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Jpn. J. Protozool. Vol. 38, No. 2. (2005) 171

Review

On the origin of mitochondria and Rickettsia-related eukaryotic endo- symbionts

B. Franz Lang1*, Henner Brinkmann1, Liisa B. Koski1, Masahiro Fujishima2, Hans-Dieter Görtz3 and Gertraud Burger1

1Program in Evolutionary Biology, Canadian Institute for Advanced Research; Centre Robert Cedergren, Département de Biochimie, Université de Montréal, 2900 Boulevard Edouard- Montpetit, Montréal, Québec, H3T 1J4, Canada. 2Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan. 3Abteilung Zoologie, Biologisches Institut, Universität Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany.

SUMMARY sence of genes for oxidative phosphorylation, the TCA cycle, and many other metabolic pathways, Resent insights into the origin and early evo- but the presence of several pathogenesis-related lution of mitochondria come from two approaches: genes and a high number of bacterial IS elements. the investigation of mtDNAs from minimally de- Phylogenetic analyses with multiple protein se- rived (primitive) mitochondriate , in quences place H. obtusa basally to the Rickettsia- particular , and of genomes from Ehrlichia-Wolbachia assemblage of bacterial intracellular α-proteobacterial symbionts. Of par- pathogens. This leads us to postulate that H. ob- ticular interest in this context is Holospora obtusa, tusa is the closest bacterial relative of mitochon- an intracellular bacterial endosymbiont that resides dria known to date. and replicates in the somatic nucleus of its eu- karyotic host, the Paramecium caudatum. Currently we have sequenced close to 50% of the INTRODUCTION ~ 1.7 Mbp H. obtusa genome, revealing the ab- One of the major advancements in under- standing eukaryotic evolution was the discovery that mitochondria evolved from an endosymbiotic α-Proteobacterium, and that mitochondrial DNA *Corresponding author (mtDNA) is a relict bacterial genome. Most E-mail: [email protected] mtDNAs have lost earmarks of their bacterial ori- Received: 28 June 2005. gin, with a notable exception in jakobid flagellates 172 Mitochondrial origins

Table 1: Biological processes in mitochondria 1 DNA replication, recombination and repair 2 Transcription (a phage-like RNA polymerase, except in jakobid flagellates using an α2ββ’σ poly- merase3) RNA processing (including RNA editing and intron splicing) Translation (rRNAs, tRNAs and ribosomal proteins, EFTU3 and tmRNAs3) Protein and RNA translocation (Tom, Tim, Tob, Tat (tatA3, tatC), Sec3, Oxa1, and VDAC translocases) Mitochondrial carriers (translocators of ADP/ATP, co-factors, metabolites, substrates, etc.) Chaperones Mitochondrial morphology and division Proteases Nucleases Electron transport and ATP synthesis Lipid metabolism Nucleotide metabolism Amino acid metabolism Carbohydrate metabolism Fe-S biosynthesis and export Heme biosynthesis Ubiquinone biosynthesis Enzyme cofactor biosynthesis 1 Note that some pathways serve mitochondria-specific functions only, whereas others supply the whole cell. 2 Processes in bold involve mtDNA-encoded components. 3 MtDNA-encoded only in americana (Lang et al. 1997; Jacob et al. 2004). For a compilation of genes from completely sequenced mtDNAs see GOBASE at http://amoebidia.bcm.umontreal.ca/pg- gobase/searches/compilations.php

