Download Proteobacteria as the Origin of Eukaryotes: The Syntrophic Hypothesis

J Mol Evol (1998) 47:517–530
© Springer-Verlag New York Inc. 1998
Symbiosis Between Methanogenic Archaea and ␦-Proteobacteria as the
Origin of Eukaryotes: The Syntrophic Hypothesis
David Moreira,1 Purificación López-Garcı́a2
1
2
Laboratoire de Biologie Cellulaire (BC4), Bâtiment 444, URA CNRS 2227, Université Paris-Sud, 91405 Orsay Cedex, France
Institut de Génétique et Microbiologie, Bâtiment 409, Université Paris-Sud, 91405 Orsay Cedex, France
Received: 5 January 1998 / Accepted: 18 March 1998
Abstract. We present a novel hypothesis for the origin
of the eukaryotic cell, or eukaryogenesis, based on a
metabolic symbiosis (syntrophy) between a methanogenic archaeon (methanobacterial-like) and a ␦proteobacterium (an ancestral sulfate-reducing myxobacterium). This syntrophic symbiosis was originally
mediated by interspecies H2 transfer in anaerobic, possibly moderately thermophilic, environments. During eukaryogenesis, progressive cellular and genomic cointegration of both types of prokaryotic partners occurred.
Initially, the establishment of permanent consortia, accompanied by extensive membrane development and
close cell–cell interactions, led to a highly evolved symbiotic structure already endowed with some primitive
eukaryotic features, such as a complex membrane system
defining a protonuclear space (corresponding to the archaeal cytoplasm), and a protoplasmic region (derived
from fusion of the surrounding bacterial cells). Simultaneously, bacterial-to-archaeal preferential gene transfer
and eventual replacement took place. Bacterial genome
extinction was thus accomplished by gradual transfer to
the archaeal host, where genes adapted to a new genetic
environment. Emerging eukaryotes would have inherited
archaeal genome organization and dynamics and, consequently, most DNA-processing information systems.
Conversely, primordial genes for social and developmental behavior would have been provided by the ancient
myxobacterial symbiont. Metabolism would have been
Correspondence to: P. López-Garcı́a; e-mail: [email protected]
issued mainly from the versatile bacterial organotrophy,
and progressively, methanogenesis was lost.
Key words: Eukaryogenesis — Syntrophy — Symbiosis — Eukaryotic evolution — Interspecies H2 transfer — Methanogenic archaea — Histones — ␦Proteobacteria — Myxobacteria — Anaerobic consortium
Introduction
Evolutionary biology has entered an era characterized by
the widespread use of phylogenetic reconstruction techniques based on protein and nucleic acid sequence comparison. The most striking breakthrough was the socalled ‘‘Woesian revolution,’’ whose origin dates back to
1977 when, after comparison of ribosomal RNA (rRNA)
oligonucleotide catalogs, a tripartite division of the living
world into primary kingdoms, Eubacteria, Archaebacteria, and Eukaryotes, was claimed (Woese and Fox 1977).
These lineages were later reclassified as the domains
Bacteria, Archaea, and Eucarya, respectively (Woese et
al. 1990). Additional work on protein sequence comparison further supported the phylogenetic distinctness of the
three groups. Subsequently, sequence comparison of ancestral paralogous genes, i.e., those derived from genes
duplicated before the separation of the three domains,
such as the elongation factors EF-Tu (1␣) and EF-G (2)
and ATPase V and F genes, was used by some authors to
root the tree of life in the bacterial branch (Gogarten et
518
al. 1989; Iwabe et al. 1989). Such classification and rooting have become widely accepted and represent the
prevalent viewpoint on biological evolution, although its
validity remains controversial (Forterre et al. 1993;
Doolittle and Brown 1994).
Those ideas have essential implications regarding one
of the major unsolved questions in contemporary biology: the origin of the eukaryotic cell or eukaryogenesis.
Notwithstanding Woese’s original speculation of a very
primitive last common ancestor, a ‘‘progenote,’’ from
which the three domains would have been directly derived (Woese 1987), the Gogarten and Iwabe rooting
implies two things: first, that the last common ancestor
(or cenancestor) was prokaryotic and, second, that eukaryotes were direct descendants of a common archaeal–
eukaryal ancestor (Gogarten et al. 1989; Iwabe et al.
1989; Woese 1990). Despite the scientific success of this
so-called ‘‘archaeal model,’’ alternative hypotheses addressing the question of eukaryotic origins have been
proposed. Detailed discussion of these is beyond the
scope of this work; however, we briefly summarize them
in order to provide a general framework as a base to
formulate a different model for eukaryogenesis.
Hypotheses for the Origin of Eukaryotes: State of
the Art and Criticisms
A completely independent hypothesis was originally
stated by Reanney (1974) and Darnell (1978), who proposed that the cenancestor was a eukaryote itself. Nevertheless, these authors did not explain how the transition
from this ancestor to prokaryotic cells proceeded. The
idea was recently revived by Forterre, who argued that
the results shown by Woese and others are artifacts derived from the inadequacy of the phylogenetic methods
employed (Forterre 1996). He hypothesized that prokaryotes issued from a eukaryote-like ancestor by a reductive
process as a consequence of adaptation to hyperthermophilic environments (Forterre 1995). However, these
ideas fail to explain the evolution of the extremely complex eukaryotic cellular organization directly from precellular systems. If eukaryotes evolved directly from precellular systems, they must have relied on a continuous
supply of prebiotic organic substrates over a long period
of time, since bacterial-derived chloroplasts appeared
later in evolution. It is highly unlikely that any prebiotic
‘‘soup’’ provided a regular source of organic nutrients up
to a eukaryote-like stage of organismal evolution. In addition, eukaryotic life seems incompatible with the high
temperature regimes of the archaean Earth, for which
increasing evidence exists (Knauth 1992; Schwartzman
and Shore 1996). The antagonism with high-temperature
regimes could be circumvented by alluding to a relatively moderate thermophilic environment compatible
with higher temperature limits for eukaryotes (Brock
1986). However, the essential advantage of a hot origin
hypothesis is the implication that the first living beings
were autotrophic, relying only on chemolithotrophic resources. Finally, a eukaryotic ancestor is at odds with the
Precambrian fossil record, which suggests that the most
ancient fossils (3500 million years ago) are prokaryotic
(Schopf 1993), whereas the earliest eukaryotic fossils
date at 1800 to 2100 million years ago (Han and Runnegar 1992).
