- Email: [email protected]
Endosymbiotic Gene Transfer
C
H
A
P
T
E
R
27
Endosymbiotic Gene Transfer: A Special Case of Horizontal Gene Transfer Germane to Endosymbiosis, the Origins of Organelles and the Origins of Eukaryotes Katrin Henze, Claus Schnarrenberger and William Martin
Endosymbiotic gene transfer describes the process through which chloroplasts and mitochondria relinquished the majority of their genes to the nucleus while not having surrendered the majority of proteins integral to the eubacterial nature of their metabolism. It is a special case of lateral gene transfer that was very important for the establishment of biochemical compartmentation during the evolution of eukaryotic cells. Many examples of endosymbiotic gene transfer in the history of higher plant genes for chloroplast-cytosolic isoenzymes of the Calvin cycle and glycolysis have been considered in the past few years. The data indicate that nuclear genes for almost all glycolytic enzymes of the eukaryotic cytosol were acquired from eubacteria early in eukaryotic evolution, probably in the course of the endosymbiotic event that gave rise to the origin of mitochondria. The genes for enzymes that are possessed both by the symbiont and host during a given endosymbiosis are redundant. In such cases, it is observed that the intruding gene contributed by the symbiont had a very high likelihood of successful gene transfer. But the protein products of genes that were transferred from symbiont to host chromosomes have been surprisingly often rerouted to compartments other than that from Horizontal Gene Transfer ISBN: 0-12-680126-6
which the genes were donated. Because of this, the localization of a protein within the eukaryotic cell is not per se an indicator of the evolutionary origin of its gene. The evolutionary history of the enzymes of central carbon metabolism in eukaryotes and the role of lateral gene transfer in that process is summarized. These results are considered in light of the ability of competing alternative theories for the origins of eukaryotes to account for the observations.
INTRODUCTION Genes encoded in organellar genomes attest beyond all reasonable doubt to the eubacterial ancestry of chloroplasts (Martin et al., 1998) and mitochondria (Gray et al., 1999). But how does endosymbiotic theory account for the origin of organellar proteins that are not encoded in organellar DNA? Their fate is commonly explained with the help of a scenario elegantly argued by Weeden (1981) that is known as the “product specificity corollary” (also known as the “gene transfer corollary”) to endosymbiotic theory. It posits that during the course of eukaryotic history, the majority of genes for proteins integral to organellar metabolism were
351
369
Copyright © 2002 by Academic Press. All rights of reproduction in any form reserved.
352
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
be expected for a given eukaryotic nuclear gene, one has to have a biological model for the origins of eukaryotes that generates such predictions. What was the host? Biologists have a number of different suggestions, but no generally accepted answer to that question, but there are a few important clues that can serve as a guideline. Discovery of the archaebacterial nature of components of the nuclear genetic apparatus (Pühler et al., 1989; Ouzounis and Sander, 1992; Rowlands et al., 1994; Langer et al., 1995) and the results of extensive molecular phylogenetic work have slowly unveiled the host that acquired the mitochondrion as a descendant (in part or in whole) of an archaebacterium (Zillig et al., 1989; Ouzounis and Sander, 1992; Brown and Doolittle, 1995, 1997; Langer et al., 1995; Doolittle, 1996; Ribiero and Golding, 1998; Rivera et al., 1998). Under the simplest set of premises possible, this means that the host cell that acquired the mitochondrion was an archaebacterium (Doolittle, 1996; Martin and Müller, 1998). However, many other more complicated models involving more than two symbiotic partners to give rise to a mitochondrionbearing eukaryote have been elaborated. It is worthwhile briefly to recapitulate a few of these models to see what sorts of predictions they generate, in particular about the evolution of molecules that occur in the cytosol of contemporary eukaryotes. Probably the most popular model up until a few years ago is what is best described as the achezoa model (Cavalier-Smith 1987a,b), also sometimes called the “classical hypothesis” (Doolittle, 1980, 1998). Under this view, a primitively amitochondriate, nucleus-bearing cell (an archezoon) is suggested to have arisen from an archaebacterial ancestor in a process that involved the origin of typical eukaryotic features (cytoskeleton, endocytosis, nucleus and mitosis). A member of that ancient group acquired the mitochondrion through endosymbiosis, while others (the suspectedly most primitive eukaryotes) remained amitochondriate up to the present day. This model has not withstood the scrutiny of molecular phylogeneticists, who have found that all amitochondriate eukaryotes studied to date possessed a mitochondrion in
transferred to the nucleus where they became integrated into the regulatory hierarchy of the nucleus and acquired a transit peptide, so that the functional gene products could be reimported into the organelle of their genetic origin on a daily basis since. This is a reasonable and logical scenario that satisfyingly explains why organelles have retained so much of their biochemically eubacterial heritage while having relinquished to the nucleus the majority of genes necessary to have done so. Though ultimately incorrect in the majority of specific cases in which it has been tested (Martin and Schnarrenberger, 1997; Martin, 1998), primarily due to the unfulfilled predictions that it generates about the origin of cytosolic enzymes, the gene transfer corollary does manage adequately to account for several organelle-to-nucleus gene transfer events (Martin and Cerff, 1986; Baldauf and Palmer, 1990; Brennicke et al., 1993; Martin et al., 1993). However, the simple phrase “organelle-tonucleus gene transfer” raises a different, much more difficult and in our view much more pressing question, namely: what is the origin of the nuclear genome under endosymbiotic theory? When Weeden (1981) formulated the gene transfer corollary, it was not known whether chloroplasts and mitochondria were descendants of endosymbionts or not, and his considerations generated testable predictions that could help to muster evidence in favor (or in disfavor) of that view (Gray and Doolittle, 1982). A salient element of that reasoning was that nuclear-encoded organellar enzymes should reflect the evolutionary history of the symbiont while cytosolic enzymes in eukaryotes should reflect the evolutionary history of the host (Weeden, 1981; Gray and Doolittle, 1982). Although we now know with certainty that plastids and mitochondria are descendants of free-living eubacteria, and although we are reasonably (but not completely) confident that the origin of mitochondria preceeded the origin of chloroplasts in evolution, it is perhaps surprising that endosymbiotic theory is just as much in the dark about the origin of the host that acquired mitochondria today as it was 20 years ago. In other words, in order to have a null hypothesis for the patterns of similarity to
370
ENDOSYMBIOTIC GENE TRANSFER
their evolutionary past, but lost the organelle subsequently (Clark and Roger, 1995; Germot et al., 1997; Embley and Hirt, 1998; Martin and Müller, 1998; Gray et al., 1999; Roger, 1999; Tovar et al., 1999; Müller, 2000; Philippe et al., 2000), the evidence for which is founded in nuclear genes in these amitochondriate organisms which reflect a mitochondrial ancestry (that is, they were transferred from the organelle to the nucleus via endosymbiotic gene transfer). Furthermore, the achezoa model clearly predicts all eukaryotes in general, and amitochondriate eukaryotes in particular, to possess archaebacterial enzymes of cytosolic metabolism, because under this model, amitochondriate protists are, on the bottom line, to be interpreted as direct descendants of archaebacteria (Cavalier-Smith, 1987b). A version of the achezoa model has been published under the name of the “ox-tox” hypothesis (Andersson and Kurland, 1999), but it is not fundamentally different from the achezoa model, rather it is very similar to 1975 formulations of the endosymbiont hypothesis (John and Whatley, 1975). For a further discussion of this matter, see Rotte et al. (2000) and Martin (2000). A second model is that of Lynn Margulis, which entails a symbiosis between a spirochete and a Thermoplasma-like archaebacterium (Sagan, 1967; Margulis, 1970, 1996) to give rise to a primitive, flagellated, mitochondrion-lacking (amitochondriate) eukaryote that subsequently acquired the mitochondrion through endosymbiosis. In the most recent formulation, that model suggests that the spirochete endosymbiont also gave rise to the nuclear compartment (Margulis et al., 2000). This model predicts that eukaryotic enzymes should, in general, be more similar to Thermoplasma enzymes than to homologues from other prokaryotes, but the sequence of the Thermoplasma genome did not bear out that prediction (Cowan, 2000). A third model that has been discussed at length in the literature is the idea that the nucleus was an endosymbiont. This model is exactly as old as the notion that chloroplasts descend from cyanobacteria, because the first paper thoroughly to argue the latter case (almost 100 years ago) also argued the former, albeit more briefly (Mereschkowsky, 1905). The
353
idea that the nuclear compartment is the remnant of an ancient endosymbiosis suffered extinction for many decades (Mereschkowsky, 1905), but it was resuscitated in 1994 (Lake and Rivera, 1994), was rejuvenated in 1996 (Gupta and Golding, 1996), came more or less fully back to life in 1998 (Lopez-Garcia and Moreira, 1999) and has even been claimed to receive direct support from data (Horiike et al., 2001). In these formulations, the nucleus is viewed as an archaebacterial symbiont that took up residence in a eubacterial host. In Margulis’s formulation, a spirochete symbiont (that became the flagellum and the nucleus) took up residence in an archaebacterial host. It should be noted that there are several severe cell biological problems (discussed in Martin, 1999b; Rotte and Martin, 2001) with this model that are usually overlooked by its advocates, most notably (1) that the nucleus is not bounded by a double membrane, like chloroplasts and mitochondria, but rather by a folded single membrane, (2) that the nuclear membrane disintegrates at mitosis (viable chloroplasts and mitochondria never lose their surrounding membranes), and (3) the way that the cytosol is separated from the nucleus has no similarity whatsoever to the way that free-living prokaryotes are separated from their environment. Perhaps most importantly, “nucleosymbiotic” models derive an archezoon (a nucleus-bearing but amitochondriate eukaryote), whereby – as mentioned above – available data indicate that all eukaryotes, including those that lack mitochondria, once possessed mitochondria in their evolutionary past, but lost them secondarily. Therefore, these models predict eukaryotes that never possessed mitochondria (achaezoa) to be found, descendants of the stem that acquired mitochondria. Furthermore, with regard to their statements about the cytosol, these models would ultimately predict eukaryotes to possess DNA both in the nucleus and in the cytosol (in addition to genomes in chloroplasts and mitochondria). The fourth, and arguably simplest, current model to derive a mitochondrion-bearing eukaryote is called the “hydrogen hypothesis” (Martin and Müller, 1998). It posits that the host cell of the mitochondrial symbiosis was an
371
354
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
archaebacterium, specifically an autotrophic and H2-dependent archaebacterium, with an energy metabolism possibly similar to that found in modern methanogens. It accounts for the common ancestry of mitochondria and hydrogenosomes – the H2-producing organelles of ATP-synthesis in anaerobic eukaryotes (Müller, 1988, 1993; Embley and Hirt, 1998). It posits that eukaryotes acquired their heterotrophic lifestyle, including the genes and enzymes necessary for that lifestyle, from the mitochondrial symbiont via endosymbiotic gene transfer from the heterotrophic symbiont’s (eubacterial) to the autotrophic host’s (archaebacterial) chromosomes (Martin and Müller, 1998). Under this model, the origins of mitochondria/hydrogenosomes and the origins of eukaryotes are identical and the nucleus is a subsequent eukaryotic invention, the origin of which probably relates mechanistically to the origin of eubacterial lipid synthesis in eukaryotes (Martin, 1999b). This model explicitly predicts eukaryotes to have a eubacterial glycolytic pathway in the cytosol as the result of specifically selected endosymbiotic gene transfer from symbiont to host prior to the evolution of a mitochondrial protein import machinery.