(Lang et al. 1997; Jacob et al. 2004). According to grated to the nucleus during early steps of endo- current estimates, mitochondria were acquired symbiosis. The total number of nuclear genes that more than one billion years ago, which makes in- are involved in mitochondrial biogenesis has been ferences of early steps in mitochondrial evolution a estimated at 700-800 proteins in yeast (see Table 1 non-trivial problem. Not surprisingly, the nature of for biological processes in which mitochondria of the partners implicated in mitochondrial endosym- yeast and other eukaryotes are involved). This biosis, the driving forces and timing of their fu- estimate is based on mass spectrometry of proteins sion, and initial stages of reductive mitochondrial from isolated mitochondria (Sickmann et al. 2003) genome evolution, remain vague. and sub-cellular localization studies (Kumar et al. Contemporary mtDNAs have retained only 5 2002; Huh et al. 2003), combined with genomic – 100 from the original set of several hundred to approaches (Prokisch et al. 2004). However, these several thousand genes residing in bacterial ge- numbers come with a high degree of imprecision nomes. One portion of these genes was lost in the (Reichert and Neupert 2004). In addition, only a proto-mitochondrial genome, another one has mi- small fraction of yeast mitochondrial proteins can Jpn. J. Protozool. Vol. 38, No. 2. (2005) 173 be placed with confidence in phylogenetic analy- ses; consequently, it remains unknown how many of the nucleus-encoded mitochondrial proteins derive from the α-proteobacterial ancestor, the eukaryotic host, or other sources (Burger and Lang 2003). To address the question of mitochondrial ori- gins, we have begun to investigate the genome of the bacterium Holospora obtusa, an endosymbiont of the ciliate Paramecium caudatum (Görtz et al. 1990). The objective of our study is to conduct genome comparisons between intracellular versus free-living on one hand, and endosymbi- otic bacteria versus organelles on the other. Figure 1: Genome size and coding content of mito- In the present review, we will give a brief up- chondria and Rickettsia. Circles and lines represent date on the diversity of mitochondrial genomes circular and linear genome shapes, respectively. For genomes >60 kbp, the portion coding for genes with across eukaryotes and discuss the early evolution- known function (red) is distinguished from that in- ary events that led to the establishment of mito- cluding intergenic regions and ORFs (purple). chondria. Then, we will present a preliminary analysis of the Holospora obtusa genome se- quence, and conclude with a phylogenetic analysis from mitochondria because they import the pro- of this species. teins mentioned above (Williams and Keeling 2003). Taken together, this raises the possibility Distribution of mitochondria across eukaryotes that eukaryotes without mitochondria may never Not all eukaryotes contain mitochondria. Ex- have existed. Alternatively, their descendants ceptions, although rare, are species that live in might have not survived the increase of oxygen anoxic environments, such as concentration on Earth that was due to the emer- (), and (diplo- gence and proliferation of photosynthetic eukaryo- monads), Entamoeba and Mastigamoeba (pelo- tes. bionts), (), Micro- sporidia (fungi) and Neocallimastix (fungi). Sev- Typical features of mitochondrial genomes – a eral of these species have most likely lost func- brief overview tional mitochondria secondarily rather than being The more mitochondrial genetic systems are primitively amitochondriate, because they contain analyzed, the less holds the concept of a 'typical' nucleus-encoded genes that are most likely of mi- mtDNA. Even so, certain features are frequently tochondrial origin (e.g., the ADP/ATP carrier (van found across a broad taxonomical range of eu- der Giezen et al. 2002; Voncken et al. 2002) and karyotes, as briefly summarized in the following the 60 and 70 kDa heat-shock proteins (van der (for more details and references, see (Gray et al. Giezen et al. 2003)). In addition, a growing num- 1998; Lang et al. 1999; Gray et al. 2004)). ber of amitochondriate eukaryotes are reported to A+T content. With few exceptions, mitochondrial contain organelles (hydrogenosomes, cryptosomes genomes feature a high A+T content (60-80%, or mitosomes), which are believed to be derived compared to nuclear) a property often used for 174 Mitochondrial origins