The remaining hypotheses are based on the currently
accepted idea of a prokaryotic cenancestor from which
the prokaryotic domains, i.e., Archaea and Bacteria, were
the primary offshoots. The above-mentioned archaeal
model, which entails the direct evolution of the eukaryotic nuclear genome from a common archaeal–eukaryal
ancestor, could be classified as ‘‘autogenous.’’ This
model has not always been supported by protein sequence comparison. Some phylogenies, especially those
based on elongation factors EF-1␣ and EF-2 (Rivera and
Lake 1992; Baldauf et al. 1996), but also those based on
rRNA sequence parsimony analysis (Lake 1988), argued
in favor of a specific sisterhood between eukaryotes and
crenarchaea (kingdom Crenarchaeota), or ‘‘eocytes,’’ according to the nomenclature of Rivera and Lake (1992).
The other archaeal lineage (kingdom Euryarchaeota)
would be closer to Bacteria (especially Gram-positive
bacteria), and the paraphyly of Archaea would be inferred (Lake 1988; Gupta and Singh 1992; Rivera and
Lake 1992; Gupta and Golding 1993; Doolittle and
Brown 1994). However, some of these results have been
interpreted as artifacts derived from undetected hidden
paralogies and presumptive horizontal gene transfers
(Hilario and Gogarten 1993; Brown et al. 1994; Roger
and Brown 1996). Bias in species sampling could also
account for some molecular characteristics claimed to be
shared only by eukaryotes and crenarchaea; for instance,
several of these have recently been detected in some
methanogenic euryarchaea, including Methanopyrus
kandleri (Lake and Rivera 1996; Feng et al. 1997). In
addition, limitations in current phylogeny reconstruction
methods may constitute a major pitfall in this kind of
analysis, since very often the approximations and assumptions utilized are quite unrealistic. These methods
are especially sensitive to different evolutionary rates
among the organisms studied, a problem known as ‘‘unequal rate effect’’ (Felsenstein 1978; Lake 1991).
As a result of all these apparent contradictions, hypotheses appealing to an archaeal–bacterial partnership
at the origin of eukaryotes have been on the rise. These
could be considered ‘‘chimeric’’ or ‘‘heterogenous,’’
and may be classified into three subgroups according to
the kind of relationship that putatively occurred between
the two partners: fusion, phagotrophy, or symbiosis. The
fusion model postulates that the eukaryotic nuclear genome is a chimera resulting from the amalgamation of an
archaeon and a bacterium (Zillig et al. 1989; Zillig
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1991). Among other mechanistic difficulties, this hypothesis fails to explain the origin of the nuclear membrane. A previous model attempting to overcome this
problem had already been proposed by Cavalier-Smith,
suggesting that the origin of this structure was the fusion
of cytoplasmic cisternae (Cavalier-Smith 1980) in a transient intermediate between an archaeon and a Grampositive bacterium (Cavalier-Smith 1987). In addition,
fusion models are based on events never observed in
nature, i.e., cell fusion between lineages as phylogenetically distant as archaea and bacteria. Phagotrophic models propose the engulfment of one of the partners by the
other. Thus, Lake and Rivera (1994) suggested that a
bacterium engulfed a crenarchaeon (eocyte), which
would have originated the nucleus (‘‘eocyte hypothesis’’). Similarly, Gupta and Golding (1996) postulated
the engulfment of an eocyte by a Gram-negative bacterium lacking a cell wall. In these models, the nuclear
envelope and the endoplasmic reticulum would be remnants of the eocytic plasma membrane and the bacterial
phagotrophic vesicle membrane, respectively. In a related but quite different model, Sogin (1991) proposed
the engulfment of an archaeon by a protoeukaryote still
endowed with RNA-based metabolism. However, it is
difficult to conceive the persistence of such ancient protoeukaryotes having a primitive and inefficient RNAbased metabolism in a world already inhabited by ecologically competitive DNA-based prokaryotes. However,
this hypothesis overcomes the main objection for the rest
of the phagotrophic models, which is the inability for
bacteria to accomplish phagocytosis.
Considering these major drawbacks, the most realistic
models could be those based on symbiotic relationships
between archaea and bacteria, i.e., the symbiotic models.
Symbiosis does not depend on single, perhaps unlikely,
events, such as cell fusion or phagocytosis, but offers the
possibility of coevolution (eventually cointegration) over
long periods of time. To date (see also Note Added in
Proof), the only symbiotic model described is the serial
endosymbiotic theory (SET) proposed by Margulis
(1970, 1993), which recapitulates some of the pioneering
ideas of Mereschkowsky (1905). This hypothesis postulates that eukaryotes evolved from the symbiosis between an archaeon (a thermoacidophilic Thermoplasmalike organism) and a bacterium (a spirochete). In this
way, the archaeon acquired swimming motility, while
the spirochete obtained useful metabolites (Margulis
1996). Subsequent endosymbiotic incorporations of ␣proteobacteria and cyanobacteria originated mitochondria and plastids, respectively. This hypothesis does not
favor the endosymbiotic origin of the nucleus. Although
this kind of motile symbiosis is known to occur between
eukaryotic protista and spirochetes (Cleveland and Grimstone 1964; Bloodgood and Fitzharris 1976), it has never
been observed between archaea and spirochetes, which,
in addition, are not known to thrive in extreme acido-
philic environments. T. acidophilum lacks a cell wall and
was the first archaeal species where putative ‘‘histone’’
proteins were characterized (Searcy and DeLange 1980).
Those were regarded as crucial eukaryotic-like traits, and
some others followed (Searcy 1987), all of which made
this archaeon a candidate of choice for that proposal. But
in the end, its histone-like proteins turned out to be much
more closely related to bacterial HU proteins rather than
to eukaryotic histones (DeLange et al. 1981; Drlica and
Rouvière-Yaniv 1987; Grayling et al. 1994).