from a variety of sources were collected from GenBank and subjected to phylogenetic analysis (Figure 27.1). The tree for RPI, which is a relatively short enzyme of about 220 amino acids, does not provide very sharp resolution, but it does reveal that the enzymes from plants, animals, and fungi are clearly more similar to eubacterial homologues than they are to archaebacterial homologues (Figure 27.1A). In spinach, there is only one isoenzyme of RPI known; cell fractionation has shown it to be located in the plastid (Schnarrenberger et al., 1995). The subcellular localization of the two Arabidopsis enzymes inferred from the genome sequence is currently unknown. The plant enzyme does not branch specifically with the cyanobacterial homologue, nor does the enzyme from animals and fungi branch specifically with proteobacterial homologues, as one might expect if these enzymes were donated to eukaryotes from the ancestors of plastids and mitochondria, respectively. This can be due to a number of factors, including the simple phylogenetic resolution problems involved with a protein that is only 220 amino acids long or, alternatively, to horizontal gene transfer between free-living eubacteria subsequent to the origins of organelles (see below). It is noteworthy that the plant enzyme does not branch with its homologues from animals and yeast, suggesting that the eubacterial donor of the plant gene was a different bacterium from that which donated the gene to the heterotrophic eukaryotes sampled. The tree for PGluM (Figure 27.1B) reveals that the eukaryotic genes are derived from a single acquisition from a eubacterial donor, and that the higher plant chloroplast-cytosol isoenzymes are related by a gene duplication in the plant lineage, as has been observed for the majority of chloroplast-cytosol isoenzymes studied to date (Martin and Schnarrenberger, 1997), and in contrast to the predictions from the product specificity corollary (Weeden, 1981). Archaebacterial homologues of PGluM were not identified, but further searching revealed that eubacterial PGluM is related to phosphomannomutase (PMM) which does have homologues in archaebacterial genomes (Figure 27.1B). The plant PMM genes surveyed are descendants of
RESULTS AND DISCUSSION Evolution of two further enzymes of central sugar phosphate metabolism in higher plants In previous work, we have studied the evolution of several enzymes involved in sugar phosphate metabolism in the chloroplast and cytosol of higher plants (Henze et al., 1994; Martin and Schnarrenberger, 1997; Martin and Herrmann, 1998) and of the cytosolic homologues in several protists (Henze et al., 1995, 1998, 2001; Wu et al., 2001). Two enzymes whose gene origins have been unclear are ribose-5-phosphate isomerase (RPI), that catalyzes the freely reversible isomerization of ribose-5-phosphate and ribulose-5-phosphate, and phosphoglucomutase (PGluM), that catalyzes the freely reversible interconversion of glucose-1-phosphate and glucose-6-phosphate. Homologues for both genes
372
ENDOSYMBIOTIC GENE TRANSFER
355
FIGURE 27.1 Protein phylogenies for (A) ribose-5-phosphate isomerase and (B) phosphoglucomutase. The RPI tree was inferred using ProtML (Adachi and Hasegawa, 1996) with local rearrangements and the JTT-F matrix starting from the neighbor-joining topology. The PGluM tree was inferred with the neighbor-joining method (Saitou and Nei, 1987) from the Dayhoff distance matrix using Phylip (Felsenstein, 1993). Bootstrap proportions >80 are indicated at branches.
eubacterial PMM genes, but again, not specifically related to the cyanobacterial homologue (see below). Notably, Figure 27.1B shows that the PGluM sequence from the amitochondriate protist Entamoeba histolytica (Ortner et al., 1997) is also an acquisition from eubacteria. In this
respect it is important to recall that although Entamoeba histolytica does not have either mitochondria or hydrogenosomes, it was recently discovered to possess a small, double membranebounded organelle in the cytosol – termed the mitosome – that is most easily interpreted as an
373
356
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
intermediate stage in the evolutionary process of mitochonrion and hydrogenosome reduction (Tovar et al., 1999).
and Doolittle, 1997), glucose-6-phosphate dehydrogenase (G6PDH) (Wendt et al., 1999) and 6-phosphogluconate dehydrogenase (6PGDH) (Krepinsky et al., 2001). One enzyme in the chloroplast, enolase (ENO) (Hannaert et al., 2000) is more similar to archaebacterial homologues than to eubacterial homologues. In those cases where the chloroplast enzyme does not reflect a cyanobacterial origin, the nuclear gene for the enzyme arose through duplication of a preexisting nuclear gene in the higher plant lineage. But as mentioned above, in all cases except enolase those pre-existing nuclear genes were themselves acquisitions from eubacteria. In the cytosol, almost without exception, all of the enzymes of central carbon metabolism in higher plants are acquisitions from eubacteria. The exception is again enolase, which reflects an archaebacterial origin (Hannaert et al., 2000). Another is phosphoglycerate kinase, where a cyanobacterial enzyme has taken up residence in the cytosol. Very importantly, if one were to prepare Figure 27.2 for yeast or humans, the cytosolic portion of the figure would be exactly the same, except that PPi-dependent PFK would be missing, both PGK and 6GPDH would be blue (instead of green), the oxidative pentose phosphate cycle would be in the cytosol, and transketolase (TKL) would be blue in fungi and red in animals. Yet even more importantly, for those glycolytic enzymes that have been studied from amitochondriate protists (Markos et al., 1993; Henze et al., 1995, 1998; Keeling and Doolittle, 1997) a similar picture emerges, namely that amitochondriate protists also possess eubacterial enzymes of the glycolytic pathway (Martin and Müller, 1998). Specific examples include glucokinase from Trichomonas vaginalis (Wu et al., 2001), PPi-PFK from Trichomonas vaginalis and Giardia lamblia (Mertens et al., 1998), aldolase from Giardia lamblia (Henze et al., 1998) and from Trichomonas vaginalis (Henze and Müller, unpublished), TPI from Giardia lamblia (Keeling and Doolittle, 1997; Martin, 1998), GAPDH from Trichomonas vaginalis (Markos et al., 1993), Entamoeba histolytica and Giardia lamblia (Markos et al., 1993; Henze et al., 1995), and PGluM from Entamoeba histolytica (Figure 27.1B).
The evolution of compartmentalized sugar phosphate metabolism in higher plants Figure 27.2 (see color plates) summarizes the observations from a large number of individual gene phylogenies published over the past years concerning the enzymes of compartmentalized sugar phosphate metabolism in higher plants. Importantly, all of the enzymes represented in the figure, with the exception of the large subunit of Rubisco, are encoded in the nucleus. Color coding indicates whether the nuclear encoded enzyme is (1) more similar to cyanobacterial homologues (green), (2) more similar to eubacterial (but not specifically cyanobacterial) homologues (blue), or (3) more similar to archaebacterial homologues (red). For the enzymes shaded in gray, no statement is currently possible. The results summarized in the figure have been discussed in further detail elsewhere (Martin and Schnarrenberger, 1997; Henze et al., 1998; Martin and Herrmann, 1998; Nowitzki et al., 1998; Wu et al., 2001) In general, what we observe is the following. In the chloroplast, many of the enzymes of carbohydrate metabolism are indeed encoded by nuclear genes that were acquired from the cyanobacterial antecedent of plastids, as the product specificity corollary predicts. Examples are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Martin et al., 1993), phosphoglycerate kinase (PGK) (Brinkmann and Martin, 1996; Martin and Schnarrenberger, 1997), transketolase (TKL) (Martin, 1998) and glucose-6-phosphate isomerase (GPI) (Nowitzki et al., 1998). But there are many exceptions to this rule. Notably, many of the enzymes localized in the plastid compartment are encoded by genes that were acquired from eubacteria, but – on the basis of available data – not from cyanobacteria. Examples are fructose-1,6-bisphosphatase (FBP) (Martin et al., 1996), triosephosphate isomerase (TPI) (Henze et al., 1994; Schmidt et al., 1995; Keeling
374
ENDOSYMBIOTIC GENE TRANSFER
The endosymbiont hypothesis in its various formulations and the archezoa hypothesis is at a loss to account for the observation that eukaryotes have a eubacterial glycolytic pathway in their cytosol. By contrast, the hydrogen hypothesis specifically predicts eukaryotes to have a eubacterial glycolytic pathway because the mech anism that it posits to have associated the mitochondrial (hydrogenosomal) symbiont with its H2-dependent host entails the acquisition by the autotrophic host of the symbiont’s heterotrophic lifestyle (glycolysis in particular) through endosymbiotic gene transfer, a special case of horizontal gene transfer (Martin and Müller, 1998). It is noteworthy that the only enzyme of the glycolytic pathway in eukaryotes that seems to be a direct inheritance from the archaebacterial host lineage, enolase, is more similar to homologues from methanogens than to homologues from other eukaryotes (Hannaert et al., 2000), in line with the prediction of the hydrogen hypothesis that the host lineage should ultimately reflect a methanogenic ancestry (Martin and Müller, 1998). This view is also supported by that finding that methanogens are the only prokaryotes that possess true histones (Sandman and Reeve, 1998). Also in line with the hydrogen hypothesis is the finding that some protists have even preserved glycolytic enzymes in their mitochondria (Liaud et al., 2000). Furthermore in line with the predictions of the hydrogen hypothesis is the finding that a highly characteristic enzyme of hydrogenosomes, pyruvate:ferredoxin oxidoreductase (Müller, 1993), is found in the mitochondria of Euglena gracilis and that the mitochondrial and hydrogenosomal enzymes share a common eubacterial origin (Rotte et al., 2001).
Horizontal gene transfer between freeliving eubacteria adds an additional level of complexity Since the first edition of this book was published, it has become apparent that horizontal gene transfer between free-living eubacteria is a very widespread process, at least in evolutionarily recent times (Lawrence and Ochman, 1998; Doolittle, 1999; Eisen 2000; Ochman et al., 2000). Since horizontal gene transfer is well known
357
to occur at appreciable rates among free-living prokaryotes today, we should assume it also to have occurred in the distant past. This simple logic is essential to keep in mind when considering the origin of eukaryotic genes that were acquired from eubacterial symbionts through endosymbiotic gene transfer (Martin and Schnarrenberger, 1997; Martin, 1999a). The reason is because very many of the eukaryotic genes studied to date that clearly come from eubacteria do not branch specifically with their homologues from cyanobacteria and α-proteobacteria, the antecedents of chloroplasts and mitochondria. The interpretation of such findings requires a bit of thought. For example, in a recent survey of several thousand Arabidopsis genes, all of 20 different sequenced prokaryotic genomes sampled would have appeared to have donated genes to the Arabidopsis lineage, if the gene phylogenies are interpreted at face value (Rujan and Martin, 2001). In other words, for all 20 prokaryotic genomes in that study (16 eubacteria and four archaebacteria), at least one gene tree was observed where the Arabidopsis nuclear gene branched with a homologue from the given genome. At face value, that would suggest that all 20 lineages of prokaryotes donated genes to Arabidopsis. Had 40 different prokaryotic genomes been studied, then 40 different donors to the Arabidopsis lineage would have been implied, if the trees are taken at face value. But if we think things through in full and consider the process of horizontal gene transfer between free-living prokaryotes as a continuum back through time, it becomes immediately evident that it is unrealistic to expect all plant nuclear genes that were donated by the antecedent of plastids to branch with homologues from contemporary cyanobacteria, and it is unrealistic to expect all genes that were donated to the eukaryotic lineage by the antecedent of mitochondria to branch with α-proteobacterial homologues. This is shown in Figure 27.3 (see color plates), which was modified from Martin (1999a). All available evidence indicated a single origin of plastids (Martin et al., 1998; Moreira et al., 2000) and mitochondria (Gray et al., 1999). When the cyanobacterium that gave rise to plastids about 1.5 billion years ago (Doolittle, 1997) entered into
375
358
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
its symbiosis, it contained exactly one genome’s worth of genes (plus a plasmid or two, probably). Its free-living relative among the cyanobacteria that existed then contained exactly the same set of genes. But the cyanobacterium that became the plastid was cut off from gene exchange with other free-living prokaryotes, because it was genetically isolated within the eukaryotic cell, a condition that persisted for another 1.5 billion years to the present. By contrast, its free-living relatives were free to exchange their genes with other cyanobacteria and with other non-cyanobacterial prokaryotes for another 1.5 billion years. Exactly the same reasoning applies to the α-proteobacterium that gave rise to mitochondria, except that the freeliving cousins of the mitochondrial (hydrogenosomal) symbiont had even more time, about 2 billion years (Doolittle, 1997), to exchange genes with other prokaryotes, whereas the mitochondrial symbiont did not. Given that, let us consider the blazing speed (in terms of geological time) with which E. coli, as a well-studied example, has exchanged its genes with other eubacteria (Lawrence and Ochman, 1998). Lawrence and Ochman (1998) estimated that E. coli has acquired foreign DNA at a rate of about 16 kb per million years over the last 100 million years, and by inference, that about the same amount of DNA has been lost from its genome in return. The values for other prokaryotes may be similar (Doolittle, 1999; Eisen, 2000; Ochman et al., 2000). If that rate were to be projected back in time into the depths of history where mitochondria and plastids arose, then the current E. coli genome would not share any genes with the genome that existed in ancestors of E. coli that lived 1 billion years ago (Martin, 1999a). Thus, it is extremely unlikely that any freeliving prokaryote contains exactly the same set of genes as either the plastid symbiont and or the mitochondrial symbiont (Martin and Schnarrenberger, 1997; Martin 1999a; Rujan and Martin, 2001). For exactly the same reasons, it is extremely likely that many of the genes that are currently found in eukaryotic genomes and that are clearly acquisitions from eubacteria, probably come from the ancestors of mitochondria (hydrogenosomes) and the ancestors of
plastids, even though they do not appear to stem from α-proteobacteria and cyanobacteria respectively. Conversely, one can expect some (or many) genes that were donated to the nucleus from mitochondria and chloroplasts not to branch with α-proteobacterial or cyanobacterial homologues. Rather, they will branch with any number of eubacterial groups, depending upon where these ancestrally donated genes have ended up in contemporary eubacteria. This is the essence of what is shown in Figure 27.3 (Martin, 1999a; Rujan and Martin, 2001).
