Archaebacteria and the Three Domains

peter at carrot.mcb.uconn.edu peter at carrot.mcb.uconn.edu
Fri Apr 15 17:04:49 EST 1994

I am tempted to post our whole paper from BioSystems in response to 
the recent postfrom L.A. Moran [<Co9JK6.6yu at gpu.utcc.utoronto.ca>
lamoran at gpu.utcc.utoronto.ca], but I think I should stick with the main
points and add some newer findings.

Yes, there certainly are V- (or rather A- for archaebacterial) ATPases in
some (but certainly not in many) eubacteria.

Thermus (and as I heard from K. H. Schleifer) some close relatives have
only the A-type ATPase.  (It is curious that Thermus also has a
"eukaryotic type" of malic acid dehydrogenase, Iwabe et al. '89 and
Zillig al. '92). Schleifer's, Yoshida's and my group looked for the F-
ATPase which this organism should have according to its phylogenetic
position (based mainly on 16S rRNA), but so far no F-ATPase was found in
Thermus.  For what it's worth: Thermotoga which according to 16S rRNA
forms a deeper branch has an F-ATPase; and in our analyses of F-ATPases
it so far forms the deepest branch among the F-ATPases (total of 50+ F-
ATPases).  In phylogenetic analyses the subunits of the Thermus A-ATPase
subunits group within the group of the archaebacterial ATPases.
Enterococcus has both, a normal F-ATPase (eubacterial) and a Sodium
pumping ATPase that is of the vacuolar/archaebacterial type.  It has been
claimed that this ATPase is more like the eukaryotic enzyme, however,
closer inspection shows it to be archaebacterial, some features in the
quaternary structure of the enzyme (i.e., the larger proteolipid type)
apparently evolved twice independently.  Considering this finding of
archaebacterial ATPases found in two separate (i.e. not closely related
groups of eubacteria) there are two explanations:

1: the last common ancestor already had both an A/V-type and an F-type
ATPase (both already had the paralogous catalytic and non catalytic
subunits) (Forterre, 1992).  Under this assumption we still can use the
paralogous subunits and the F-ATPase as an outgroup; one then can
consider the V/A-ATPases as a marker for organismal evolution:  The root
of the tree of life would be located inside the archaebacterial "domain";
the eubacteria would have evolved (and possibly not as a monophyletic
group) from mesophylic archaebacteria.  What makes this scenario unlikely
are the many independent losses of the F- and the V/A-ATPases in the
different groups: most of the eubacteria would have lost the A/V-type
ATPases, most of the archaebacteria would have lost the F-ATPases.  This
would have had to occur many times independently, because the two groups
of eubacteria that have the A/V type do not group together, and they do
not group at the base of the eubacterial domain.  Transport ATPases in
eubacteria have been studied extensively, I think it is unlikely that we
will find many more cases of A/V ATPases in eubacteria (but we and others
certainly keep on looking).  The other argument relies on other molecular
markers to provide restrains concerning possible organismal phylogenies
(see L.A. Morans original posting and Hilario and Gogarten, 1993).  A
third argument against this scenario is the phylogeny of the F-ATPases
(see below) and a fourth that both the V/A and the F-ATPases appear to
have evolved as proton pumping ATPases, why would the last common
ancestor have had two multisubunit enzymes that do exactly the same

2. Thermus and Entorococcus got their A-type ATPases twice independently
by means of horizontal gene transfer (defined broadly to also include the
formation of a chimera).
The second point is the "finding" of F-ATPases (or rather of a DNA that
encodes a catalytic F-ATPase subunit) in an archaebacterium.
All archaebacteria that have been studied in this respect have an A-type
ATPase (whose primary structure is very similar to the V-ATPases,
however, with respect to quaternary structure and with respect to
function it is more similar to the F-ATPases.  Methanococcus,
Methanosarcina, Halobacterium, Sulfolobus and Thermoplasma all have this
type of ATPase (A-ATPase, characterized by the sequence of its subunits).
Based on biochemical characteristics it had been suggested that
Sulfolobus, Halobacteria and Methanosarcina have an F-type ATPase, but it
might be that the A-type ATPase has biochemical features more similar to
the F than to the V-ATPase.  The latter explanation is favored by
Schaefer's group ('92) working on Sulfolobus and Mukohata's group working
on Halobacterium (92).
The F-ATPase encoding DNA fragment has been obtained by PCR from
Methanosarcina (which has a sequenced and biochemically well
characterized proton pumping A(/V)-type ATPase).  This F-ATPase fragment
from Methanosarcina groups within the eubacterial domain as shown in the
composite tree given in L.A. Moran's posting.  Under hypothesis 1
(paralogous genes, see above) this would suggest that the archaebacteria
evolved from a eubacterial ancestor.  The grouping is reminiscent of the
HSP70 and glutamine synthetase phylogenies.  If we take this grouping as
reflecting the organismal evolution, we would have to conclude that the
eubacterial A-ATPases were obtained by horizontal gene transfer (or
fusion of lineages).  Thus A/V and F- ATPases interpreted under
hypothesis 1 contradict each other, one still needs to invoke horizontal
gene transfer and a surprising multiplicity in convergent losses of