If we dismiss this (quite unlikely) Thermoplasma–
spirochete symbiosis as the origin of eukaryotes, does
any kind of primary symbiosis exist that could have
paved the way to eukaryotic evolution? A look at the
present-day archaeal–bacterial symbioses may help answer this question.
Contemporary Symbioses Between Archaea
and Bacteria
The existence of symbiosis has not been extensively explored within the domain Archaea. Most characterized
symbioses between archaea and bacteria are related to
the process of ‘‘interspecies H2 transfer,’’ which is metabolically advantageous for both partners. The bacterial
partner ferments organic compounds producing more
oxidized organic molecules and H2 as end products. This
H2, together with environmental CO2, is assimilated by
the archaeal partner, a methanogen, to form methane in
an energy-conserving reaction. The methanogen acts as a
sink of H2, thus increasing the velocity of the otherwise
less efficient bacterial fermentative reactions. This kind
of mutualistic symbiosis is known as syntrophy and it is
frequently found in anaerobic natural communities (for a
review, see Fenchel and Finlay 1995). Generally, a close
proximity between partners is required to avoid loss of
the volatile H2. This requirement leads to the establishment of well-integrated consortia. A most striking example is the case of the so-called ‘‘Methanobacillus omelianskii,’’ which, 50 years after its discovery, was
shown to be not a single organism but a pair of dissimilar
species in close contact: a chemotrophic bacterium
known as S-organism and the methanogenic archaeon
Methanobacterium bryantii (Bryant et al. 1967). When
isolated, each organism grows poorly on its own. For this
reason they were classified as obligate syntrophs.
Many more examples of this kind of syntrophic symbiosis between methanogens and bacteria are presently
known. They involve a variety of methanogenic archaea
and a more restricted diversity of bacteria, usually members of the ␦ subdivision of the Proteobacteria, which
groups the vast majority of the sulfate-reducing bacteria
(Woese 1987). Some of these bacteria, e.g., species of
the genera Desulfovibrio and Syntrophobacter, can develop syntrophic relationships with methanogens (Tatton
520
et al. 1989). Also, some nonstrict sulfate-reducing ␦proteobacterial genera are specialized in obligate syntrophic symbioses with methanogens, such as Pelobacter
and Syntrophus (Zinder 1993).
The existence of such intimate relationships raises an
intriguing and exciting question: Might a methanogenic
archaeon/␦-proteobacterium syntrophic symbiosis have
triggered eukaryogenesis?
A Syntrophic Symbiosis at the Origin of Eukaryotes
We hypothesize that eukaryotes emerged from a syntrophic symbiotic event involving a methanogenic archaeon
and a ␦-proteobacterium in an anaerobic, likely moderately thermophilic, context. The former would have provided the basic genome and nucleic acid metabolism, and
the latter, most metabolic capabilities. Presumably, the
eukaryotic endoplasmic reticulum and nuclear membrane arose, at least in part, from archaeal membranes or
membrane components, while the plasmic membrane derived mostly from a bacterial membrane.
We try now to justify this proposal briefly by first
analyzing some key eukaryotic-like features of the respective candidate partners.
The Archaeal Partner
Since the recognition of Archaea as a distinct lineage,
archaeal research has been prompted largely by the discovery of many essential features in common with eukaryotes but absent from bacteria. Transcriptional machinery similarities were among the first to be
documented (Zillig et al. 1978, 1979). Subsequent work,
including completion of the first archaeal genome sequences, allowed the identification of homologies in replication and translation as well (Bult et al. 1996; Klenk et
al. 1997; Olsen and Woese 1997; Smith et al. 1997).
Most authors interprete these as evidence of archaeal–
eukaryal sisterhood rather than specific bacterial loss
from a complex ancestor. Many of these shared features
would equally support either an autogenous or any bacterial–archaeal chimeric (heterogenous) hypothesis. In
this section, we do not recapitulate all these characteristics, extensively revised elsewhere (Klenk and Doolittle
1994; Langer et al. 1995; Thomm 1996; Belfort and
Weiner 1997; Dennis 1997; Brown and Doolittle 1997;
Edgell and Doolittle 1997; Reeve et al. 1997). Alternatively, we focus on certain essential traits shared specifically by eukaryotes and archaea belonging to the kingdom Euryarchaeota (particularly a subgroup of
euryarchaeal methanogens), which might reveal an intimate phyletic relationship between both (a common history). These are the presence of histones and nucleosomes, DNA topoisomerases, and lipids.
A classical eukaryotic hallmark is the organization of
chromatin in nucleosome arrays. Eukaryotic nucleosomes are formed by a histone core, consisting of an
octamer built upon an (H3–H4)2 tetramer plus two H2A–
H2B dimers, around which DNA wraps (Kornberg 1977;
Luger et al. 1997). The tetramer interacts first with DNA,
and it is critical to determine nucleosome positioning,
whereas H2A–H2B dimers assemble later and may have
a role in transcriptional regulation. Likely, H2A and H2B
evolved after H3–H4, allowing further DNA condensation (Ramakrisnan 1995) and increasing the regulatory
repertoire (Luger et al. 1997). Histone homologues specifically related, by sequence and structural data, to the
highly conserved H3–H4 have been discovered in Archaea, but exclusively in the euryarchaeal branch (Grayling et al. 1994; Starich et al. 1996; Pereira et al. 1997;
Reeve et al. 1997; Zlatanova 1997). No core histone has
been discovered so far in Bacteria or in the crenarchaea,
in spite of intense search and of the fact that these proteins are very abundant in the cell. They have been found
in the hyperthermophilic Thermococcales (Sandman et
al. 1994; Ronimus and Musgrave 1996), and in several
hyperthermophilic and mesophilic methanogens from the
orders Methanococcales and Methanobacteriales (Grayling et al. 1994; Darcy et al. 1995; Pereira et al. 1997)
(Fig. 1). However, archaeal histones are not present in
the related Thermoplasma acidophilum, which contains
instead a bacterial HU-like protein, HTa (DeLange et al.
