Since Dr. van den Broek's posting "Is Carl Woese losing a
Kigngdom", and because a number of replies posted in response,
are in reference to our work, I think it would be useful to
clearly define the issues.
The two main tenets of the current "three domain
phylogenetic view" are:
(i) Archaebacteria (or Archaea) constitutes a monophyletic domain
which is completely distinct from the rest of the bacteria.
(ii) Eukaryotic nuclear genome has directly descended from an
Archaebacterial ancestor.
However, both of these tenets, which were initially proposed
based on either 16S rRNA data or elongation factor (EF-1,EF-2)
sequences are not supported by other recent well characterized
phylogenies.
In terms of the relationship within the prokaryotes, if one
examines all available protein sequences two common patterns are
observed. (a) For a number of proteins that in general are not
highly conserved in all prokaryotes (e.g. EF-1 /Tu, EF-2/G, RNA
polymerase II and III subunits, GroEL/Tcp-1, etc.), the
phylogenies based on such sequences generally indicate a distinct
grouping of archaebacteria and eubacteria. (b) On the other hand
for several other protein sequences that are highly conserved
viz. Hsp70, glutamine synthetase, glutamate dehydrogenase,
FGARAT, FtsZ, etc., a specific relationship of archaebacteria to
the gram-positive group of bacteria is observed. Such a
relationship is supported by different phylogenetic methods as
well as by the signature sequences in these proteins. Further for
a number of these gene sequences where sequence information from
adequate number of archaebacterial species is available (viz.
Hsp70, glutamate dehydrogenase), the various archaebacterial
species do not form a monophyletic group as analysed by different
phylogenetic methods. Instead they show a polyphyletic branching
within the gram-positive bacteria. These results do not support
the first tenet of the three domain hypothesis. It is of interest
in this regard that before Woese's proposal that "the
archaebacteria constitutes the third form of life",
archaebacteria were traditionally classified with the gram-
positive group of bacteria and many of them show a gram-positive
staining characteristic. A close relationship between
archaebacteria and gram-positive bacteria has also been suggested
by Cavalier-Smith based on other characteristics.
It should also be pointed out that in the phylogenetic trees
based on 16S rRNA on which the current prokaryotic phylogeny is
almost entirely based, both gram-positive and gram-negative
bacteria show polyphyletic branching within each other. However,
most of the critical nodes pointing to such relationships
generally have very low bootstrap scores ( 30-50%) and hence are
statistically not supported. In contrast, phylogenies and
signature sequences based on a number of protein sequences (viz.
Hsp70, GroEL)indicate a clear distinction between gram-positive
and gram-negative bacteria and such a relationship is
statistically strongly supported. For some proteins such as GDH,
FGARAT, FtsZ, common sequence signatures are seen between
archaebacteria, gram-positive bacteria and cyanobacteria.
However, this is a reflection of the fact that within gram-
negative bacteria, cyanobacteria constitute one of the deepest
branching groups. Thus within the prokaryotes, the gram-positive
bacteria show specific relationship on one hand to archaebacteria
and on the other hand to gram-negative bacteria.
This relationship could be indicated in the following form;
Archaebacteria <-> Gram +ve bacteria -> Gram -ve bacteria
(The double headed arrow indicates the polyphyletic nature of the
two groups)
However, it need be emphasized that the above division
between the prokaryotic groups is not very rigid, but constitutes
an evolutionary continuum. It is expected that species will be
found and some examples (e.g. Deinococcus) already exist which
represent intermediates in the above transitions. Thus the
evolutionary relationship between various prokaryotes
(i.e.archaebacteria and eubacteria) could be readily explained by
normal evolutionary mechanisms without evoking any unusual event.
By contrast to the evolutionary relationship within the
prokaryotes, if one examines the evolutionay relationship of the
eukaryotic nuclear genes (i.e. excluding the organelle genes) to
the prokaryotes then two different type of relationships is
observed.