CONCLUSION Horizontal gene transfer is a very powerful process in evolution when it comes to shaping the gene content of genomes, both prokaryotic and eukaryotic. Plastids and mitochondria donated many genes to eukaryotic genomes. Some of the products of those genes are reimported as proteins into the organelle, many others are not – rather they are imported into other organelles or localized in the cytosol or the nucleus, which is not an organelle. Contrary to what was believed 20 years ago (and is sometimes still believed today), the compartmentation of a protein is not a good indicator of its evolutionary origin. Molecular phylogenetics generally has a hard time coming to grips with the problem of horizontal gene transfer because it makes it more difficult to draw direct inferences about the evolutionary past from the phylogenies of contemporary genes. But the situation may not be so dire. If we do not allow ourselves to digress into the realm of descriptive hypothesis-free science, that is if we have good biological theories with which to interpret molecular phylogenies and which we can test with molecular phylogenies, then horizontal gene transfer will probably enrich, rather than hamper, our understanding of life’s history, in particular the origin of eukaryotic genes.
ACKNOWLEDGMENTS We thank Miklos Müller for numerous stimulating discussions and Marianne Limpert
376
ENDOSYMBIOTIC GENE TRANSFER
for help in preparing the manuscript. Generous financial support from the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
REFERENCES Adachi, J. and Hasegawa, M. (1996) Computer Science Monographs, No. 28. MOLPHY Version 2.3: Programs for Molecular Phylogenetics Based on Maximum Likelihood, Institute of Statistical Mathematics, Tokyo. Andersson, S.G.E. and Kurland, C.G. (1999) Origins of mitochondria and hydrogenosomes. Curr. Opin. Microbiol. 2: 535–541. Baldauf, S. and Palmer, J.D. (1990) Evolutionary transfer of the chloroplast tufA gene to the nucleus. Nature 344: 262–265. Brennicke, A., Grohmann, L., Hiesel, R. et al. (1993) The mitochondrial genome on its way to the nucleus: different stages of gene transfer in higher plants. FEBS Letters 325: 140–145. Brinkmann, H. and Martin, W. (1996) Higher plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement. Plant Mol. Biol. 30: 65–75. Brown, J.R. and Doolittle, W.F. (1995) Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications. Proc. Natl Acad. Sci. USA 92: 2441–2445. Brown, J.R. and Doolittle, W.F. (1997) Archaea and the prokaryote-to-eukaryote transition. Microbiol. Mol. Biol. Rev. 61: 456–502. Cavalier-Smith, T. (1987a) Eukaryotes with no mitochondria. Nature 326: 332–333. Cavalier-Smith, T. (1987b) The origin of eukaryote and archaebacterial cells. Ann. NY Acad. Sci. 503: 17–54. Clark, C.G. and Roger, A.J. (1995) Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proc. Natl Acad. Sci. USA 92: 6518–6521. Cowan, D. (2000) Use your neighbor’s genes. Nature 407: 466–467. Doolittle, W.F. (1980) Revolutionary concepts in evolutionary biology. Trends Biochem. Sci. 5: 146–149. Doolittle, W.F. (1996) Some aspects of the biology of cells and their possible evolutionary significance. In Evolution of Microbial Life (eds, D. Roberts, P. Sharp, G. Alserson and M. Collins), pp. 1–21, 54th Symp. Soc. Gen. Microbiol., Cambridge University Press, Cambridge. Doolittle, W.F. (1997) Fun with genealogy. Proc. Natl Acad. Sci. USA 94: 12751–12753. Doolittle, W.F. (1998) A paradigm gets shifty. Nature 392: 15–16. Doolittle, W.F. (1999) Phylogenetic classification and the universal tree. Science 284: 2124–2128. Eisen, J. (2000) Horizontal gene transfer among microbial genomes: new insights from complete genome analysis. Curr. Opin. Genet. Dev. 10: 606–611.
359
Embley, T.M. and Hirt, R.P. (1998) Early branching eukaryotes? Curr. Opin. Genet. Dev. 8: 655–661. Embley, T.M. and Martin, W. (1998) A hydrogen-producing mitochondrion. Nature 396: 517–519. Felsenstein, J. (1993) Phylip (Phylogeny Inference Package) Manual, version 3.5c. Distributed by the author. University of Washington, Seattle, Department of Genetics. Germot, A., Philippe, H. and Le Guyader, H. (1997) Evidence for loss of mitochondria in microsporidia from a mitochondrial-type HSP70 in Nosema locustae. Mol. Biochem. Parasitol. 87: 159–168. Gray, M.W. and Doolittle, W.F. (1982) Has the endosymbiont hypothesis been proven? Microbiol. Rev. 46: 1–42. Gray, M.W., Burger, G. and Lang, B.F. (1999) Mitochondrial evolution. Science 283: 1476–1481. Gupta, R.S. and Golding, G.B. (1996) The origin of the eukaryotic cell. Trends Biochem. Sci. 21: 166–171. Hannaert, V., Brinkmann, H., Nowitzki, U. et al. (2000) Enolase from Trypanosoma brucei, from the amitochondriate protist Mastigamoeba balamuthi, and from the chloroplast and cytosol of Euglena gracilis: pieces in the evolutionary puzzle of the eukaryotic glycolytic pathway. Mol. Biol. Evol. 17: 989–1000. Hashimoto, T., Sanchez, L.B., Shirakura, T. et al. (1998) Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny. Proc. Natl Acad. Sci. USA 95: 6860–6865. Henze, K., Schnarrenberger, C., Kellermann, J. and Martin, W. (1994) Chloroplast and cytosolic triosephosphate isomerases from spinach: purification, microsequencing and cDNA cloning of the chloroplast enzyme. Plant Mol. Biol. 26: 1961–1973. Henze, K., Badr, A., Wettern, M. et al. (1995) A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution. Proc. Natl Acad. Sci. USA 92: 9122–9126. Henze, K., Morrison, H.G., Sogin, M.L. and Müller, M. (1998) Sequence and phylogenetic position of a class II aldolase gene in the amitochondriate protist, Giardia lamblia. Gene 222: 163–168. Henze, K., Suguri, S., Moore, D.V. et al. (2001) Cyanobacterial relationships of the glycolytic enzymes of Giardia intestinalis, glucokinase and glucose-6phosphate isomerase. Gene, in press. Horiike, T., Hamada, K., Kanaya, S. and Shinozawa, T. (2001) Origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria revealed is revealed by homology hit analysis. Nature Cell Biol. 3: 210–214. John, P. and Whatley, F.R. (1975) Paracoccus denitrificans and the evolutionary origin of the mitochondrion. Nature 254: 495–498. Keeling, P.J. and Doolittle, W.F. (1997) Evidence that eukaryotic triosephosphate isomerase is of alphaproteobacterial origin. Proc. Natl Acad. Sci. USA 94: 1270–1275. Keeling, P.J. and Palmer, J.D. (2000) Parabasalian flagellates are ancient eukaryotes. Nature 405: 635–637.
377
360
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
somes: A case study of functional redundancy in ancient pathways through endosymbiosis. Curr. Genet. 32: 1–18. Martin, W., Brinkmann, H., Savona, C. and Cerff, R. (1993) Evidence for a chimaeric nature of nuclear genomes: eubacterial origin of eukaryotic glyceraldehyde-3phosphate dehydrogenase genes. Proc. Natl Acad. Sci. USA 90: 8692–8696. Martin, W., Mustafa, A.-Z., Henze, K. and Schnarrenberger, C. (1996) Higher plant chloroplast and cytosolic fructose1,6-bisphophosphatase isoenzymes: origins via duplication rather than prokaryote-eukaryote divergence. Plant Mol. Biol. 32: 485–491. Martin, W., Stoebe, B., Goremykin, V. et al. (1998) Gene transfer to the nucleus and the evolution of chloroplasts. Nature 393: 162–165. Mereschkowsky, C. (1905) Über Natur und Ursprung der Chromatophoren im Pflanzenreiche. Biol. Centralbl. 25: 593–604 (English translation in Martin, W. and Kowallik, K.V. (1999) Eur. J. Phycol. 34: 287–295). Mertens, E., Ladror, U.S., Lee, J.A. et al. (1998) The pyrophosphate-dependent phosphofructokinase of the protist, Trichomonas vaginalis, and the evolutionary relationships of protist phosphofructokinases. J. Mol. Evol. 47: 739–750. Moreira, D., Le Guyader, H. and Philippe, H. (2000) The origin of red algae and the evolution of chloroplasts. Nature 405: 69–72. Müller, M. (1988) Energy metabolism of protozoa without mitochondria. Annu. Rev. Microbiol. 42: 465–488. Müller, M. (1993) The hydrogenosome. J. Gen. Microbiol. 139: 2879–2889. Müller, M. (2000) A mitochondrion in Entamoeba histolytica? Parasitol. Today 16: 368–369. Nowitzki, U., Flechner, A., Kellermann, J. et al. (1998) Eubacterial origin of eukaryotic nuclear genes for chloroplast and cytosolic glucose-6-phosphate isomerase: sampling eubacterial gene diversity in eukaryotic chromosomes through symbiosis. Gene 214: 205–213. Ochman, H., Lawrence, J.G. and Groisman, E.S. (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405: 299–304. Ortner, S., Binder, M., Schiener, O. et al. (1997) Molecular and biochemical characterization of phosphoglucomutases from Entamoeba histolytica and Entamoeba dispar. Mol. Biochem. Parasitol. 90: 121–129. Ouzounis, C. and Sander, C. (1992) TFIIB, an evolutionary link between the transcription machineries of archaebacteria and eukaryotes. Cell 71: 189–190. Philippe, H., Germot, A. and Moreira, D. (2000) The new phylogeny of eukaryotes. Curr. Opin. Gen. Dev. 10: 596–601. Plaxton, W.C. (1996) The organization and regulation of plant glycolysis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 185–214. Pühler, G., Leffers, H., Gropp, F. et al. (1989) Archaebacterial DNA dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome. Proc. Natl Acad. Sci. USA 86: 4569–4573. Ribiero, S. and Golding, G.B. (1998) The mosaic nature of the eukaryotic nucleus. Mol. Biol. Evol. 15: 779–788.