1981; Grayling et al. 1994). The presence of histones in
these archaea and in eukaryotes establishes an intriguing
link between both groups of organisms. Plausible explanations of the occurrence of this essential eukaryotic
character in some archaea are (i) independent loss of
histones inherited from a histone-endowed common archaeal–eukaryal ancestor (or even cenancestor), but retention in eukaryotes and some archaea for unknown
reasons, and (ii) horizontal transfer. However, the most
parsimonious possibility may be that histones evolved in
the euryarchaeal branch and eukaryotes simply inherited
them. From this viewpoint, eukaryotes would have
implemented and optimized histone function, may be
more related to transcriptional regulation at the beginning. Indeed, a parallel origin of the nucleosome core
and eukaryotic transcription from archaea has already
been formulated based on the similarities between the
protein families H3, H4, archaeal histones, and the central domain of subunits A and B of the CCAAT-binding
factor (CBF), a transcription factor associated with eukaryotic promoters (Ouzounis and Kyrpides 1996).
A second euryarchaeal–eukaryotic connection concerns DNA topoisomerase distribution. These universal
proteins are required to disentangle DNA strands or duplexes and are, therefore, indispensable for cellular processes such as replication, transcription, recombination,
and regulation of DNA supercoiling (Drlica 1992; Wang
1996). Depending on their ability to cut one or both DNA
strands, they are classified into two mechanistic types, I
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Fig. 1. Distribution of histones, reverse gyrase, and gyrase in an rRNA-based archaeal tree and possible phylogenetic position of the archaeal
syntrophic partner. The tree (adapted from Woese et al. 1990; Stetter 1996) is idealized, so that branch lengths are not necessarily proportional to
phylogenetic distances. Thick lines indicate thermophilic lineages; asterisks, moderately thermophilic species.
and II, each one of which is subdivided into two phylogenetic families (Wang 1996; Bergerat et al. 1997). The
presence of at least one type I and one type II enzyme
member seems to be necessary for any organism. In this
regard, and strictly speaking, the only archaeal trace present in eukaryotes is a protein involved in meiotic recombination that is homologous to one of the two subunits of topoisomerase VI (Topo VI) (Bergerat et al.
1997). Topo VI is the prototype of a recently recognized
type II phylogenetic family, which is widespread among
the archaea but not found in bacteria. Nevertheless, another interesting observation comes from the distribution
in archaea of the two supercoil-introducing activities
known to date, gyrase and reverse gyrase. Reverse gyrase has attracted much attention because of its ability to
introduce positive supercoils in DNA and because it appears to be a marker of hyperthermophily. It was first
thought to be only archaeal. However, it was found not
only in all hyperthermophilic archaea, but also in hyperthermophilic bacteria (for reviews, see Duguet 1995;
Forterre et al. 1996). Thus, it is present in hyperthermophilic methanogens, but not in mesophilic or thermophilic (such as Methanobacterium thermoautotrophicum,
growing optimally at 65°C) species (Fig. 1). On the other
hand, gyrase, a type II DNA topoisomerase specifically
introducing negative supercoils, is present in all bacteria.
It may be an original bacterial feature, since it is the only
type II enzyme in the deep-branching Thermotoga maritima (Guipaud et al. 1997) and likely in many other
bacteria (Huang 1996). It also appears in some euryar-
chaea (Archaeoglobus and haloarchaea; see Fig. 1), presumably by horizontal import from bacteria (Forterre et
al. 1994; Klenk et al. 1997; López-Garcı́a 1998). Neither
gyrase nor reverse gyrase is present in eukaryotes, which
possess only topoisomerases with relaxing activities. If
we consider, for instance, the rRNA phylogenetic tree, a
sort of ‘‘living temperature gradient’’ may be established
along the euryarchaeal branch, from the deep-branching
hyperthermophiles to the mesophilic halophiles (Fig. 1).
Roughly, reverse gyrase is present at the base of the
branch, coincident with hyperthermophily, whereas gyrase appears toward the tip. Interestingly, M. thermoautotrophicum, located in the middle of this gradient, is
devoid of the genes coding for both supercoilintroducing activities (Smith et al. 1997). Although this
is not a conclusive piece of evidence, it is interesting to
note that this organism is endowed with only relaxing
topoisomerases, which, in combination with histones,
highly recalls the situation found in eukaryotic cells.
One major argument against eukaryotic membranes
being derived from archaeal ones concerns lipids. Archaeal lipids are mainly isoprenoid glycerol ethers, in
contrast to the bacterial and eukaryal ester-based linkages. However, the presence of ether lipids is not restricted to archaea, being present also in hyperthermophilic bacteria of the genera Thermotoga and Aquifex
(De Rosa et al. 1989; Huber et al. 1992). Only the simultaneous occurrence of ether linkages, the isopranic
nature of the aliphatic chains, and the chirality of glycerol (2,3-sn-glycerol instead of 1,2-sn stereochemistry)
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Table 1.
General lipid occurrence and distribution in Archaeaa
‘‘Core’’ lipids
Polar lipids (headgroups)
Extreme thermophiles
Methanogens
Halophiles
Caldarchaeol-based
Nonitol caldarchaeol
(with or without rings)
Phosphoinositolb
Archaeol-based
Caldarchaeol (no rings)-basedb
Archaeol-based only
Phosphoamino polar headgroups
(serine and ethanolamine, mostly)b
Unsulfated sugar groups
(glucose, galactose)
Phosphoglycerol-derived
Sugars (glucose, galactose,
usually unsulfated)
a
b
Sulfated/unsulfated glycosyl groups
(glucose, mannose, galactose)
Archaeol, diphytanylglycerol diether; caldarchaeol, dibiphytanyldiglycerol tetraether. Based on Kates (1993).