My conclusion is that one has to consider more than one molecular
phylogeny to get a complete picture of the early organismal evolution,
and that the organismal evolution is not a tree but a net.  Given that
many different characters suggest the archaebacteria as a group distinct
from the Eubacteria (and that some of these molecular markers are
involved with the basic cellular functions of transcription and
translation) it seems safe to me to conclude that a (not necessarily the)
major component of the archaebacterial cell groups separate from the
eubacteria and this component of the archaebacteria also contributed part
of the eukaryotic nucleocytoplasm.

Some other comments:

L.A Moran wrote: > I had a little trouble under-
>standing Margulis' (1993) paper. Did you read it? She seems to be saying
>that all bacteria on the planet are just one species that constantly
>exchange genes between individuals. When various bacteria fuse, new
>are formed, including eukaryotes.

I agree with Lynn Margulis in that the fusion of - and the exchange of
genetic information between organisms is more important than usually
recognized in the interpretation of molecular phylogenies.  I also agree
that it is difficult to define a bacterial species; however, their
certainly are different bacterial lineages whose evolution and phylogeny
can and should be studied.

>Your paper was quite a surprise. You readily admit that there are now

I take that as a compliment.

> To me this strongly
>suggests that many bacteria contain both types of gene (or perhaps their
>common ancestor did and one has been lost in individual species).
>If this is true, and the archaebacteria do not form a separate
>group, then you will get the trees that you have published. Both the
>V-ATPases and the F-ATPases would form independent trees with eubacteria
>and archaebacteria interspersed.

But one should find the same topology for both F and V/A-ATPases.  This
is not what we found.

>This seems to be circular reasoning. Your original dendrogram of ATPase
>sequences is always cited as one of the two papers that most obviously
>support the Three Domain hypothesis.

That's news to me, we didn't get the paper published for over a year
because it did not fit the "domain" thinking at that time (three
"equally" distant ....progenote).  As far as I know it is cited as
providing a root to the tree of life and as suggesting that an
archaebacterium like organism provided a share to the eukaryotic
Besides, the original paper included sequences from a single
archaebacterium (which certainly makes it difficult to show the
archaebacteria as monophyletic).

>Do you believe that it is still proper to cite Gogarten et al. (1989) as
>one of the main bits of evidence in support of Three Domains and the
>relationship of archaebacteria and eukaryotes?

The latter yes (qualified), the former: I would not and have not regarded
it as the main bit of information to prove the monophyly of
archaebacteria.  In most of the analyses that I have done the
archaebacterial ATPases appear as a paraphyletic group (with the
mesophylic Halobacteria and Methanosarcina closer to the eukaryotes).
Again, in either case I would not consider a single molecular phylogeny
as representative of the organismal evolution, which I do not expect to
be very tree like.

>I realize that you have rebutted their suggestion by hypothesizing one
>or more horizontal genes transfers to explain the data but to me their
>schematic dendrogram in Figure 6 looks more convincing.

It certainly looks nice, however, as I tried to point out it is at odds
with the data.  Under this hypothesis the F-ATPases and the V/A-ATPases
should result in the same phylogeny - they don't.  You still need at
least one case of horizontal gene transfer + a lot of convergent losses
of genes.  See the above discussion.
Peter Gogarten

Schaefer, G. and Meyering-Vos, M. 1992, F-type or V-type? The chimeric
nature of the archaebacterial ATPsynthase. Biochim. Biophys. Acta 1101,

Ihara, K. and Mukohata, Y. 1992, The ATP synthase of Halobacterium
salinarium is an archaebacterial type as revealed from the amino acid
sequences of its two major subunits. Arch. Biochem. Biophys. 286, 111-116

Other citations as in previous postings.

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