Origin of Eukaryotic Cell Nuclei by Symbiosis of Archaea in Bacteria supported by the newly clarified origin of functional genes

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1 Genes Genet. Syst. (2002) 77, p Origin of Eukaryotic Cell Nuclei by Symbiosis of Archaea in Bacteria supported by the newly clarified origin of functional genes Tokumasa Horiike, Kazuo Hamada, Takao Shinozawa* Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Tenjincho 1-5-1, Kiryu, Gunma , Japan, (Received 10 June 2002, accepted 7 October 2002) In the previous report, we demonstrated the origin of eukaryotic cell nuclei as the symbiosis of Archaea in Bacteria by the newly developed Homology-Hit Analysis. In that case, we counted yeast Open Reading Frames (ORFs) showing the highest similarity to a bacterial ORF as orthologous ORFs (Orthologous ORFs were produced by speciation from a common ancestor, and have the highest similarity to each other.) by comparing whole ORFs of yeast with those of individual bacteria. However, we could not count all yeast ORFs showing the highest similarity to a bacterial ORF in functional categories of yeast. Therefore, the origin of ORFs in the functional categories of yeast could not be inferred strictly. Here, we have improved the method for detecting orthologous ORFs. In this method, we count the numbers of ORF with the highest similarity between individual yeast functional categories and individual bacteria as orthologous ORFs. By this method, it was possible to detect the correct orthologous ORFs and to infer the origins of the functional categories in eukaryotic cells. As a result, two categories, assembly of protein complexes and DNA repair were newly judged to be of Archaeal origin, while five categories, lipid (fatty-acid and isoprenoid) metabolism, protein folding and stabilization, signal transduction, organization of the plasma membrane and organization of the cytoplasm, were newly judged to be of Bacterial origin. On the other hand, the origins of two categories (meiosis and cellular import, which were determined in the previous analysis) could not be judged. It is considered that functional categories related to the nucleus have origins common to Archaea, while those related to the cytoplasm have origins common to Bacteria. From these data including the origin of plasma membrane, it was further clarified that cell nucleus originated by the symbiosis of Archaea in Bacteria. INTRODUCTION Estimation of the origin of the Eukaryote has been mainly tried by the phylogenic analysis of the rrna and protein genes (Lake, 1988; Lake and Rivera, 1994; Gupta et al., 1994; Moreira and Lopez-Garcia, 1998). By these researchs, ORF groups such as replication, transcription and translation have been generously supposed to be Archaea origin and the ORF group related to the energy is Bacteria origin. Also, other ORF groups were suggested to be originating from both forming a mosaic structure in eukaryotic genome. However, the numbers of genes used and also their functional varieties were small. Also, disadvantage of this analysis is that it is not Edited by Naruya Saitou * Corresponding author. shinozawa@bce.gunma-u.ac.jp possible to judge whether the selected genes are really representing one or not (Philippe et al., 1999). Using the BLAST (Altschul et al., 1997) and FASTA (Pearson et al., 1998) programs, the similarity of ORF (Open Reading Frame) groups between three domains (Eukarya, Archaea and Bacteria) was estimated at one threshold (Koonin et al., 1997; Andrade et al., 1999). Therefore, their similarities at other thresholds could not be clarified. Previously, in order to clarify the origin of the functional categories of yeast by Homology-Hit Analysis, we counted the numbers of orthologous ORFs between yeast and individual bacteria (Orthologous genes have common ancestor and high similarity each other, and were produced by speciation.)(horiike et al., 2001). In that analysis, we were able to determine the origins of 20 categories. The results suggested that yeast ORFs

2 370 T. HORIIKE et al. related to the nucleus share their origins with Archaeal ORFs, while ORFs related to the cytoplasm share their origins with Bacterial ORFs. Our results provided an evidence that eukaryotic cell nuclei arose by the symbiosis of Archaea in Bacteria. However, we could not determine the origin of 23 yeast categories. Here, we improved the method to determine the origin of these categories. In the previous analysis, orthologous ORFs showing the highest degree of similarity between whole yeast ORFs and the ORFs of each bacterium were detected, and then the yeast orthologous ORFs were classified into each functional category (Fig. 1a). In this case, we counted not all yeast ORFs in categories showing the highest similarity to a bacterial ORF. The ORFs showing the highest similarity to bacterial ORFs in each category, but not highest similarity in total yeast ORFs, have not been counted. These ORFs may be produced by gene duplication. Gene duplication produces various genes with homologies to the original gene, and belonging to many other functional categories. Therefore, we detected the orthologous ORFs in each functional category and counted their numbers, because the origin of the Fig. 1. Schemes showing the previous and present analyses.