Important traits (molecules or synthetic pathways) found also in eukaryotes (see text).
can be considered of phylogenetic relevance (Gambacorta et al. 1994). Archaeal lipids are diverse and complex (Table 1). This turns out to be especially true for
lipids in methanogenic species, whose variability is derived mainly from the phosphodiester-bonded, watersoluble alcoholic residues, in contrast to the lipid structure in extreme halophiles or sulfur-dependent archaea,
whose diversity is due to the sugar residues of glycolipids (Koga et al. 1993).
Glucosaminyl archaetidylinositol was the first polar
lipid reported to share a characteristic with eukaryalspecific lipids. It is the major either polar lipid in Methanosarcina barkeri, and it consists of GlcNp(␣1-6)-myoinositol 1-phosphate as the polar head group, linked via
a phosphodiester bond to an archaeol (2,3-di-Ophytanyl-sn-glycerol). Interestingly, this polar group is
identical to the conserved core structure of glycosylated
phosphatidylinositol, which serves as a membrane protein anchor in eukaryotes (Nishihara et al. 1992).
Another differential feature of archaeal lipids is the
occurrence of tetraether lipids in some archaeal members, including methanogens and sulfur-dependent archaea (but not Methanopyrus or Thermococcus species)
(Table 1). It has been demonstrated recently that the
synthesis of tetraether from diether lipids in T. acidophilum, a close relative of methanogens (Olsen et al. 1994),
is inhibited by a potent inhibitor of eukaryotic steroid
biosynthesis acting on squalene epoxidase. Both biosynthetic enzymes appear to have structural similarity. Since
squalene epoxidase is located in the endoplasmic reticulum, it has already been suggested that this structure
originated from cytoplasmic protoarchaeal membranes
(Yamagishi et al. 1996).
Although inositol and tetraether lipids are present in
some sulfur-dependent archaea and in methanogens, they
have not been detected in extreme halophiles. Glycerol
and amino compounds have been found as polar head
groups only in methanogens (Koga et al. 1993) (Table 1).
Taken together, these data suggest that there is a restricted range of methanogenic archaea (centered around
Methanobacteriales) as putative candidates to have set up
a syntrophic eukaryogenetic symbiosis.
The Bacterial Partner
The ␦ subdivision of the Proteobacteria clusters three
groups that are phenotypically different, the dissimilatory sulfate-reducing bacteria, the myxobacteria, and the
bdellovibrios (Oyaizu and Woese 1985). All these
groups are able to perform a broad variety of energetic
metabolisms, and they may have evolved their respiratory chains from that of an anaerobic sulfate-reducing
ancestor (Woese 1987). Some genera within this subdivision exhibit gliding motility, and the myxobacteria and
the bdellovibrios also exhibit complex developmental
and cell division cycles (Reichenbach and Dworkin
1992; Shimkets and Woese 1992).
Myxobacteria are of special interest with regard to the
problem of eukaryogenesis since, indeed, they display a
surprising number of similarities with eukaryotes at the
molecular level. The most remarkable are (a) the presence of serine–threonine kinases (previously thought to
be unique to Eukarya) (Muñoz-Dorado et al. 1991); (b) a
protein involved in spore morphogenesis that shares
some structural properties with calmodulin and with vertebrate ␤ and ␥ crystallins (Wistow et al. 1985; Inouye et
al. 1993); (c) an intercellular signaling factor (factor C)
homologous to vertebrate 17 ␤-hydroxysteroid dehydrogenase (Baker 1994), which is related to the existence of
steroids in this group (Kohl et al. 1983); (d) the presence
of a phosphatidyl-inositol cycle with the possible participation of a G protein in the process (Benaı̈ssa et al. 1994)
(inositol phosphates are second messengers in eukaryotic
signal transduction); (e) the existence of reverse transcriptase and retron elements (Rice et al. 1993); (f) the
presence of a GTPase involved in motility homologous
to the Ras/Rab/Rho superfamily of small eukaryotic
GTPases (Hartzell 1997); and (g) the existence of a transcriptional factor homologous to the eukaryotic highmobility group (Y) proteins (Murillo 1997).
In addition, myxobacteria display interesting similarities to some eukaryotic groups at the level of primary and
secondary metabolism. They secrete a great variety of
hydrolytic enzymes and antibiotics, likely in relation to
their ecological role as predators of other microorgan-
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Fig. 2. Schematic representation of eukaryogenesis from syntrophic
symbiosis between methanogenic archaea and ancestral sulfaterespiring ␦-proteobacteria. Initially, a syntrophic consortium is established (A) where each methanogenic cell (black) is surrounded by
several bacterial partners endowed with double membranes. Extensive
membrane development for efficient interspecies H2 transfer and in-
creased cell–cell interactions progressively occur (B), leading eventually to bacterial cytoplasmic fusion (C). Ultimately, redundant membranes are lost (D), an endoplasmic reticulum develops, and nuclear
pores appear (E). The process is accompanied by gene transfer, gene
replacement, and bacterial genome extinction. Mitochondria may have
been acquired at the latest stages. See text.
isms or scavengers of organic matter (Dworkin 1996).
This ability, together with pigment production, like
melanin (Burchard and Dworkin 1966), and their complex developmental cycle, explains why botanists and
microbiologists classified them as fungi for almost a century (reviewed by Reichenbach and Dworkin 1992).
Taken together, all these data allowed some authors to
conclude that ‘‘there is little doubt that the myxobacteria
have played some kind of role in the evolution of eukaryotic multicellularity’’ (Dworkin 1996). We think
that this ‘‘kind of role’’ may have been indeed played by
ancestral sulfate-reducing fermentative myxobacteria at
the very origin of eukaryotes.
both partners (Schink 1992). The production of mucus,
slime, and other extracellular materials by some syntrophic species greatly enhances the cohesion of these structures (Fenchel and Finlay 1995).
In this way, the physical juxtaposition of the syntrophic partners facilitates the interspecies H2 transfer,
which would be strongly improved by subsequent development of coupled membrane invaginations and extrusions. Interestingly, methanobacteria are able to develop
extensive membrane invaginations forming internal
membrane systems. Indeed, they have been referred to as
‘‘methanochondria,’’ due to their mitochondrial-like appearance under the microscope (Zeikus and Bowen
1975). They are also able to form membrane extrusions,
and even the production of branched cellular extensions
by an apparent cell wall-less species, Methanoplasma
elizabethii, was reported (Rose and Pirt 1981). These
kinds of membrane invaginations and extrusions are also
widespread among the proteobacteria, improving considerably diverse metabolic capacities with the increase in
cell surface (Leive 1973).