Figure 2: Mitochondrial genomes from diverse eukaryotic groups. Genetic maps of mtDNAs (see also (O'Brien et al. 2003) and http://amoebidia.bcm.umontreal.ca/pg-gobase/searches/map.php) from an (Anderson et al. 1981), a (Forget et al. 2002) and three (Burger et al. 1995; Lang et al. 1997; Lang et al. 2002), dem- onstrating the wide range of coding capacity and organization (the mtDNA of Hyaloraphidium although linear, is depicted as a circle to facilitate comparisons). Black rectangles, identified genes; grey rectangles, ORFs; white rec- tangles with grey-shaded sections, introns and intronic open reading frames; rectangles on outer/inner ring, tran- scription is clockwise/counterclockwise. Gene names in black belong to the basic gene complement common to ani- mal mtDNAs; green, ORFs; blue, extra genes present in various protists and ; red, extra genes specific to jako- bid flagellates. Jpn. J. Protozool. Vol. 38, No. 2. (2005) 175 physically separating mtDNA from other DNAs the extent of gene migration to the nucleus (Adams within an organism. and Palmer 2003; Burger and Lang 2003). Size. The medium size of mtDNA is ~50 kbp, which corresponds to ~1% of the typical size of an The most bacteria-like mtDNA is found in Re- α-proteobacterial genome from which it is derived. clinomonas americana Yet deviations from the medium size by an order In past years, the eubacterial ancestry of mito- of magnitude are quite common (Fig. 1). With chondria has become strikingly obvious through only 6 kbp, the mtDNA of the primate and bird the study of minimally derived protists, such as the parasite Plasmodium is the smallest known, while recently recognized jakobid Reclinomo- the largest ones occur in land plants, measuring nas americana (Lang et al. 1997) (Fig. 2). Among from 180 to 2,400 kbp (Ward et al. 1981). the most eubacteria-like features of jakobid Shape. MtDNA molecules (referred to in the fol- mtDNAs are putative ribosome binding motifs lowing as chromosomes) come in three major to- (Shine-Dalgarno sequences) upstream of protein- pologies (for a review, see (Bendich 1996)). In coding genes, multi-subunit RNA polymerase, most organisms, mitochondrial chromosomes are Sec-dependent protein import, transfer messenger circular-mapping but linear in structure, made up RNA (tmRNA) involved in the release of stalled of several tandemly concatenated copies. True ribosomes, and the elongation factor Tu (Lang et (monomeric) circular or linear mtDNAs have been al. 1997; Jacob et al. 2004). Neither of these has shown to occur in only a limited number of phy- been found so far in mtDNAs of other eukaryotes. logenetically disparate taxa such as fungi, verte- brates and (e.g., (Forget et al. 2002; Mitochondrial versus bacterial genomes Nosek and Tomaska 2003; Marande et al. 2005) Genomes of free-living bacteria usually con- and references therein). tain several thousand genes, compared to only ~ Chromosome number and ploidy. Mitochondrial 500 to 1000 in endosymbiotic bacteria. For exam- DNA typically consists of a single kind of mole- ple, in the obligatory pathogen Haemophilus influ- cule in numerous copies, ranging from ~10 to enza, the causative agent of meningitis, 636 genes ~1000 per cell, i.e., a dozen or more per organelle could be assigned to a function and a further 363 and often more than one organelle per cell. are predicted to code for proteins of unknown Gene content. Invariantly, mtDNA-encoded com- function (TIGR http://pohh.phy.ncu.edi/tw/cdy/ ponents participate in processes that take place in USS/). The gene content of mitochondrial ge- mitochondria, including respiration, oxidative nomes is yet an order of magnitude smaller than phosphorylation and translation; less frequently in those of pathogenic/endosymbiotic bacteria. Com- protein import/maturation and RNA processing, pletely absent from mtDNAs are genes involved in and rarely in transcription (Table 1, Fig. 2). Again, several basic cellular processes such as DNA repli- at the extremes are Plasmodium and its relatives cation, recombination, motility, metabolism, and with as few as five genes (three of which code for signal transduction (Fig. 2, Table 1). proteins and two for