For a number of gene sequences (the majority of which are
transcription/translation related, e.g. EF-1 , EF-2, RNA
polymerase II and III subunits, GroEL/Tcp-1, ATP synthase and
genes, isoleucine t-RNA synthetase, etc.) the eukaryotic homologs
show a close relationship to the archaebacteria. Since some of
the genes which were initially examined showed this sort of
relationship,it led to the belief (which has now become one of
the main tenent of the three domain dogma) that the eukaryotic
cells have directly descended from an archaebacterial ancestor.
On the other hand for a number of highly conserved protein
sequences (e.g. Hsp70, glutamine synthetase, glutamate
dehydrogenase, FGARAT, etc.) a very different sort of
relationship is observed. In these cases the eukaryotic homologs
show a closer relationship to the gram-negative bacteria, and in
some cases (viz. Hsp70) are clearly derived from gram-negative
bacteria.
It should be mentioned that by contrast to the
archaebacteria which have contributed only to the nuclear genome,
the gram-negative bacteria have also contributed the eukaryotic
organeller (viz. mitochondria and chloroplasts) genomes as well.
Thus, very often eukaryotic cells have multiple homologs which
are derived from gram-negative bacteria. For most sequences, due
in part to poor characterization, the information to clearly
distinguish between different homologs is generally lacking. The
presence of multiple genes within a cell also raises the
possibility of genetic exchange between the nuclear and the
organelle genes. Due to these concerns, establishing that a
particular eukaryotic nuclear gene is of eubacterial origin has
proven a more difficult task. However, in the case of Hsp70
sequences, which constitute the most conserved protein known to
date, and where all of the euaryotic homologs have been
extensively characterized and clearly distinguished from each
other based on numerous sequence signatures, it has been possible
to establish beyond any doubt that the eukaryotic nuclear genes
for this protein are derived from gram-negative bacteria and that
this relationship is not due to any sort of horizontal transfer
or genetic exchange between the sequences.
Roger in his posting has criticized some of our results as
resulting from (i) comparison of NON-HOMOLOGOUS sequences,(ii)
resulting from lateral gene transfers and (iii) limited sampling
of sequences from each group. Our detailed response to Roger's
criticisms will be appearing in the October issue of TIBS.
However, briefly for the present discussion, for the hsp70
phylogeny, which represents at present the best characterized
protein phylogeny covering all life forms, none of the above
criticisms apply, and it clearly shows that the eukaryotic
nuclear homologs are derived from gram-negative bacteria rather
than archaebacteria. In comparison to Hsp70, some of the other
protein phylogenies that we have examined (viz. glutamine
synthetase, glutamate dehydrogenase, aspartate aminotransferase,
etc.) are not as clear cut (for reasons stated above and
discussed in the literature). However, for these protein
sequences as well, the eukaryotic homologs are clearly more
closely related to the gram-negative bacteria than to the
archaebacteria. These observations thus do not support and
challenge the second tenet of the three domain hypothesis that
the eukaryotic cells are direct descendent of archaebacteria.
To explain these mutually discordant phylogenies, we have
proposed (an extension of Zillig's model) that the ancestral
eukaryotic cell arose by a unique endosymbiotic fusion event
involving an archaebacteria (eocyte, based on Lake's data on EF-1
and EF-2 sequences) and a gram-negative bacteria. Following the
fusion event, at an early stage, an assortment of genes from the
two fusion partners took place and the resulting cell acquired
and characteristics from each of the two parents. (see our recent
TIBS article May, 1996, for further details of the model).
The question may be asked as to why should the chimeric
model be preferred over direct descendance of eukaryotic cells
from archaebacteria? Well, there are several reasons for doing
so:
(a) It is the simplest and most parsimonius model to explain all
of the genes/proteins phylogenies.
(b) It readily explains why certain characteristics of
eukaryotic cells (e.g. components of transcription and
translation machinery) are similar to archaebacteria, while
others are clearly derived from eubacteria (e.g. ester-
linked straight chain membrane lipids, fatty acid
synthetase)
(c) It provides a plausible explanation for the origin of the
eukaryotic cell nucleus and endomembrane systems (Discussed
in our recent TIBS article ) and is supported by the
observed duplication of chaperones genes (e.g. Hsp70,
Hsp90)which accompanied this event.