Once this putative consortium of methanoarchaea and
anaerobic ancestral sulfate-reducing myxobacteria was
established, a further step would involve bacterial membrane fusion around the methanogenic core (Fig. 2). This
would have been facilitated by the intimate relationships
already existing between the bacterial cells surrounding
the methanogen, in an analogous way to contemporary
myxobacterial interactions. The result would be a methanogen embedded within a continuous layer of bacterial
cells with a complex membrane interphase. This arrangement may be regarded as similar to the final situation of
The Eukaryogenetic Pathway
Formation of Stable Consortia and Close
Cell–Cell Interactions
As depicted schematically in Fig. 2, the first eukaryogenetic step would consist of the consolidation of a permanent syntrophic consortium integrated by a core methanogenic archaeon entirely wrapped by ␦-proteobacterial
cells. This situation can actually be found in present-day
microbial flocs, such as those formed by Methanobacterium and Syntrophobacter species (Dubourgier et al.
1988; Thiele et al. 1988; Fenchel and Finlay 1995). Flocs
are generally composed of large numbers of both cell
types, sustaining a maximum efficiency by the adoption
of mosaic arrangements allowing extensive mixing of
524
a putative phagocytosis of an archaeon by a bacterium,
although not requiring the necessity of such a very unlikely event.
Membrane Development and Membrane Loss
A later eukaryogenetic state would imply loss of one of
the plasma membranes (Fig. 2). Membrane loss occurs in
nature; for instance, phagocytotic membranes engulfing
some cellular endosymbionts, including mitochondria
and plastids (Gray 1992). Even the outer membrane of
Gram-positive bacteria could have been lost if, as suggested, the presence of two cell membranes is an ancient
character in bacteria (Blobel 1980; Rachel et al. 1990).
Two possibilities can be considered. One involves the
loss of the external membrane of bacterial cells, i.e., the
bacterial membrane in closest contact with the methanogen. Hence, a single eukaryotic plasma membrane could
be generated. The preservation of the archaeal membrane, currently used as an argument against a putative
archaeal origin of the eukaryotic nucleus, could in this
way explain some archaeal-like features of nuclear and
endoplasmic reticulum membranes (see above). If this
turns out to be the case, substitution of ether-linked lipids
by bacterial ester-linked ones would have been required,
while some polar lipids and metabolic pathways would
have been conserved. However, these similarities do not
argue specifically in favor of this possibility, since a
similar situation could result if the archaeal membrane
were lost, but some of the genes involved were conserved, and their products began to interact with bacterial
membranes. This suggestive possibility would entail
elimination of the unique membrane of the methanogenic
archaeon, while maintaining useful membrane components and their associated synthetic pathways. In such a
scenario, it might be speculated that the archaeal membrane, completely encircled by a continuous layer of bacterial ones, became redundant and was lost, as well as the
outer bacterial membranes exposed to the environment.
In any case, the final situation would have corresponded to a methanogenic core wrapped by a double
membrane with a complex system of invaginations and
extrusions. This eukaryotic-like membrane arrangement
could be considered preadaptative, having evolved initially with a function (increase in contact surface between syntrophic partners) quite different from that of
present-day eukaryotic membranes. The central compartment surrounded by a double membrane could be considered as a protonucleus plus a protoendoplasmic reticulum.
Gene Flow, Gene Replacement, and Eukaryotic
Genome Emergence
We suggest that, during this cointegrative process, a bidirectional flow of genetic material between both part-
ners took place. Since single archaeal cells were surrounded by several bacterial ones, horizontal transfer
from bacterial to archaeal genomes would have been
more important. Two critical consequences must have
followed. First, transfer of some housekeeping genes
from one species to the other led to an irreversible stabilization of the symbiotic consortium. Second, the transferred bacterial genes met a new genetic environment in
the archaeal genome, and therefore, they had to adapt to
it by acquisition of some (eukaryotic-like) archaeal features such as TATA boxes or histone-mediated regulation. This symbiotic gene transfer could be compared to
that undergone by mitochondrial and plastid genomes
towards the nuclear genome (Gray 1992). In some cases,
the complete genome of a symbiont can be transferred in
a so-called ‘‘genome extinction’’ process (Palmer 1997).
Interesting examples are the hydrogenosomes (Müller
1993) and some secondary plastid endosymbionts
(Palmer and Delwiche 1996). Obviously, each transferred gene had to acquire a targeting signal ensuring
efficient posttranslational transport of the encoded protein to the donor symbiont. Such targeting signals generally consist of short sequences, which seem to be easily
incorporated, as testified by all mitochondrial and plastid-derived nuclear genes. This may happen in a short
period of time, as has been documented for coxII. This
gene is normally transcribed from mitochondrial DNA,
but in some legumes it was recently transferred to the
nucleus, where it acquired a classical sequence to target
the protein back into the mitochondrion (Nugent and
Palmer 1991).
Logically, bacterial genes having homologues within
the host archaeal genome should also have been transferred, and these genes may have often replaced the host
copies. This is a well-known process for some eukaryotic
genes of mitochondrial or plastidial origin, and is referred to as symbiotic gene replacement (Martin et al.
1993; Martin and Schnarrenberger 1997). Obviously, the
probability of a successful gene replacement was higher
when the transferred gene encoded a protein that was not
involved in a large number of interactions with other
proteins, as could be the case for many enzymes of different metabolic pathways. In contrast, host genes encoding proteins interacting with many others were unlikely to be replaced by the transferred homologues. This
would also be in agreement with the finding that eukaryotic replication, transcription, and translation are archaeal-like, whereas some eukaryotic metabolic genes
are more akin to those of bacteria (Feng et al. 1997;
Brown and Doolittle 1997).
