blocks, so many citadels to conquer. The
immediate road block, of course, was the mighty "procaryote" ─
which seemed to deny everything that biology is ─ organism,
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The Archaea: an Invitation to Evolution
ecology, evolution. From the late 1970's onward I perceived
defanging the procaryote as absolutely essential if microbiology (and
biology) were to be set right again ─ and devoted much of my energy
to it.
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