Krepinsky, K., Plaumann, M., Martin, W. and Schnarrenberger, C. (2001) Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach: cyanobacterial genes for chloroplast and cytosolis isoenzymes encoded in eukaryotic chromosomes. Eur. J. Biochem. 268: 2678–2686. Lake, J.A. and Rivera, M.C. (1994) Was the nucleus the first endosymbiont? Proc. Natl Acad. Sci. USA 91: 2880–2881. Langer, D., Hain, J., Thuriaux, P. and Zillig, W. (1995) Transcription in Archaea: similarity to that in Eukarya. Proc. Natl Acad. Sci. USA 92: 5768–5772. Lawrence, J.G. and Ochman, H. (1998) Molecular archaeology of the Escherichia coli genome. Proc. Natl Acad. Sci. USA 95: 9413–9417. Liaud, M.-F., Lichtlé, C., Apt, K. et al. (2000) Compartmentspecific isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic pathway. Mol. Biol. Evol. 17: 213–223. Lopez-Garcia, P. and Moreira, D (1999) Metabolic symbiosis at the origin of eukaryotes. Trends Biochem. Sci. 24: 88–93. Margulis, L. (1970) Origin of Eukaryotic Cells, Yale University Press, New Haven, CT. Margulis, L. (1996) Archaeal–eubacterial mergers in the origin of Eukarya: phylogenetic classification of life. Proc. Natl Acad. Sci. USA 93:1071–1076. Margulis, L., Dolan, M.F. and Guerrero, R. (2000) The chimeric eukaryote: origin of the nucleus from the karyomastigont in amitochondriate protists. Proc. Natl Acad. Sci. USA 97: 6954–6959. Markos, A., Miretsky, A. and Müller, M. (1993) A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote Trichomonas vaginalis. J. Mol. Evol. 37: 631–643. Martin, W. (1998) Endosymbiosis and the origins of chloroplast-cytosol isoenzymes: a revision of the gene transfer corollary. In Horizontal Gene Transfer: Implications and Consequences (eds, M. Syvanen and C. Kado), pp. 363–379, Chapman and Hall, London. Martin, W. (1999a) Mosaic bacterial chromosomes – a challenge en route to a tree of genomes. BioEssays 21: 99–104. Martin, W. (1999b) A briefly argued case that mitochondria and plastids are descendants of endosymbionts, but that the nuclear compartment is not. Proc. R. Soc. Lond. B 266: 1387–1395. Martin, W. (2000) Primitive anaerobic protozoa: the wrong host for mitochondria and hydrogenosomes? Microbiology 146: 1021–1022. Martin, W. and Cerff, R. (1986) Prokaryotic features of a nucleus encoded enzyme: cDNA sequences for chloroplast and cytosolyic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba). Eur. J. Biochem. 159: 323–331. Martin, W. and Herrmann, R.G. (1998) Gene transfer from organelles to the nucleus: How much, what happens and why? Plant Physiol. 118: 9–17. Martin, W. and Müller, M. (1998) The hydrogen hypothesis for the first eukaryote. Nature 392: 37–41. Martin, W. and Schnarrenberger, C. (1997) The evolution of the Calvin cycle from prokaryotic to eukaryotic chromo-
378
ENDOSYMBIOTIC GENE TRANSFER
Rivera, M.C., Jain, R., Moore, J.E. and Lake, J.A. (1998) Genomic evidence for two functionally distinct gene classes. Proc. Natl Acad. Sci. USA 95: 6239–6244. Roger, A.J. (1999) Reconstructing early events in eukaryotic evolution. Am. Nat. 154: 146–163. Rotte, C. and Martin, W. (2001) Endosymbiosis does not explain the origin of the nucleus. Nature Cell. Biol. 8: E173. Rotte, C., Henze, K., Müller, M. and Martin, W. (2000) Origins of hydrogenosomes and mitochondria. Curr. Opin. Microbiol. 3: 481–486. Rotte, C., Stejskal, F., Zhu, G. et al. (2001) Pyruvate:NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a fusion of pyruvate:ferredoxin oxidoreductase and NADPH-cytochrome P450 reductase. Mol. Biol. Evol. 18: 710–720. Rowlands, T., Baumann, P. and Jackson, S.P. (1994) The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. Science 264: 1326–1329. Rujan, T. and Martin, W. (2001) How many genes in Arabidopsis come from cyanobacteria? An estimate from 386 protein phylogenies. Trends Genet., in press Sagan, L. (1967) On the origin of mitosing cells. J. Theoret. Biol. 14: 225–274. Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425. Sandman, K. and Reeve, J. (1998) Origin of the eukaryotic nucleus. Science 280: 501–503. Schmidt, M., Svendsen, I. and Feierabend, J. (1995) Analysis of the primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves by protein
361
and cDNA sequencing indicates a eukaryotic origin of its gene. Biophys. Acta 1261: 257–264. Schnarrenberger, C., Flechner, A. and Martin, W. (1995) Enzymatic evidence for a complete oxidative pentose phosphate pathway in chloroplasts and an incomplete pathway in the cytosol of spinach leaves. Plant Physiol. 108: 609–614. Tovar, J., Fischer, A. and Clark, C.G. (1999) The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica. Mol. Microbiol. 32: 1013–1021. Van de Peer, Y., Rensing, S., Maier, U.G. and De Wachter, R. (1996) Substitution rate calibration of small subunit RNA identifies chlorarachniophyte endosymbionts as remnants of green algae. Proc. Natl Acad. Sci. USA 93: 7744–7748. Weeden, N.F. (1981) Genetic and biochemical implications of the endosymbiotic origin of the chloroplast. J. Mol. Evol. 17: 133–139. Wendt, U.K., Hauschild, R., Lange, C. et al. (1999) Evidence for functional convergence of redox regulation in G6PDH isoforms of cyanobacteria and higher plants. Plant Mol. Biol. 40: 487–494. Wu, G., Henze, K. and Müller, M. (2001) Evolutionary relationships of the glucokinase from the amitochondriate protist, Trichomonas vaginalis. Gene 264: 265–271. Zauner, S., Fraunholz, M., Wastl, J. et al. (2000) Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph. Proc. Natl Acad. Sci. 97: 200–205. Zillig, W., Klenk, H.-P., Palm, P. et al. (1989) Did eukaryotes originate by a fusion event? Endocyt. Cell Res. 6: 1–25.
379
Color profile: Generic CMYK printer profile Composite Default screen
00
100
5
95
5
75
5
25
5
0
00
100
5
95
5
75
5
25
FIGURE 27.2 (See following page for caption.)
5
0
D:\WORK\Harcourt\Syvanen + Kado\Syvanen + Kado (Plates).vp 06 November 2001 15:42:07
Color profile: Generic CMYK printer profile Composite Default screen
00
5
5
5
00
5
5
5
100
FIGURE 27.2 (On previous page.) Summary of gene phylogenies for enzymes of compartmentalized sugar phosphate metabolism in plants. Suggested evolutionary origins for the nuclear genes are color-coded. Enzymes regulated through the thioredoxin system are indicated. Many enzymes in the figure are allosterically regulated, but no allosteric regulation is indicated here. Enyzme abbreviations are: FBA fructose-1,6-bisphosphate aldolase; FBP fructose-1,6-bisphosphatase; GAPDH glyceraldehyde-3-phosphate dehydrogenase; PGK 3-phosphoglycerate kinase; PRI ribose-5-phosphate isomerase; PRK phosphoribulokinase; Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase; RPE ribulose-5-phosphate 3-epimerase; SBP sedoheptulose-1,7-bisphosphatase; TKL transketolase; TPI triosephosphate isomerase; TAL transaldolase; GPI glucose-6-phosphate isomerase; G6PDH glucose-6-phosphate dehydrogenase; 6GPDH 6-phosphogluconate dehydrogenase; PGluM phosphoglucomutase; PGM phosphoglyceromutase; PFK phosphofructokinase (pyrophosphate and ATP-dependent); ENO enolase; PYK pyruvate kinase; PDC pyruvate dehydrogenase complex (E1, E2, E3 components), T translocator. PDC is a multienzyme complex, but only one set of components is drawn here. Note that chloroplast isoenzymes of PGM, ENO, and PYK have not been demonstrated in spinach leaves, but for convenience we have included those enzymes in this figure, since they have been well characterized in the plastids of other higher plants (Plaxton, 1996). For further details see Martin and Schnarrenberger (1997), Martin and Herrmann (1998), Nowitzki et al. (1998) and Krepinsky et al. (2001).
FIGURE 27.3 (Opposite.) A tree of genomes. Each prokaryotic genome is represented as a single colored line, different colors symbolize different groups of prokaryotes. A working hypothesis for the origin of eukaryotic genes as outlined primarily in Martin and Müller (1998) and taking lateral gene transfer into account (modified from Martin, 1999a). The figure extends symbiotic associations (merging of genomes into the same cellular confines is indicated by the merging of colored lines) to include schematic indication of several independent secondary symbioses for the acquisition of plastids during eukaryotic history (Martin et al., 1998; Zauner et al., 2000). Importantly, among eukaryotes, colored lines indicate merely that prokaryotic genomes existed at one time within the cellular confines of a given eukaryotic lineage, not that they have persisted to the present as an independently compartmented genome (because most genes from organelles are transferred to the nucleus). For example some eukaryotes with secondary symbionts are schematically indicated with six lines, but only have four distinctly compartmented genomes. Similarly, eukaryotes that lack mitochondria apparently possessed such organelles in the past but only have one genome – that in the nucleus (for example Giardia, Trichomonas, and Entamoeba, see text). Furthermore it is important to note that mitochondrion-lacking eukaryotes have arisen through loss of the organelle in many independent lineages (Embley and Martin, 1998; Hashimoto et al., 1998; Roger, 1999). In the enlargement, lateral gene transfer between eubacteria prior to – and implicitly, but not shown, subsequent to – the origin of mitochondria (blue lines) and plastids (green lines) is schematically represented. Genomes are represented as heavy lines, individual gene transfer events (regardless of possible numbers of genes involved) as thin lines. The phylogeny of eukaryotes indicated is mostly schematic, containing various elements from van der Peer et al. (1996), Keeling and Palmer (2000), Martin et al. (1998) and other sources, and is not intended to be close to correct. The various schematic plastid organizational types are shown merely to indicate that dramatic differences in organelle structure can arise from a single ancestral symbiotic state.
95
75
25
5
0
100
95
75
25
5
0
D:\WORK\Harcourt\Syvanen + Kado\Syvanen + Kado (Plates).vp 06 November 2001 15:42:07
Color profile: Generic CMYK printer profile Composite Default screen
00
100
5
95
5
75
Euglena
Diatoms Rhodophytes
Chlorophytes
5
25
5
0
Cyanophora Heterotrophic Eukaryotes with Hydrogenosomes, or Mitochondria, or no ATP-Producing Organelles at all
Pro
teo
bac
ter
Pro
Eukaryotes
ia
teo
bac
ter
CO2
ia
Substrate
Eubacteria
Glucose H2O
Archaebacteria
Pyruvate
ATP ATP
ATP H2O CO2
Anaerobic Respiration
Substrate
ATP H2O CO2
Secondary Symbiosis
H2 H2 Glucose H2 CO2 ATP ATP Acetate ATP ATP H H2 2 CH4 Facultative Anaerobic H2-dependent Autotrophic Host Symbiont Glucose
ATP
Glucose
H2O
H2 Anaerobic CO2 Fermentation Acetate
Substrate
Glucose Pyruvate
CO2
Glucose ATP Pyruvate
ATP
ATP
O2
Substrate
O2 Respiration
O2
Origins of Mitochondria Origins of Plastids
00
100
Horizontal Gene Transfer between free-living Eubacteria
5
5
5
95
75
25
5
0
D:\WORK\Harcourt\Syvanen + Kado\Syvanen + Kado (Plates).vp 06 November 2001 15:42:08
H
A
P
T
E
R
27
Endosymbiotic Gene Transfer: A Special Case of Horizontal Gene Transfer Germane to Endosymbiosis, the Origins of Organelles and the Origins of Eukaryotes Katrin Henze, Claus Schnarrenberger and William Martin
Endosymbiotic gene transfer describes the process through which chloroplasts and mitochondria relinquished the majority of their genes to the nucleus while not having surrendered the majority of proteins integral to the eubacterial nature of their metabolism. It is a special case of lateral gene transfer that was very important for the establishment of biochemical compartmentation during the evolution of eukaryotic cells. Many examples of endosymbiotic gene transfer in the history of higher plant genes for chloroplast-cytosolic isoenzymes of the Calvin cycle and glycolysis have been considered in the past few years. The data indicate that nuclear genes for almost all glycolytic enzymes of the eukaryotic cytosol were acquired from eubacteria early in eukaryotic evolution, probably in the course of the endosymbiotic event that gave rise to the origin of mitochondria. The genes for enzymes that are possessed both by the symbiont and host during a given endosymbiosis are redundant. In such cases, it is observed that the intruding gene contributed by the symbiont had a very high likelihood of successful gene transfer. But the protein products of genes that were transferred from symbiont to host chromosomes have been surprisingly often rerouted to compartments other than that from Horizontal Gene Transfer ISBN: 0-12-680126-6
which the genes were donated. Because of this, the localization of a protein within the eukaryotic cell is not per se an indicator of the evolutionary origin of its gene. The evolutionary history of the enzymes of central carbon metabolism in eukaryotes and the role of lateral gene transfer in that process is summarized. These results are considered in light of the ability of competing alternative theories for the origins of eukaryotes to account for the observations.