Such a putative gene flow, and eventually gene replacement, toward the archaeal symbiont would explain
eukaryotic genome’s birth more easily than fusion models of eukaryogenesis do (Zillig et al. 1989). Whereas, as
stated by Roger and Brown (1996), it is very difficult to
explain how two very different genetic systems (archaeal
525
and bacterial) could be successfully integrated by such a
radical event as cell fusion, a progressive integration by
transfer of discrete amounts of genetic information, as
occurred in the case of mitochondria and plastids, seems
much more feasible. One simple and efficient genetic
transfer mechanism to be considered, although not exclusive, would be conjugation, which, indeed, can efficiently mobilize DNA between distantly related prokaryotes (Mazodier and Davies 1991) and even between
individuals from different domains, including eukaryotes
(Buchanen-Wollaston et al. 1987; Heineman and Sprague 1989). Notably, conjugative plasmids and conjugation are widespread in Archaea (Rosenshine et al. 1989;
Schleper et al. 1995; Zillig et al. 1996) and also in Proteobacteria, including the ␦ subdivision (Argyle et al.
1992; Wall et al. 1993).
All these processes of gene uptake would have been
accompanied by an increase in genome size and, ultimately, genome partition and linearization. These phenomena occur in present-day prokaryotes. Indeed, some
(relatively large) prokaryotic genomes are integrated by
more than one large replicon (chromosomes and/or giant
plasmids) (Allardet-Servent et al. 1993; Michaux et al.
1993; Choudhary et al. 1994; Netolitzky 1995), and
many bacteria have linear chromosomes and plasmids
(Hinnebusch and Tilly 1993; Casjens et al. 1995). In
halophilic euryarchaea, some of which carry the largest
known genomes among the Archaea, large extrachromosomal elements (up to approximately 700 kb) are widespread too (López-Garcı́a et al. 1996). Remarkably,
myxobacterial genomes are the largest found in prokaryotes (around 9–10 mbp) (Chen et al. 1990; Neumann et
al. 1992), which might be required for their complex
social and developmental biology. Such values are comparable to those of free-living unicellular eukaryotes
such as Saccharomyces cerevisiae (13 mbp) and the microalga Galdieria sulphuraria (10 mbp) (Coetzee et al.
1987; Moreira et al. 1994).
Thus, finally, a developed integrated consortium already endowed with a complex large genome would
have emerged. Metabolic, social, and developmental
functions would have had a bacterial origin, whereas
genetic information-processing activities, less susceptible to the interspecific transfer, would have been archaeal-like. This is the overall case in contemporary eukaryotic genomes.
Metabolism
Initially, the metabolic activities of the eukaryogenetic
syntrophic consortium would have been greatly dependent on interspecific H2 transfer via intimate cell-to-cell
interactions. General metabolism would have mostly relied on the bacterial H2-releasing fermentation of organic
substrates, followed by archaeal H2-dependent methanogenesis. The end products were diverse oxidized organic
molecules and methane. Likely, the consortium displayed broad metabolic abilities, contributed mainly by
the bacterial partner. In fact, most ␦-proteobacteria (both
sulfate-reducing and myxobacteria) are able to decompose a great diversity of organic compounds (Reichenbach and Dworkin 1992; Widdel and Hansen 1992). Interestingly, myxobacteria produce extracellular enzymes
hydrolyzing several macromolecules (Reichenbach and
Dworkin 1992), which might have already evolved in the
putative ancestral sulfate-reducing myxobacterial partner. Consequently with these capabilities, these consortia
would have thrived in anaerobic (maybe microaerobic)
environments, gaining energy from the fermentation of a
variety of organic compounds and, possibly, playing the
ecological role of organotrophic scavengers, as some eukaryotes do today.
During the eukaryogenetic process, a dramatic metabolic change must have occurred, leading to the complete
loss of methanogenesis, absent in contemporary eukaryotes. This change can be explained in several ways. On
the one hand, adaptation to an efficient saprophytism
(even phagotrophism after cytoskeleton development,
likely favored by size increase) made methanogenesis no
more selectively advantageous, and it was finally lost.
The methanogenic partner was, however, already essential to the consortium as recipient of the bacterial genome. On the other hand, the acquisition of the mitochondrial endosymbiont undoubtedly caused substantial
changes in the protoeukaryotic metabolism. All known
eukaryotes appear to have harbored mitochondria once
(Germot et al. 1996; Roger et al. 1996, 1998). Therefore,
mitochondrial symbiosis must have been a very early
event in the history of eukayotes, perhaps as ancient as
they are. At this point, it is tempting to speculate that the
mitochondrial symbiosis driving force might have been
methane production by the syntrophic consortium itself.
Most methanotrophic bacteria, which derive energy from
methane oxidation under aerobic or anaerobic conditions, belong to the Proteobacteria, and some genera
(such as Methylocystis, Methylosinus, and Methylobacterium) to the ␣ subdivision (Lidstrom 1991; Hanson and
Hanson 1996). Methanotrophs are ubiquitous; some species associate with methanogens to form consortia under
anoxic conditions (Hoehler et al. 1995), and some are
intracellular symbionts of diverse invertebrates (Cavanaugh 1985, 1993). Furthermore, methanotrophs develop
internal membrane systems strikingly similar to those of
mitochondria (Davies and Whittemburg 1970; Takeda
and Tanaka 1980). If ancestral eukaryotes were methane
producers, the first mitochondrial endosymbionts could
have been anaerobic or microaerophilic methanotrophs
of the ␣-Proteobacteria. Taking into account the fact that
mitochondrial acquisition is a very ancient event, perhaps even prior to the massive accumulation of atmospheric O2, the original endosymbiont’s metabolism may
have been different from the present-day aerobic one.
526
The common evolutionary origin of aerobic mitochondria and anaerobic hydrogenosomes may actually suggest this (Bui et al. 1996). A mitochondrial transition to
aerobic respiration would have been subsequent, as an
adaptative consequence to increasing environmental O2
concentrations. Methanogenesis was, thus, ultimately
lost in favor of a much more productive aerobic metabolism, and old-fashioned methanogenic (proto)eukaryotes
might have been completely outcompeted by the novel
efficient organotrophs.