rRNAs), and jakobid flagel- As discussed in detail elsewhere (see (Burger lates with nearly 100 genes (including 70 proteins and Lang 2003) and references therein), gene loss and 25-26 tRNAs). The number of mtDNA- and elevated genomic A+T content in bacteria encoded tRNAs is most variable across eukaryotes appear to progress concurrently with the transition (0 to >30). The reason for the up to 20-fold differ- from a free-living to an intracellular lifestyle. In- ence in mitochondrial gene content is variation in terestingly, the trends of genome evolution associ- 176 Mitochondrial origins ated with intracellularity are shared by bacterial harbor a variety of bacteria that enter pathogens of different phylogenetic affiliation: α- their host via the food . Some of these bac- (Rickettsia, Wolbachia, Ehrlichia), terial species further invade the cytoplasm as well β-Proteobacteria (Neisseria), γ-Proteobacteria as the micro- and the macronucleus where they (Haemophilus, Buchnera, Wigglesworthia), Fir- reside and reproduce. About 200 ciliate species micutes (Mycoplasma), the Chlamydia group, and have been recorded to host intracellular bacteria; Spirochetes (Borrelia). Obviously, by adapting to the Paramecium genus alone harbors close to 60 intracellular environments, these organisms have different bacterial taxa (reviewed in (Fokin et al. undergone convergent evolution. A similar conver- 2004)). gence is found in mitochondria and plastids. The specific host of Holospora obtusa is the ciliate Paramecium caudatum, on which the bacte- Do mitochondria originate from endosymbiotic rium depends for propagation. Previous studies or free-living α-Proteobacteria? have uncovered the complex cycle of Phylogenetic analyses provide clear evidence Holospora, including the invasion of the somatic that mitochondria originated from within α-Proteo- (macro-) nucleus and its interactions with and bacteria (e.g., (Andersson et al. 1998; Burger et al. modulation of the host’s subcellular structures 1999)), a bacterial assemblage that includes free- upon infection (Görtz et al. 1990). In the course of living species such as Magnetospirillum and transition from the infectious to the reproductive Rhodobacter, facultative symbionts/pathogens of bacterial form, patterns of protein expression plants such as Rhizobium and Agrobacter, and change considerably. So far, more than 20 bacte- intracellular obligatory animal pathogens of the rial proteins that are specifically involved in infec- Rickettsia group including the causative agents of tion have been characterized by immunological typhus and spotted fever. However, phylogenetic methods and N-terminal microsequencing (e.g., reconstructions, even when including mitochon- (Görtz et al. 1990; Wiemann and Görtz 1991; drial data from the minimally derived , do Dohra et al. 1997; Dohra et al. 1998; Nakamura et not resolve with confidence which of these α- al. 2004); Fujishima and Görtz, unpublished re- proteobacterial groups is most closely related to sults). mitochondria. To address this question with a We have decided to sequence the complete broader dataset, we began sequencing the genome genome of Holospora for two reasons, (i) to facili- of the α-proteobacterial Holospora obtusa, an in- tate identification of genes implicated in infection tracellular endosymbiont of the ciliate Parame- and (ii) to revisit the question about the phyloge- cium caudatum. In the following, we briefly de- netic position of this endosymbiont. At the time of scribe this bacterium, its life style and genome, writing, we have sequenced close to 50% of the before going on to revisit the ancestry of mito- 1.7 mbp genome size estimated by Timofeyeva chondria using these new data. AS, Rautian M and Görtz HD (unpublished). With an A+T-content of ~ 65%, the Holospora genome size is only moderately A+T rich, compared to RESULTS AND DISCUSSION other endosymbiotic bacteria, which have A+T contents up to 75% (reviewed in (Wernegreen The highly reduced genome of the endosymbi- 2002)). otic bacterium Holospora obtusa As of now, more than 353 genes have been identified in the Holospora genome, including Jpn. J. Protozool. Vol. 38, No. 2. (2005) 177