INTRODUCTION Genes encoded in organellar genomes attest beyond all reasonable doubt to the eubacterial ancestry of chloroplasts (Martin et al., 1998) and mitochondria (Gray et al., 1999). But how does endosymbiotic theory account for the origin of organellar proteins that are not encoded in organellar DNA? Their fate is commonly explained with the help of a scenario elegantly argued by Weeden (1981) that is known as the “product specificity corollary” (also known as the “gene transfer corollary”) to endosymbiotic theory. It posits that during the course of eukaryotic history, the majority of genes for proteins integral to organellar metabolism were
351
369
Copyright © 2002 by Academic Press. All rights of reproduction in any form reserved.
352
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
be expected for a given eukaryotic nuclear gene, one has to have a biological model for the origins of eukaryotes that generates such predictions. What was the host? Biologists have a number of different suggestions, but no generally accepted answer to that question, but there are a few important clues that can serve as a guideline. Discovery of the archaebacterial nature of components of the nuclear genetic apparatus (Pühler et al., 1989; Ouzounis and Sander, 1992; Rowlands et al., 1994; Langer et al., 1995) and the results of extensive molecular phylogenetic work have slowly unveiled the host that acquired the mitochondrion as a descendant (in part or in whole) of an archaebacterium (Zillig et al., 1989; Ouzounis and Sander, 1992; Brown and Doolittle, 1995, 1997; Langer et al., 1995; Doolittle, 1996; Ribiero and Golding, 1998; Rivera et al., 1998). Under the simplest set of premises possible, this means that the host cell that acquired the mitochondrion was an archaebacterium (Doolittle, 1996; Martin and Müller, 1998). However, many other more complicated models involving more than two symbiotic partners to give rise to a mitochondrionbearing eukaryote have been elaborated. It is worthwhile briefly to recapitulate a few of these models to see what sorts of predictions they generate, in particular about the evolution of molecules that occur in the cytosol of contemporary eukaryotes. Probably the most popular model up until a few years ago is what is best described as the achezoa model (Cavalier-Smith 1987a,b), also sometimes called the “classical hypothesis” (Doolittle, 1980, 1998). Under this view, a primitively amitochondriate, nucleus-bearing cell (an archezoon) is suggested to have arisen from an archaebacterial ancestor in a process that involved the origin of typical eukaryotic features (cytoskeleton, endocytosis, nucleus and mitosis). A member of that ancient group acquired the mitochondrion through endosymbiosis, while others (the suspectedly most primitive eukaryotes) remained amitochondriate up to the present day. This model has not withstood the scrutiny of molecular phylogeneticists, who have found that all amitochondriate eukaryotes studied to date possessed a mitochondrion in
transferred to the nucleus where they became integrated into the regulatory hierarchy of the nucleus and acquired a transit peptide, so that the functional gene products could be reimported into the organelle of their genetic origin on a daily basis since. This is a reasonable and logical scenario that satisfyingly explains why organelles have retained so much of their biochemically eubacterial heritage while having relinquished to the nucleus the majority of genes necessary to have done so. Though ultimately incorrect in the majority of specific cases in which it has been tested (Martin and Schnarrenberger, 1997; Martin, 1998), primarily due to the unfulfilled predictions that it generates about the origin of cytosolic enzymes, the gene transfer corollary does manage adequately to account for several organelle-to-nucleus gene transfer events (Martin and Cerff, 1986; Baldauf and Palmer, 1990; Brennicke et al., 1993; Martin et al., 1993). However, the simple phrase “organelle-tonucleus gene transfer” raises a different, much more difficult and in our view much more pressing question, namely: what is the origin of the nuclear genome under endosymbiotic theory? When Weeden (1981) formulated the gene transfer corollary, it was not known whether chloroplasts and mitochondria were descendants of endosymbionts or not, and his considerations generated testable predictions that could help to muster evidence in favor (or in disfavor) of that view (Gray and Doolittle, 1982). A salient element of that reasoning was that nuclear-encoded organellar enzymes should reflect the evolutionary history of the symbiont while cytosolic enzymes in eukaryotes should reflect the evolutionary history of the host (Weeden, 1981; Gray and Doolittle, 1982). Although we now know with certainty that plastids and mitochondria are descendants of free-living eubacteria, and although we are reasonably (but not completely) confident that the origin of mitochondria preceeded the origin of chloroplasts in evolution, it is perhaps surprising that endosymbiotic theory is just as much in the dark about the origin of the host that acquired mitochondria today as it was 20 years ago. In other words, in order to have a null hypothesis for the patterns of similarity to
370
ENDOSYMBIOTIC GENE TRANSFER
their evolutionary past, but lost the organelle subsequently (Clark and Roger, 1995; Germot et al., 1997; Embley and Hirt, 1998; Martin and Müller, 1998; Gray et al., 1999; Roger, 1999; Tovar et al., 1999; Müller, 2000; Philippe et al., 2000), the evidence for which is founded in nuclear genes in these amitochondriate organisms which reflect a mitochondrial ancestry (that is, they were transferred from the organelle to the nucleus via endosymbiotic gene transfer). Furthermore, the achezoa model clearly predicts all eukaryotes in general, and amitochondriate eukaryotes in particular, to possess archaebacterial enzymes of cytosolic metabolism, because under this model, amitochondriate protists are, on the bottom line, to be interpreted as direct descendants of archaebacteria (Cavalier-Smith, 1987b). A version of the achezoa model has been published under the name of the “ox-tox” hypothesis (Andersson and Kurland, 1999), but it is not fundamentally different from the achezoa model, rather it is very similar to 1975 formulations of the endosymbiont hypothesis (John and Whatley, 1975). For a further discussion of this matter, see Rotte et al. (2000) and Martin (2000). A second model is that of Lynn Margulis, which entails a symbiosis between a spirochete and a Thermoplasma-like archaebacterium (Sagan, 1967; Margulis, 1970, 1996) to give rise to a primitive, flagellated, mitochondrion-lacking (amitochondriate) eukaryote that subsequently acquired the mitochondrion through endosymbiosis. In the most recent formulation, that model suggests that the spirochete endosymbiont also gave rise to the nuclear compartment (Margulis et al., 2000). This model predicts that eukaryotic enzymes should, in general, be more similar to Thermoplasma enzymes than to homologues from other prokaryotes, but the sequence of the Thermoplasma genome did not bear out that prediction (Cowan, 2000). A third model that has been discussed at length in the literature is the idea that the nucleus was an endosymbiont. This model is exactly as old as the notion that chloroplasts descend from cyanobacteria, because the first paper thoroughly to argue the latter case (almost 100 years ago) also argued the former, albeit more briefly (Mereschkowsky, 1905). The
353
idea that the nuclear compartment is the remnant of an ancient endosymbiosis suffered extinction for many decades (Mereschkowsky, 1905), but it was resuscitated in 1994 (Lake and Rivera, 1994), was rejuvenated in 1996 (Gupta and Golding, 1996), came more or less fully back to life in 1998 (Lopez-Garcia and Moreira, 1999) and has even been claimed to receive direct support from data (Horiike et al., 2001). In these formulations, the nucleus is viewed as an archaebacterial symbiont that took up residence in a eubacterial host. In Margulis’s formulation, a spirochete symbiont (that became the flagellum and the nucleus) took up residence in an archaebacterial host. It should be noted that there are several severe cell biological problems (discussed in Martin, 1999b; Rotte and Martin, 2001) with this model that are usually overlooked by its advocates, most notably (1) that the nucleus is not bounded by a double membrane, like chloroplasts and mitochondria, but rather by a folded single membrane, (2) that the nuclear membrane disintegrates at mitosis (viable chloroplasts and mitochondria never lose their surrounding membranes), and (3) the way that the cytosol is separated from the nucleus has no similarity whatsoever to the way that free-living prokaryotes are separated from their environment. Perhaps most importantly, “nucleosymbiotic” models derive an archezoon (a nucleus-bearing but amitochondriate eukaryote), whereby – as mentioned above – available data indicate that all eukaryotes, including those that lack mitochondria, once possessed mitochondria in their evolutionary past, but lost them secondarily. Therefore, these models predict eukaryotes that never possessed mitochondria (achaezoa) to be found, descendants of the stem that acquired mitochondria. Furthermore, with regard to their statements about the cytosol, these models would ultimately predict eukaryotes to possess DNA both in the nucleus and in the cytosol (in addition to genomes in chloroplasts and mitochondria). The fourth, and arguably simplest, current model to derive a mitochondrion-bearing eukaryote is called the “hydrogen hypothesis” (Martin and Müller, 1998). It posits that the host cell of the mitochondrial symbiosis was an
371
354
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
archaebacterium, specifically an autotrophic and H2-dependent archaebacterium, with an energy metabolism possibly similar to that found in modern methanogens. It accounts for the common ancestry of mitochondria and hydrogenosomes – the H2-producing organelles of ATP-synthesis in anaerobic eukaryotes (Müller, 1988, 1993; Embley and Hirt, 1998). It posits that eukaryotes acquired their heterotrophic lifestyle, including the genes and enzymes necessary for that lifestyle, from the mitochondrial symbiont via endosymbiotic gene transfer from the heterotrophic symbiont’s (eubacterial) to the autotrophic host’s (archaebacterial) chromosomes (Martin and Müller, 1998). Under this model, the origins of mitochondria/hydrogenosomes and the origins of eukaryotes are identical and the nucleus is a subsequent eukaryotic invention, the origin of which probably relates mechanistically to the origin of eubacterial lipid synthesis in eukaryotes (Martin, 1999b). This model explicitly predicts eukaryotes to have a eubacterial glycolytic pathway in the cytosol as the result of specifically selected endosymbiotic gene transfer from symbiont to host prior to the evolution of a mitochondrial protein import machinery.