Discussion and Perspectives
We have reported here an alternative hypothesis for the
origin of eukaryotes in an attempt to overcome some of
the unsolved questions in current models, many of which
were artificially born to explain particular contradictions
in protein phylogenies. We believe that the idea that
eukaryotes derived from syntrophic symbiosis between
certain methanogenic archaea and ancestral anaerobic
sulfate-respiring ␦-proteobacteria provides a coherent
molecular and cellular evolutionary scenario compatible
with actual phenomena in nature.
This evolutionary syntrophic symbiotic process presumably took place under anaerobic (or microaerobic),
and possibly moderately thermophilic (Schwartzman and
Shore 1996), conditions, as can be found in many present-day methanogenic consortia (Zinder and Koch
1984). The inferred primeval eukaryotic anaerobic nature
could be in agreement with the reported existence of
eukaryotic fossils predating the massive oxygen release
to the atmosphere (Han and Runnegar 1992; Canfield
and Teske 1996).
In addition to the compatibility with observations
from microbial ecology, this eukaryogenetic pathway accommodates a continuous evolution, allowing a parsimonious explanation of the existence of many eukaryotic
characters in common with archaea or bacteria, in many
cases with specific groups within them. This is seen in
the case of histone- and nucleosome-based genome organization shared with the euryarchaea and of many molecular features of the complex social behavior and developmental cycles of some ␦-proteobacteria.
Curiously, as mentioned in a previous section, certain
molecular phylogenies may favor a closer proximity of
eukaryotes and crenarchaea (Rivera and Lake 1992;
Baldauf et al. 1996). In addition to possible unrecognized
paralogies, horizontal transfers, or biased sampling, unequal rate effects may mask their real evolutionary relationships, especially since very different evolutionary
rates often occur among eukaryotes and archaea. Certain
insertion/deletion sequences are more difficult to explain. The example often cited corresponds to an oligopeptide stretch of 11 amino acids shared by EF-1␣ sequences from crenarchaea and eukaryotes, which is used
as evidence for their specific sisterhood (Rivera and
Lake 1992). However, it has been noted that this insertion/deletion is not a stable character. In fact, among the
Euryarchaeota, this particular region has changed at least
three times (Creti et al. 1994). Therefore, an alternative
explanation would be that this oligopeptide stretch represents an ancestral archaeal character (perhaps present
in the euryarchaeon that we propose to be involved in the
eukaryogenetic pathway) that has been lost several independent times in the euryarchaeal branch. Additionally,
although the EF-1␣ and EF-2 analysis indicates that the
eukaryote–crenarchaea relationship seems the best supported, other possibilities cannot be statistically rejected
(Baldauf et al. 1996). In any case, an essential consequence of the proposed syntrophic hypothesis is that the
resulting eukaryotic genome would have had, at least
partly, a high evolutionary rate. Acceleration of evolutionary rates appears to be a common phenomenon
among symbiotic partners (Lutzoni and Pagel 1997; Matic et al. 1997), and indeed, some eukaryotic genes, such
as rRNA genes, have evolved rapidly (De Rijk et al.
1995; Baldauf et al. 1996). Besides, the generation of
innovative properties is an intrinsic, even conclusive,
characteristic of symbiosis (Smith 1989; Margulis and
Fester 1993). Although mosaic evolution and differential
gene loss can complicate our understanding of life’s evolutionary history, the differential presence of a significant number of relevant characters, such as the common
occurrence of histones and associated pathways of genomic regulation in euryarchaea and eukaryotes, may constitute a valuable phylogenetic tool.
This syntrophic eukaryogenetic hypothesis is capable
of being tested. First, some nuclear eukaryotic genes are
thought to have been acquired via endosymbiotic gene
replacement by mitochondrial genes (Martin and Schnarrenberger 1997). Perhaps a closer investigation of some
of them could reveal more similarity to their ␦-proteobacterial than to their ␣-proteobacterial counterparts.
Even if many phylogenetic signals permitting discrimination between both possibilities could have been extinguished after such a long evolutionary period, having
␦-proteobacterial genomes sequenced may be very useful. Unfortunately, few ␦-proteobacterial gene sequences
are presently available to perform a meaningful analysis.
Second, evidence may come from the analysis of present-day consortia. It would be most interesting to analyze in more detail the molecular biology of obligatory
symbiosis and try to detect, for instance, recent gene
horizontal transfers or how, if detected, they may be
taking place. Finally, a different look at the fossil record
(especially of prokaryotic consortia) might reveal important information concerning one of the most exciting
intelectual challenges in evolutionary biology, the
enigma of the origin of eukaryotes.
Acknowledgments. We wish to thank David Musgrave and Henner
Brinkmann for critical reading of the manuscript. D.M. is a postdoc-
527
toral fellow of the spanish Ministerio de Educación y Ciencia, and
P.L.G. is a postdoctoral fellow of the European Community (Biotechnology Program).
Note Added in Proof. During the reviewing process of the manuscript, a new symbiotic hypothesis for the origin of eukayotes was
proposed founded on metabolic grounds: the hydrogen hypothesis
(Martin and Müller 1998). In this case, an anaerobic H2-dependent
autotrophic archaeon (possibly a methanogen) would have established
a syntrophic relationship with a bacterium (␣-proteobacterium-like)
generating H2 as a waste product of heterotrophic metabolism. The
archaeon would have finished by engulfing the bacterial symbiont to
increase contact surface. The bacterium would have subsequently
evolved to originate either hydrogenosomes or mitochondria in different present-day eukaryotes. Although both ‘‘hydrogen’’ and ‘‘syntrophic’’ hypotheses are complementary in several aspects (especially
some metabolic details of the symbiosis and archaeal molecular features), they differ substantially in the bacterial partnership. In our proposal, mitochondria would derive from an independent, maybe simultaneous, symbiotic event. Interestingly, both hypotheses concur in the
suggestion of an anaerobic metabolism for the origin of protomitochondrial symbiosis.
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