~200 genes of known function (coding for 181 and Burger G, unpublished). These genes code for distinct proteins and 18 structural RNAs). The components of the general secretion pathway, lipo- remaining ~150 hypothetical proteins (ORFs) have protein carriers, and permeases. The Holospora no recognizable counterparts in other organisms. genome also contains a class of ~ 40 nearly identi- The majority of assigned genes participate in basic cal insertion elements (classified as transpo- cellular processes (replication and repair, transcrip- sase_11; pfam01609) that are most similar to tion and translation, ATP synthesis), a wide variety counterparts in Legionella pneumophila ((Chien et of transport functions (ABC transporters) and a al. 2004); locus CAH17112)) and Francisella tu- few metabolic pathways (e.g., lipid, heme, glucan, larensis ((Larsson et al. 2005); locus nucleotide and alanine biosynthesis). Most path- YP_169173)). Finally, we identified numerous ways considered essential for autotrophy appear to other repeat elements, some of which have simi- be completely missing, except, curiously, biotin larities to transposase-containing IS sequences of synthesis (note that biotin synthesis is lacking in bacteria, and others that appear to be Holospora- Rickettsia). We speculate that the provision of specific. biotin constitutes one of the benefits that Parame- cium receives from its association with Holospora. Holospora obtusa is only distantly related to Other potential benefits for the host cell are longer Rickettsiales survival of infected Paramecium at low tempera- Initial phylogenetic analyses with small sub- tures (10 ºC) and longer survival and increased cell unit rRNA (SSU-rRNA) sequences affiliated motility at 37 ºC, which has been associated with Holospora obtusa with Caedibacter caryophila over-expression of Hsp70 and/or GroEL (Hori and (another Paramecium endosymbiont; (Springer et Fujishima 2003; Fujishima et al. 2005). al. 1993)), which together emerge from within the Intriguingly, we found in the Holospora ge- Rickettsia lineage of α-Proteobacteria (e.g., nome no genes for respiratory chain complexes, (Springer et al. 1993; Gray and Spencer 1996; which in aerobic organisms serve to drive efficient Horn et al. 1999)). However, these results have to ATP synthesis via oxidative phosphorylation. It be met with reservations because the statistical appears that Holospora relies on ‘energy parasit- support for this placement with SSU-rRNA data is ism’ by ATP import via its ADP/ATP transporter non-significant (< 95% bootstrap support; likeli- system (Linka et al. 2003). This feature distin- hood ratio tests were not performed). In addition, guishes Holospora from Rickettsia and its relatives the clustering of Rickettsia- and Holospora- who have retained the oxidative phosphorylation relatives may be solely due to their elevated A+T system (e.g., (Andersson et al. 1998; Renesto et al. content, contrasting with the low A+T content of 2005)), as well as the TCA cycle and the glycoly- free-living α-Proteobacteria. Nucleotide sequence sis III pathway (pyruvate → acetate). The loss of datasets of highly unbalanced A+T content are respiration and oxidative phosphorylation in known to be prone to a strong phylogenetic arti- Holospora is not surprising, given that this endo- fact, whose effect is similar to that caused by un- symbiont resides and reproduces in the host’s nu- equal evolutionary rates known as long-branch cleus where oxygen supply is limited. attraction (Felsenstein 1978). In fact, long-branch We have discovered additional genes in the attraction is itself a major concern, as all symbi- Holospora genome whose products play a role in otic/pathogenic bacteria have highly accelerated host invasion and interact with the host’s subcellu- evolutionary rates as a consequence of their adap- lar components (Fujishima M, Görtz HD, Lang BF tation to an intracellular life style. In combination, 178 Mitochondrial origins