from a variety of sources were collected from GenBank and subjected to phylogenetic analysis (Figure 27.1). The tree for RPI, which is a relatively short enzyme of about 220 amino acids, does not provide very sharp resolution, but it does reveal that the enzymes from plants, animals, and fungi are clearly more similar to eubacterial homologues than they are to archaebacterial homologues (Figure 27.1A). In spinach, there is only one isoenzyme of RPI known; cell fractionation has shown it to be located in the plastid (Schnarrenberger et al., 1995). The subcellular localization of the two Arabidopsis enzymes inferred from the genome sequence is currently unknown. The plant enzyme does not branch specifically with the cyanobacterial homologue, nor does the enzyme from animals and fungi branch specifically with proteobacterial homologues, as one might expect if these enzymes were donated to eukaryotes from the ancestors of plastids and mitochondria, respectively. This can be due to a number of factors, including the simple phylogenetic resolution problems involved with a protein that is only 220 amino acids long or, alternatively, to horizontal gene transfer between free-living eubacteria subsequent to the origins of organelles (see below). It is noteworthy that the plant enzyme does not branch with its homologues from animals and yeast, suggesting that the eubacterial donor of the plant gene was a different bacterium from that which donated the gene to the heterotrophic eukaryotes sampled. The tree for PGluM (Figure 27.1B) reveals that the eukaryotic genes are derived from a single acquisition from a eubacterial donor, and that the higher plant chloroplast-cytosol isoenzymes are related by a gene duplication in the plant lineage, as has been observed for the majority of chloroplast-cytosol isoenzymes studied to date (Martin and Schnarrenberger, 1997), and in contrast to the predictions from the product specificity corollary (Weeden, 1981). Archaebacterial homologues of PGluM were not identified, but further searching revealed that eubacterial PGluM is related to phosphomannomutase (PMM) which does have homologues in archaebacterial genomes (Figure 27.1B). The plant PMM genes surveyed are descendants of
RESULTS AND DISCUSSION Evolution of two further enzymes of central sugar phosphate metabolism in higher plants In previous work, we have studied the evolution of several enzymes involved in sugar phosphate metabolism in the chloroplast and cytosol of higher plants (Henze et al., 1994; Martin and Schnarrenberger, 1997; Martin and Herrmann, 1998) and of the cytosolic homologues in several protists (Henze et al., 1995, 1998, 2001; Wu et al., 2001). Two enzymes whose gene origins have been unclear are ribose-5-phosphate isomerase (RPI), that catalyzes the freely reversible isomerization of ribose-5-phosphate and ribulose-5-phosphate, and phosphoglucomutase (PGluM), that catalyzes the freely reversible interconversion of glucose-1-phosphate and glucose-6-phosphate. Homologues for both genes
372
ENDOSYMBIOTIC GENE TRANSFER
355
FIGURE 27.1 Protein phylogenies for (A) ribose-5-phosphate isomerase and (B) phosphoglucomutase. The RPI tree was inferred using ProtML (Adachi and Hasegawa, 1996) with local rearrangements and the JTT-F matrix starting from the neighbor-joining topology. The PGluM tree was inferred with the neighbor-joining method (Saitou and Nei, 1987) from the Dayhoff distance matrix using Phylip (Felsenstein, 1993). Bootstrap proportions >80 are indicated at branches.
eubacterial PMM genes, but again, not specifically related to the cyanobacterial homologue (see below). Notably, Figure 27.1B shows that the PGluM sequence from the amitochondriate protist Entamoeba histolytica (Ortner et al., 1997) is also an acquisition from eubacteria. In this
respect it is important to recall that although Entamoeba histolytica does not have either mitochondria or hydrogenosomes, it was recently discovered to possess a small, double membranebounded organelle in the cytosol – termed the mitosome – that is most easily interpreted as an
373
356
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
intermediate stage in the evolutionary process of mitochonrion and hydrogenosome reduction (Tovar et al., 1999).
and Doolittle, 1997), glucose-6-phosphate dehydrogenase (G6PDH) (Wendt et al., 1999) and 6-phosphogluconate dehydrogenase (6PGDH) (Krepinsky et al., 2001). One enzyme in the chloroplast, enolase (ENO) (Hannaert et al., 2000) is more similar to archaebacterial homologues than to eubacterial homologues. In those cases where the chloroplast enzyme does not reflect a cyanobacterial origin, the nuclear gene for the enzyme arose through duplication of a preexisting nuclear gene in the higher plant lineage. But as mentioned above, in all cases except enolase those pre-existing nuclear genes were themselves acquisitions from eubacteria. In the cytosol, almost without exception, all of the enzymes of central carbon metabolism in higher plants are acquisitions from eubacteria. The exception is again enolase, which reflects an archaebacterial origin (Hannaert et al., 2000). Another is phosphoglycerate kinase, where a cyanobacterial enzyme has taken up residence in the cytosol. Very importantly, if one were to prepare Figure 27.2 for yeast or humans, the cytosolic portion of the figure would be exactly the same, except that PPi-dependent PFK would be missing, both PGK and 6GPDH would be blue (instead of green), the oxidative pentose phosphate cycle would be in the cytosol, and transketolase (TKL) would be blue in fungi and red in animals. Yet even more importantly, for those glycolytic enzymes that have been studied from amitochondriate protists (Markos et al., 1993; Henze et al., 1995, 1998; Keeling and Doolittle, 1997) a similar picture emerges, namely that amitochondriate protists also possess eubacterial enzymes of the glycolytic pathway (Martin and Müller, 1998). Specific examples include glucokinase from Trichomonas vaginalis (Wu et al., 2001), PPi-PFK from Trichomonas vaginalis and Giardia lamblia (Mertens et al., 1998), aldolase from Giardia lamblia (Henze et al., 1998) and from Trichomonas vaginalis (Henze and Müller, unpublished), TPI from Giardia lamblia (Keeling and Doolittle, 1997; Martin, 1998), GAPDH from Trichomonas vaginalis (Markos et al., 1993), Entamoeba histolytica and Giardia lamblia (Markos et al., 1993; Henze et al., 1995), and PGluM from Entamoeba histolytica (Figure 27.1B).
The evolution of compartmentalized sugar phosphate metabolism in higher plants Figure 27.2 (see color plates) summarizes the observations from a large number of individual gene phylogenies published over the past years concerning the enzymes of compartmentalized sugar phosphate metabolism in higher plants. Importantly, all of the enzymes represented in the figure, with the exception of the large subunit of Rubisco, are encoded in the nucleus. Color coding indicates whether the nuclear encoded enzyme is (1) more similar to cyanobacterial homologues (green), (2) more similar to eubacterial (but not specifically cyanobacterial) homologues (blue), or (3) more similar to archaebacterial homologues (red). For the enzymes shaded in gray, no statement is currently possible. The results summarized in the figure have been discussed in further detail elsewhere (Martin and Schnarrenberger, 1997; Henze et al., 1998; Martin and Herrmann, 1998; Nowitzki et al., 1998; Wu et al., 2001) In general, what we observe is the following. In the chloroplast, many of the enzymes of carbohydrate metabolism are indeed encoded by nuclear genes that were acquired from the cyanobacterial antecedent of plastids, as the product specificity corollary predicts. Examples are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Martin et al., 1993), phosphoglycerate kinase (PGK) (Brinkmann and Martin, 1996; Martin and Schnarrenberger, 1997), transketolase (TKL) (Martin, 1998) and glucose-6-phosphate isomerase (GPI) (Nowitzki et al., 1998). But there are many exceptions to this rule. Notably, many of the enzymes localized in the plastid compartment are encoded by genes that were acquired from eubacteria, but – on the basis of available data – not from cyanobacteria. Examples are fructose-1,6-bisphosphatase (FBP) (Martin et al., 1996), triosephosphate isomerase (TPI) (Henze et al., 1994; Schmidt et al., 1995; Keeling
374
ENDOSYMBIOTIC GENE TRANSFER
The endosymbiont hypothesis in its various formulations and the archezoa hypothesis is at a loss to account for the observation that eukaryotes have a eubacterial glycolytic pathway in their cytosol. By contrast, the hydrogen hypothesis specifically predicts eukaryotes to have a eubacterial glycolytic pathway because the mech anism that it posits to have associated the mitochondrial (hydrogenosomal) symbiont with its H2-dependent host entails the acquisition by the autotrophic host of the symbiont’s heterotrophic lifestyle (glycolysis in particular) through endosymbiotic gene transfer, a special case of horizontal gene transfer (Martin and Müller, 1998). It is noteworthy that the only enzyme of the glycolytic pathway in eukaryotes that seems to be a direct inheritance from the archaebacterial host lineage, enolase, is more similar to homologues from methanogens than to homologues from other eukaryotes (Hannaert et al., 2000), in line with the prediction of the hydrogen hypothesis that the host lineage should ultimately reflect a methanogenic ancestry (Martin and Müller, 1998). This view is also supported by that finding that methanogens are the only prokaryotes that possess true histones (Sandman and Reeve, 1998). Also in line with the hydrogen hypothesis is the finding that some protists have even preserved glycolytic enzymes in their mitochondria (Liaud et al., 2000). Furthermore in line with the predictions of the hydrogen hypothesis is the finding that a highly characteristic enzyme of hydrogenosomes, pyruvate:ferredoxin oxidoreductase (Müller, 1993), is found in the mitochondria of Euglena gracilis and that the mitochondrial and hydrogenosomal enzymes share a common eubacterial origin (Rotte et al., 2001).
Horizontal gene transfer between freeliving eubacteria adds an additional level of complexity Since the first edition of this book was published, it has become apparent that horizontal gene transfer between free-living eubacteria is a very widespread process, at least in evolutionarily recent times (Lawrence and Ochman, 1998; Doolittle, 1999; Eisen 2000; Ochman et al., 2000). Since horizontal gene transfer is well known
357
to occur at appreciable rates among free-living prokaryotes today, we should assume it also to have occurred in the distant past. This simple logic is essential to keep in mind when considering the origin of eukaryotic genes that were acquired from eubacterial symbionts through endosymbiotic gene transfer (Martin and Schnarrenberger, 1997; Martin, 1999a). The reason is because very many of the eukaryotic genes studied to date that clearly come from eubacteria do not branch specifically with their homologues from cyanobacteria and α-proteobacteria, the antecedents of chloroplasts and mitochondria. The interpretation of such findings requires a bit of thought. For example, in a recent survey of several thousand Arabidopsis genes, all of 20 different sequenced prokaryotic genomes sampled would have appeared to have donated genes to the Arabidopsis lineage, if the gene phylogenies are interpreted at face value (Rujan and Martin, 2001). In other words, for all 20 prokaryotic genomes in that study (16 eubacteria and four archaebacteria), at least one gene tree was observed where the Arabidopsis nuclear gene branched with a homologue from the given genome. At face value, that would suggest that all 20 lineages of prokaryotes donated genes to Arabidopsis. Had 40 different prokaryotic genomes been studied, then 40 different donors to the Arabidopsis lineage would have been implied, if the trees are taken at face value. But if we think things through in full and consider the process of horizontal gene transfer between free-living prokaryotes as a continuum back through time, it becomes immediately evident that it is unrealistic to expect all plant nuclear genes that were donated by the antecedent of plastids to branch with homologues from contemporary cyanobacteria, and it is unrealistic to expect all genes that were donated to the eukaryotic lineage by the antecedent of mitochondria to branch with α-proteobacterial homologues. This is shown in Figure 27.3 (see color plates), which was modified from Martin (1999a). All available evidence indicated a single origin of plastids (Martin et al., 1998; Moreira et al., 2000) and mitochondria (Gray et al., 1999). When the cyanobacterium that gave rise to plastids about 1.5 billion years ago (Doolittle, 1997) entered into
375
358
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
its symbiosis, it contained exactly one genome’s worth of genes (plus a plasmid or two, probably). Its free-living relative among the cyanobacteria that existed then contained exactly the same set of genes. But the cyanobacterium that became the plastid was cut off from gene exchange with other free-living prokaryotes, because it was genetically isolated within the eukaryotic cell, a condition that persisted for another 1.5 billion years to the present. By contrast, its free-living relatives were free to exchange their genes with other cyanobacteria and with other non-cyanobacterial prokaryotes for another 1.5 billion years. Exactly the same reasoning applies to the α-proteobacterium that gave rise to mitochondria, except that the freeliving cousins of the mitochondrial (hydrogenosomal) symbiont had even more time, about 2 billion years (Doolittle, 1997), to exchange genes with other prokaryotes, whereas the mitochondrial symbiont did not. Given that, let us consider the blazing speed (in terms of geological time) with which E. coli, as a well-studied example, has exchanged its genes with other eubacteria (Lawrence and Ochman, 1998). Lawrence and Ochman (1998) estimated that E. coli has acquired foreign DNA at a rate of about 16 kb per million years over the last 100 million years, and by inference, that about the same amount of DNA has been lost from its genome in return. The values for other prokaryotes may be similar (Doolittle, 1999; Eisen, 2000; Ochman et al., 2000). If that rate were to be projected back in time into the depths of history where mitochondria and plastids arose, then the current E. coli genome would not share any genes with the genome that existed in ancestors of E. coli that lived 1 billion years ago (Martin, 1999a). Thus, it is extremely unlikely that any freeliving prokaryote contains exactly the same set of genes as either the plastid symbiont and or the mitochondrial symbiont (Martin and Schnarrenberger, 1997; Martin 1999a; Rujan and Martin, 2001). For exactly the same reasons, it is extremely likely that many of the genes that are currently found in eukaryotic genomes and that are clearly acquisitions from eubacteria, probably come from the ancestors of mitochondria (hydrogenosomes) and the ancestors of
plastids, even though they do not appear to stem from α-proteobacteria and cyanobacteria respectively. Conversely, one can expect some (or many) genes that were donated to the nucleus from mitochondria and chloroplasts not to branch with α-proteobacterial or cyanobacterial homologues. Rather, they will branch with any number of eubacterial groups, depending upon where these ancestrally donated genes have ended up in contemporary eubacteria. This is the essence of what is shown in Figure 27.3 (Martin, 1999a; Rujan and Martin, 2001).