(d) It provides a plausible explanation for the enormous
structural difference between the eukaryotic and prokaryotic
cell types, and the absence of any species that are
intermediates in this transition. These observations cannot
be readily explained by simple evolutionary mechanisms. By
contrast, a sudden and major evolutionary transition could
readily be explained by fusion and subsequent gene
assortment of two very different species.
(e) The inferred time of divergence of eukaryotic species from
archaebacteria and the eubacteria (about 2 By ago) based on
genetic distances between different proteins sequences
(Doolittle et al , Science, 271, 470-477, 1996) and the
fossil records, can also be satisfactorily explained by the
chimeric model.
(f) The phylogenies and signature sequences in various
eukaryotic genes also provide compelling evidence that all
extant eukaryotic species are monophyletic. This in turn
suggests that the postulated fusion event that gave rise to
the ancestral eukaryotic cell was unique and a successful
fusion between the prokaryotic parents took place only once
in the 3.8 By history of life on this planet. Had this
chance fusion event not taken place, our landscape perhaps
would have looked similar to that of Mars and its sole
inhabitants would have been prokaryotes.
Thus in contrast to the evolutionary diversity seen within
the prokaryotic or eukaryotic species, which can be readily
accounted for by normal evolutionary mechanisms, the major
evolutionary transition from prokaryote to eukaryotes and the
various characteristics of these cells (gene phylogenies,
relatedness to arhcaebacteria in some regards and to gram-
negative bacteria in others) can't be explained by such means.
These observations indicate that despite the strong views
expressed by Carl Woese
"I have such distaste for prokaryote-eukaryote
dichotomy? This is not the unifying principle that we
all once believed it to be. Quite the opposite: it is
a wall, not a bridge. Biology has been divided more
than united, confused more than enlightened, by it.
This prokaryote-eukaryote dogma has closed our minds,
retarded microbiology s development and hindered
progress in general" (Microbiol.Rev., 58,1,1994)
the basic division of living organisms into two primary
groups -
the Prokaryotes (ancestral) and the Eukaryotes (derived) is
strongly supported by all available molecular and other data.
The above observations thus challenge the two main tenets of the
current "three domain paradigm" which has resulted from Carl
Woese work. However, all of the above observations which
disagree with this view have not been dealt with by the
supporters of this paradigm. This pattern is in accordance with
what Woese has recently written
"When facts or concepts arise that challenge (a
paradigm), these tends to be ignored. If that is not
possible, they are scoffed at and otherwise communally
rejected- informally. Only as a last resort will the
paradigm formally contest the novelty that threatens it
(i.e. treat it scientifically)".
(Microbiol.Rev.,58,1,1994).
Hopefully, the discussion that has ensued by Dr. van den
broek's
posting will spur them to contest these observations and/or take
them into consideration.
For readers of this news group who may not aware of the
background to this posting, a few references to our work in this
area are given below. References to the work of others mentioned
here could be found within them.
Gupta, R.S. Golding, G.B( 1993). J. Mol. Evol. 37: 573-582.
Gupta, R.S., Aitken, K, Falah, M and Singh, B (1994). Proc. Natl.
Acad. Sci. USA 91: 2895-2899.
Gupta, R.S. and Singh, B.(1994) Current Biol., 4,1104-1114.
Falah, M. and Gupta, R.S. (1994) J. Bacteiol. 176, 7748-7753.
Golding G.B. and Gupta, R.S.(1995) Mol Biol.Evol, 12,1-6.
Gupta, R.S (1995). Mol. Microbiol. 15: 1-11.
Gupta, R.S. Golding, G.B(1996)Trends in Biochem.Sci.,21,166-171
Gupta, R.S. (1996) In Culture Collections to Improve the Quality
of Life, R.A. Samson, et al.,eds. pp. 83-90.
Gupta, R.S.(1997). Antonie von Leeuwenhoek (in press).
Radhey S. Gupta
Department of Biochemistry
McMaster University, Hamilton
Ontario, Canada.