Figure 3: Phylogenetic relationship of Holospora to other α-Proteobacteria The analysis is based on a concatenated dataset of bacterial proteins. The tree has been inferred using PhyML and the WAG+F+Γ model. Numbers at branches represent support values obtained with 100 bootstrap replicates. The scale bar denotes the estimated number of amino acid substi- tutions per site. The four eubacterial subdi- visions (α, β, γ, δ) are indicated. For more information on the selection of sequences and the phylogenetic methodology used, see (Rodríguez-Ezpeleta et al. 2005).

the effect of A+T bias plus long-branch attraction over, the analysis of multiple protein sequences might be strong enough to regroup Holospora with helps to overcome the problem of non-significant Rickettsiales in a positively misleading way. statistical support, as does the application of the Under such circumstances, it is advisable to most realistic evolutionary models (e.g., an appro- employ phylogenetic approaches that are least priate amino acid substitution matrix; among-site sensitive to long-branch attraction (such as likeli- variation of evolutionary rates modeled by a hood methods; (Delsuc et al. 2005)), and to use gamma distribution). deduced protein rather than nucleotide sequences, Lateral (or horizontal) gene transfer is another in order to reduce the impact of A+T bias. More- potential problem in bacterial phylogeny Jpn. J. Protozool. Vol. 38, No. 2. (2005) 179

(Zhaxybayeva et al. 2004). Yet, a core of genes and somewhat closer to the latter two species than that generally code for essential proteins, and that to Rickettsia. Superimposition of the bacterial tree are subunits of large complexes, are less likely shown in Fig. 3 with our bacterial/mitochondrial affected by transfers (Brochier et al. 2002). These trees predicts that H. obtusa is closer to the mito- are the genes that we chose for the present analy- chondrial divergence point than all other bacterial sis. pathogens. Fig. 3 shows a phylogenetic tree based on To test this prediction, a gene set will be re- 6,109 amino acid positions from 18 genes. The quired which is shared by H. obtusa and mitochon- proteobacterial relationships are resolved with dria, for instance nuclear-encoded mitochondrial significant support at all branches (except for a proteins with counterparts in Holospora, such as short internal branch in the γ subdivision). The ribosomal proteins, translation factors and chaper- impact of LGT seems negligible, because in multi- ons. Work is in progress by the Canada-wide col- protein phylogenies, tree resolution and support laborative EST Program (PEP; http:// values are expected to disappear with increasing amoebidia.bcm.umontreal.ca/public/pepdb/ . In contrast to the results agrm.php), to determine the expressed portion of obtained with SSU-rRNA data mentioned above, nuclear genomes from a broad range of protists. H. obtusa branches in our protein-based analysis These data will be instrumental for resolving the clearly outside the Rickettsia-Wolbachia-Ehrlichia question whether Holospora is indeed the closest lineage of bacterial pathogens. bacterial relative of mitochondria.

Divergence point of mitochondria within α- CONCLUSIONS Proteobacteria Our preliminary analyses of the H. obtusa Several studies by others suggest that mito- genome show that α-proteobacterial endosymbi- chondria are specifically associated with onts represent an unexpected broad diversity, both Rickettsiales, or even emerged from within the phylogenetically and metabolically. The complete Rickettsia lineage (e.g., (Gray and Spencer 1996; genome sequence of H. obtusa and its relatives Andersson et al. 1998; Sicheritz-Ponten et al. such as Caedibacter will not only be key to better 1998)). These conjectures need to be considered understanding eukaryotic host – pathogen interac- with caution because the corresponding analyses tions. These bacterial genome data, in combination suffer from non-significant statistical support due with EST data from minimally derived eukaryotes, to limited sequence data, or insufficient taxon sam- also promise to provide insight into the early his- pling. We previously inferred phylogenies based tory of mitochondrial endosymbiosis. on an extended set of mtDNA-encoded proteins (up to 13) that are highly conserved and present in most eukaryotic lineages, providing a total of ~ ACKNOWLEDGEMENTS 3,000 amino acid positions (e.g., (Forget et al. We thank M. Nickel, Y. Jacob and D. 2002; Lang et al. 2002; Bullerwell et al. 2003a; Tremblay for participation in H. obtusa genome Bullerwell et al. 2003b)). When including in this sequencing, and L. Forget and Z. Wang for excel- data set sequences from several α-proteobacteria lent technical assistance. This project was sup- but not Holospora (because it lacks the corre- ported by grants from the Canadian Institutes for sponding proteins), mitochondria branch between Health Research (MOP-42475, GX-15331), and by Rickettsia and Sinorhizobium/Magnetospirillum Genome Quebec and Genome Canada. Salary and 180 Mitochondrial origins interaction support from the Canadian Institute for ogy. 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