CONCLUSION Horizontal gene transfer is a very powerful process in evolution when it comes to shaping the gene content of genomes, both prokaryotic and eukaryotic. Plastids and mitochondria donated many genes to eukaryotic genomes. Some of the products of those genes are reimported as proteins into the organelle, many others are not – rather they are imported into other organelles or localized in the cytosol or the nucleus, which is not an organelle. Contrary to what was believed 20 years ago (and is sometimes still believed today), the compartmentation of a protein is not a good indicator of its evolutionary origin. Molecular phylogenetics generally has a hard time coming to grips with the problem of horizontal gene transfer because it makes it more difficult to draw direct inferences about the evolutionary past from the phylogenies of contemporary genes. But the situation may not be so dire. If we do not allow ourselves to digress into the realm of descriptive hypothesis-free science, that is if we have good biological theories with which to interpret molecular phylogenies and which we can test with molecular phylogenies, then horizontal gene transfer will probably enrich, rather than hamper, our understanding of life’s history, in particular the origin of eukaryotic genes.
ACKNOWLEDGMENTS We thank Miklos Müller for numerous stimulating discussions and Marianne Limpert
376
ENDOSYMBIOTIC GENE TRANSFER
for help in preparing the manuscript. Generous financial support from the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
REFERENCES Adachi, J. and Hasegawa, M. (1996) Computer Science Monographs, No. 28. MOLPHY Version 2.3: Programs for Molecular Phylogenetics Based on Maximum Likelihood, Institute of Statistical Mathematics, Tokyo. Andersson, S.G.E. and Kurland, C.G. (1999) Origins of mitochondria and hydrogenosomes. Curr. Opin. Microbiol. 2: 535–541. Baldauf, S. and Palmer, J.D. (1990) Evolutionary transfer of the chloroplast tufA gene to the nucleus. Nature 344: 262–265. Brennicke, A., Grohmann, L., Hiesel, R. et al. (1993) The mitochondrial genome on its way to the nucleus: different stages of gene transfer in higher plants. FEBS Letters 325: 140–145. Brinkmann, H. and Martin, W. (1996) Higher plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement. Plant Mol. Biol. 30: 65–75. Brown, J.R. and Doolittle, W.F. (1995) Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications. Proc. Natl Acad. Sci. USA 92: 2441–2445. Brown, J.R. and Doolittle, W.F. (1997) Archaea and the prokaryote-to-eukaryote transition. Microbiol. Mol. Biol. Rev. 61: 456–502. Cavalier-Smith, T. (1987a) Eukaryotes with no mitochondria. Nature 326: 332–333. Cavalier-Smith, T. (1987b) The origin of eukaryote and archaebacterial cells. Ann. NY Acad. Sci. 503: 17–54. Clark, C.G. and Roger, A.J. (1995) Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proc. Natl Acad. Sci. USA 92: 6518–6521. Cowan, D. (2000) Use your neighbor’s genes. Nature 407: 466–467. Doolittle, W.F. (1980) Revolutionary concepts in evolutionary biology. Trends Biochem. Sci. 5: 146–149. Doolittle, W.F. (1996) Some aspects of the biology of cells and their possible evolutionary significance. In Evolution of Microbial Life (eds, D. Roberts, P. Sharp, G. Alserson and M. Collins), pp. 1–21, 54th Symp. Soc. Gen. Microbiol., Cambridge University Press, Cambridge. Doolittle, W.F. (1997) Fun with genealogy. Proc. Natl Acad. Sci. USA 94: 12751–12753. Doolittle, W.F. (1998) A paradigm gets shifty. Nature 392: 15–16. Doolittle, W.F. (1999) Phylogenetic classification and the universal tree. Science 284: 2124–2128. Eisen, J. (2000) Horizontal gene transfer among microbial genomes: new insights from complete genome analysis. Curr. Opin. Genet. Dev. 10: 606–611.
359
Embley, T.M. and Hirt, R.P. (1998) Early branching eukaryotes? Curr. Opin. Genet. Dev. 8: 655–661. Embley, T.M. and Martin, W. (1998) A hydrogen-producing mitochondrion. Nature 396: 517–519. Felsenstein, J. (1993) Phylip (Phylogeny Inference Package) Manual, version 3.5c. Distributed by the author. University of Washington, Seattle, Department of Genetics. Germot, A., Philippe, H. and Le Guyader, H. (1997) Evidence for loss of mitochondria in microsporidia from a mitochondrial-type HSP70 in Nosema locustae. Mol. Biochem. Parasitol. 87: 159–168. Gray, M.W. and Doolittle, W.F. (1982) Has the endosymbiont hypothesis been proven? Microbiol. Rev. 46: 1–42. Gray, M.W., Burger, G. and Lang, B.F. (1999) Mitochondrial evolution. Science 283: 1476–1481. Gupta, R.S. and Golding, G.B. (1996) The origin of the eukaryotic cell. Trends Biochem. Sci. 21: 166–171. Hannaert, V., Brinkmann, H., Nowitzki, U. et al. (2000) Enolase from Trypanosoma brucei, from the amitochondriate protist Mastigamoeba balamuthi, and from the chloroplast and cytosol of Euglena gracilis: pieces in the evolutionary puzzle of the eukaryotic glycolytic pathway. Mol. Biol. Evol. 17: 989–1000. Hashimoto, T., Sanchez, L.B., Shirakura, T. et al. (1998) Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny. Proc. Natl Acad. Sci. USA 95: 6860–6865. Henze, K., Schnarrenberger, C., Kellermann, J. and Martin, W. (1994) Chloroplast and cytosolic triosephosphate isomerases from spinach: purification, microsequencing and cDNA cloning of the chloroplast enzyme. Plant Mol. Biol. 26: 1961–1973. Henze, K., Badr, A., Wettern, M. et al. (1995) A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution. Proc. Natl Acad. Sci. USA 92: 9122–9126. Henze, K., Morrison, H.G., Sogin, M.L. and Müller, M. (1998) Sequence and phylogenetic position of a class II aldolase gene in the amitochondriate protist, Giardia lamblia. Gene 222: 163–168. Henze, K., Suguri, S., Moore, D.V. et al. (2001) Cyanobacterial relationships of the glycolytic enzymes of Giardia intestinalis, glucokinase and glucose-6phosphate isomerase. Gene, in press. Horiike, T., Hamada, K., Kanaya, S. and Shinozawa, T. (2001) Origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria revealed is revealed by homology hit analysis. Nature Cell Biol. 3: 210–214. John, P. and Whatley, F.R. (1975) Paracoccus denitrificans and the evolutionary origin of the mitochondrion. Nature 254: 495–498. Keeling, P.J. and Doolittle, W.F. (1997) Evidence that eukaryotic triosephosphate isomerase is of alphaproteobacterial origin. Proc. Natl Acad. Sci. USA 94: 1270–1275. Keeling, P.J. and Palmer, J.D. (2000) Parabasalian flagellates are ancient eukaryotes. Nature 405: 635–637.
377
360
K. HENZE, C. SCHNARRENBERGER AND W. MARTIN
somes: A case study of functional redundancy in ancient pathways through endosymbiosis. Curr. Genet. 32: 1–18. Martin, W., Brinkmann, H., Savona, C. and Cerff, R. (1993) Evidence for a chimaeric nature of nuclear genomes: eubacterial origin of eukaryotic glyceraldehyde-3phosphate dehydrogenase genes. Proc. Natl Acad. Sci. USA 90: 8692–8696. Martin, W., Mustafa, A.-Z., Henze, K. and Schnarrenberger, C. (1996) Higher plant chloroplast and cytosolic fructose1,6-bisphophosphatase isoenzymes: origins via duplication rather than prokaryote-eukaryote divergence. Plant Mol. Biol. 32: 485–491. Martin, W., Stoebe, B., Goremykin, V. et al. (1998) Gene transfer to the nucleus and the evolution of chloroplasts. Nature 393: 162–165. Mereschkowsky, C. (1905) Über Natur und Ursprung der Chromatophoren im Pflanzenreiche. Biol. Centralbl. 25: 593–604 (English translation in Martin, W. and Kowallik, K.V. (1999) Eur. J. Phycol. 34: 287–295). Mertens, E., Ladror, U.S., Lee, J.A. et al. (1998) The pyrophosphate-dependent phosphofructokinase of the protist, Trichomonas vaginalis, and the evolutionary relationships of protist phosphofructokinases. J. Mol. Evol. 47: 739–750. Moreira, D., Le Guyader, H. and Philippe, H. (2000) The origin of red algae and the evolution of chloroplasts. Nature 405: 69–72. Müller, M. (1988) Energy metabolism of protozoa without mitochondria. Annu. Rev. Microbiol. 42: 465–488. Müller, M. (1993) The hydrogenosome. J. Gen. Microbiol. 139: 2879–2889. Müller, M. (2000) A mitochondrion in Entamoeba histolytica? Parasitol. Today 16: 368–369. Nowitzki, U., Flechner, A., Kellermann, J. et al. (1998) Eubacterial origin of eukaryotic nuclear genes for chloroplast and cytosolic glucose-6-phosphate isomerase: sampling eubacterial gene diversity in eukaryotic chromosomes through symbiosis. Gene 214: 205–213. Ochman, H., Lawrence, J.G. and Groisman, E.S. (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405: 299–304. Ortner, S., Binder, M., Schiener, O. et al. (1997) Molecular and biochemical characterization of phosphoglucomutases from Entamoeba histolytica and Entamoeba dispar. Mol. Biochem. Parasitol. 90: 121–129. Ouzounis, C. and Sander, C. (1992) TFIIB, an evolutionary link between the transcription machineries of archaebacteria and eukaryotes. Cell 71: 189–190. Philippe, H., Germot, A. and Moreira, D. (2000) The new phylogeny of eukaryotes. Curr. Opin. Gen. Dev. 10: 596–601. Plaxton, W.C. (1996) The organization and regulation of plant glycolysis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 185–214. Pühler, G., Leffers, H., Gropp, F. et al. (1989) Archaebacterial DNA dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome. Proc. Natl Acad. Sci. USA 86: 4569–4573. Ribiero, S. and Golding, G.B. (1998) The mosaic nature of the eukaryotic nucleus. Mol. Biol. Evol. 15: 779–788.
Krepinsky, K., Plaumann, M., Martin, W. and Schnarrenberger, C. (2001) Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach: cyanobacterial genes for chloroplast and cytosolis isoenzymes encoded in eukaryotic chromosomes. Eur. J. Biochem. 268: 2678–2686. Lake, J.A. and Rivera, M.C. (1994) Was the nucleus the first endosymbiont? Proc. Natl Acad. Sci. USA 91: 2880–2881. Langer, D., Hain, J., Thuriaux, P. and Zillig, W. (1995) Transcription in Archaea: similarity to that in Eukarya. Proc. Natl Acad. Sci. USA 92: 5768–5772. Lawrence, J.G. and Ochman, H. (1998) Molecular archaeology of the Escherichia coli genome. Proc. Natl Acad. Sci. USA 95: 9413–9417. Liaud, M.-F., Lichtlé, C., Apt, K. et al. (2000) Compartmentspecific isoforms of TPI and GAPDH are imported into diatom mitochondria as a fusion protein: evidence in favor of a mitochondrial origin of the eukaryotic glycolytic pathway. Mol. Biol. Evol. 17: 213–223. Lopez-Garcia, P. and Moreira, D (1999) Metabolic symbiosis at the origin of eukaryotes. Trends Biochem. Sci. 24: 88–93. Margulis, L. (1970) Origin of Eukaryotic Cells, Yale University Press, New Haven, CT. Margulis, L. (1996) Archaeal–eubacterial mergers in the origin of Eukarya: phylogenetic classification of life. Proc. Natl Acad. Sci. USA 93:1071–1076. Margulis, L., Dolan, M.F. and Guerrero, R. (2000) The chimeric eukaryote: origin of the nucleus from the karyomastigont in amitochondriate protists. Proc. Natl Acad. Sci. USA 97: 6954–6959. Markos, A., Miretsky, A. and Müller, M. (1993) A glyceraldehyde-3-phosphate dehydrogenase with eubacterial features in the amitochondriate eukaryote Trichomonas vaginalis. J. Mol. Evol. 37: 631–643. Martin, W. (1998) Endosymbiosis and the origins of chloroplast-cytosol isoenzymes: a revision of the gene transfer corollary. In Horizontal Gene Transfer: Implications and Consequences (eds, M. Syvanen and C. Kado), pp. 363–379, Chapman and Hall, London. Martin, W. (1999a) Mosaic bacterial chromosomes – a challenge en route to a tree of genomes. BioEssays 21: 99–104. Martin, W. (1999b) A briefly argued case that mitochondria and plastids are descendants of endosymbionts, but that the nuclear compartment is not. Proc. R. Soc. Lond. B 266: 1387–1395. Martin, W. (2000) Primitive anaerobic protozoa: the wrong host for mitochondria and hydrogenosomes? Microbiology 146: 1021–1022. Martin, W. and Cerff, R. (1986) Prokaryotic features of a nucleus encoded enzyme: cDNA sequences for chloroplast and cytosolyic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba). Eur. J. Biochem. 159: 323–331. Martin, W. and Herrmann, R.G. (1998) Gene transfer from organelles to the nucleus: How much, what happens and why? Plant Physiol. 118: 9–17. Martin, W. and Müller, M. (1998) The hydrogen hypothesis for the first eukaryote. Nature 392: 37–41. Martin, W. and Schnarrenberger, C. (1997) The evolution of the Calvin cycle from prokaryotic to eukaryotic chromo-
378
ENDOSYMBIOTIC GENE TRANSFER
Rivera, M.C., Jain, R., Moore, J.E. and Lake, J.A. (1998) Genomic evidence for two functionally distinct gene classes. Proc. Natl Acad. Sci. USA 95: 6239–6244. Roger, A.J. (1999) Reconstructing early events in eukaryotic evolution. Am. Nat. 154: 146–163. Rotte, C. and Martin, W. (2001) Endosymbiosis does not explain the origin of the nucleus. Nature Cell. Biol. 8: E173. Rotte, C., Henze, K., Müller, M. and Martin, W. (2000) Origins of hydrogenosomes and mitochondria. Curr. Opin. Microbiol. 3: 481–486. Rotte, C., Stejskal, F., Zhu, G. et al. (2001) Pyruvate:NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a fusion of pyruvate:ferredoxin oxidoreductase and NADPH-cytochrome P450 reductase. Mol. Biol. Evol. 18: 710–720. Rowlands, T., Baumann, P. and Jackson, S.P. (1994) The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. Science 264: 1326–1329. Rujan, T. and Martin, W. (2001) How many genes in Arabidopsis come from cyanobacteria? An estimate from 386 protein phylogenies. Trends Genet., in press Sagan, L. (1967) On the origin of mitosing cells. J. Theoret. Biol. 14: 225–274. Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425. Sandman, K. and Reeve, J. (1998) Origin of the eukaryotic nucleus. Science 280: 501–503. Schmidt, M., Svendsen, I. and Feierabend, J. (1995) Analysis of the primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves by protein
361
and cDNA sequencing indicates a eukaryotic origin of its gene. Biophys. Acta 1261: 257–264. Schnarrenberger, C., Flechner, A. and Martin, W. (1995) Enzymatic evidence for a complete oxidative pentose phosphate pathway in chloroplasts and an incomplete pathway in the cytosol of spinach leaves. Plant Physiol. 108: 609–614. Tovar, J., Fischer, A. and Clark, C.G. (1999) The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica. Mol. Microbiol. 32: 1013–1021. Van de Peer, Y., Rensing, S., Maier, U.G. and De Wachter, R. (1996) Substitution rate calibration of small subunit RNA identifies chlorarachniophyte endosymbionts as remnants of green algae. Proc. Natl Acad. Sci. USA 93: 7744–7748. Weeden, N.F. (1981) Genetic and biochemical implications of the endosymbiotic origin of the chloroplast. J. Mol. Evol. 17: 133–139. Wendt, U.K., Hauschild, R., Lange, C. et al. (1999) Evidence for functional convergence of redox regulation in G6PDH isoforms of cyanobacteria and higher plants. Plant Mol. Biol. 40: 487–494. Wu, G., Henze, K. and Müller, M. (2001) Evolutionary relationships of the glucokinase from the amitochondriate protist, Trichomonas vaginalis. Gene 264: 265–271. Zauner, S., Fraunholz, M., Wastl, J. et al. (2000) Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph. Proc. Natl Acad. Sci. 97: 200–205. Zillig, W., Klenk, H.-P., Palm, P. et al. (1989) Did eukaryotes originate by a fusion event? Endocyt. Cell Res. 6: 1–25.
379
Color profile: Generic CMYK printer profile Composite Default screen
00
100
5
95
5
75
5
25
5
0
00
100
5
95
5
75
5
25
FIGURE 27.2 (See following page for caption.)
5
0
D:\WORK\Harcourt\Syvanen + Kado\Syvanen + Kado (Plates).vp 06 November 2001 15:42:07
Color profile: Generic CMYK printer profile Composite Default screen
00
5
5
5
00
5
5
5
100
FIGURE 27.2 (On previous page.) Summary of gene phylogenies for enzymes of compartmentalized sugar phosphate metabolism in plants. Suggested evolutionary origins for the nuclear genes are color-coded. Enzymes regulated through the thioredoxin system are indicated. Many enzymes in the figure are allosterically regulated, but no allosteric regulation is indicated here. Enyzme abbreviations are: FBA fructose-1,6-bisphosphate aldolase; FBP fructose-1,6-bisphosphatase; GAPDH glyceraldehyde-3-phosphate dehydrogenase; PGK 3-phosphoglycerate kinase; PRI ribose-5-phosphate isomerase; PRK phosphoribulokinase; Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase; RPE ribulose-5-phosphate 3-epimerase; SBP sedoheptulose-1,7-bisphosphatase; TKL transketolase; TPI triosephosphate isomerase; TAL transaldolase; GPI glucose-6-phosphate isomerase; G6PDH glucose-6-phosphate dehydrogenase; 6GPDH 6-phosphogluconate dehydrogenase; PGluM phosphoglucomutase; PGM phosphoglyceromutase; PFK phosphofructokinase (pyrophosphate and ATP-dependent); ENO enolase; PYK pyruvate kinase; PDC pyruvate dehydrogenase complex (E1, E2, E3 components), T translocator. PDC is a multienzyme complex, but only one set of components is drawn here. Note that chloroplast isoenzymes of PGM, ENO, and PYK have not been demonstrated in spinach leaves, but for convenience we have included those enzymes in this figure, since they have been well characterized in the plastids of other higher plants (Plaxton, 1996). For further details see Martin and Schnarrenberger (1997), Martin and Herrmann (1998), Nowitzki et al. (1998) and Krepinsky et al. (2001).
FIGURE 27.3 (Opposite.) A tree of genomes. Each prokaryotic genome is represented as a single colored line, different colors symbolize different groups of prokaryotes. A working hypothesis for the origin of eukaryotic genes as outlined primarily in Martin and Müller (1998) and taking lateral gene transfer into account (modified from Martin, 1999a). The figure extends symbiotic associations (merging of genomes into the same cellular confines is indicated by the merging of colored lines) to include schematic indication of several independent secondary symbioses for the acquisition of plastids during eukaryotic history (Martin et al., 1998; Zauner et al., 2000). Importantly, among eukaryotes, colored lines indicate merely that prokaryotic genomes existed at one time within the cellular confines of a given eukaryotic lineage, not that they have persisted to the present as an independently compartmented genome (because most genes from organelles are transferred to the nucleus). For example some eukaryotes with secondary symbionts are schematically indicated with six lines, but only have four distinctly compartmented genomes. Similarly, eukaryotes that lack mitochondria apparently possessed such organelles in the past but only have one genome – that in the nucleus (for example Giardia, Trichomonas, and Entamoeba, see text). Furthermore it is important to note that mitochondrion-lacking eukaryotes have arisen through loss of the organelle in many independent lineages (Embley and Martin, 1998; Hashimoto et al., 1998; Roger, 1999). In the enlargement, lateral gene transfer between eubacteria prior to – and implicitly, but not shown, subsequent to – the origin of mitochondria (blue lines) and plastids (green lines) is schematically represented. Genomes are represented as heavy lines, individual gene transfer events (regardless of possible numbers of genes involved) as thin lines. The phylogeny of eukaryotes indicated is mostly schematic, containing various elements from van der Peer et al. (1996), Keeling and Palmer (2000), Martin et al. (1998) and other sources, and is not intended to be close to correct. The various schematic plastid organizational types are shown merely to indicate that dramatic differences in organelle structure can arise from a single ancestral symbiotic state.
95
75
25
5
0
100
95
75
25
5
0
D:\WORK\Harcourt\Syvanen + Kado\Syvanen + Kado (Plates).vp 06 November 2001 15:42:07
Color profile: Generic CMYK printer profile Composite Default screen
00
100
5
95
5
75
Euglena
Diatoms Rhodophytes
Chlorophytes
5
25
5
0
Cyanophora Heterotrophic Eukaryotes with Hydrogenosomes, or Mitochondria, or no ATP-Producing Organelles at all
Pro
teo
bac
ter
Pro
Eukaryotes
ia
teo
bac
ter
CO2
ia
Substrate
Eubacteria
Glucose H2O
Archaebacteria
Pyruvate
ATP ATP
ATP H2O CO2
Anaerobic Respiration
Substrate
ATP H2O CO2
Secondary Symbiosis
H2 H2 Glucose H2 CO2 ATP ATP Acetate ATP ATP H H2 2 CH4 Facultative Anaerobic H2-dependent Autotrophic Host Symbiont Glucose
ATP
Glucose
H2O
H2 Anaerobic CO2 Fermentation Acetate
Substrate
Glucose Pyruvate
CO2
Glucose ATP Pyruvate
ATP
ATP
O2
Substrate
O2 Respiration
O2
Origins of Mitochondria Origins of Plastids
00
100
Horizontal Gene Transfer between free-living Eubacteria
5
5
5
95
75
25
5
0
D:\WORK\Harcourt\Syvanen + Kado\Syvanen + Kado (Plates).vp 06 November 2001 15:42:08