3 Origin of eukaryotic cell nuclei by the symbiosis 371 ORFs in each functional category should be considered independently (Fig. 1b). If symbiosis (Archaeal cell entry into a Bacterial cell) occurred, the origin of these gene categories can be discovered using this method. MATERIALS AND METHODS ORFs from 21 unicellular organisms whose whole genome DNA sequences have been clarified were used: Saccharomyces cerevisiae (Goffeau et al., 1996), eight Archaea (Thermoplasma acidophilum (Ruepp et al., 2000), Halobacterium sp. (Ng et al., 2000), Archaeoglobus fulgidus (Klenk et al., 1997), Pyrococcus horikoshii (Kawarabayasi et al., 1998), Methanococcus jannaschii (Bult et al., 1996), Methanobacterium thermoautotrophicum (Smith et al., 1997), Aeropyrum pernix (Kawarabayasi et al., 1999), and Pyrococcus abyssi (obtained from Centre National de Sequencage, France)), and twelve Bacteria (Pseudomonas aeruginosa (Stover et al., 2000), Vibrio cholerae (Heidelberg et al., 2000), Xylella fastidiosa (Simpson et al., 2000), Escherichia coli (Blattner et al., 1997), Haemophilus influenzae (Fleischmann et al., 1995), Helicobacter pylori (Tomb et al., 1997), Aquifex aeolicus (Deckert et al., 1998), Thermotoga maritima (Nelson et al., 1999), Bacillus subtilis (Kunst et al., 1997), Mycobacterium tuberculosis (Cole et al., 1998), Synechocystis sp. strain PCC6803 (Kaneko et al., 1996), and Deinococcus radiodurans (White et al., 1999). Bacteria that are cell parasites seem to have lost genes, and to possess a biased gene composition. Therefore, parasitic bacteria were not included in this analysis. At least 50 yeast ORFs, classified into 44 categories (or sub-categories) according to their functions, were used (MIPS Saccharomyces cerevisiae-functional Catalogue Reference. National Research Center for Environment and Health, Germany, Table 1). Analyses were carried out on these categories (or sub-categories), excluding two sub-categories, mitochondrial organization and transport. Also, ORFs noted as mitochondrial, as well as those related to the tricarboxylic-acid pathway or respiration were separated from the 44 categories (or sub-categories). Then, we combined the separated ORFs to create a new category, mitochondria-related ORFs. All ORF data translated to amino acid sequence (written as ORF below) were used. In order to detect orthologous ORFs, we did following steps. 1) Gapped BLAST (Altschul et al., 1997) was used to detect yeast ORFs in each category with the highest degree of similarity to the bacterial ORFs in each bacterium. 2) Conversely, bacterial ORFs in each bacterium with the highest degree of similarity to the yeast ORFs in each category were also detected. 3) ORF pairs with the highest degree of similarity to each other were computed. In each functional category (or sub-category), the yeast ORFs of these pairs were counted as hit numbers in the category (or sub-category) to the ORFs of each bacterium. We choose E-value as the measure of similarity, because it is possible to evaluate many important points, for example, (1) identity of sequences, (2) length of matching range, (3) characters of two different amino acids in corresponding sites. We counted orthologous ORFs at each threshold (E-value). The thresholds were set at intervals of 5 from 5 to 185 as -loge. ORF hit numbers at each E-value from 5 to 185 as -loge were calculated for each bacterium. One-sided t-tests at the 5% significance level at each E-value were carried out for Archaea and Bacteria in the range that satisfied the criterion of showing a hit number of 5 or more in (at least one) bacterium. Furthermore, in order to examine (1) whether the Z i values in the first t-test were significantly greater than t na+nb-2(0. 10) (=1.734) or less than - t na+nb-2 (0. 10) (= 1.734), or did not show these tendencies, a second t-test was carried out (Horiike et al., 2001). When the ratio Z (2) /t n-1 (0.10) was greater than one and the mean of the Z (1) i values was positive, the result was judged to show homology of a yeast ORF group to be higher to Archaeal ORFs than to Bacterial ORFs. In contrast, when the ratio was above one and the mean of the Z (1) i values was negative, the homology of a yeast ORF group to Bacterial ORFs was judged to be higher than to Archaeal ORFs. RESULTS As shown in Fig. 2 (a, b), specific patterns appeared in the yeast ORF groups classified according to their functions. Using one-sided t-tests at the 5% significance level, a Z (1) i value over indicates that significantly greater hit numbers are to Archaeal ORFs than to Bacterial ORFs, and a Z (1) i value less than indicates that significantly more hit numbers are to Bacterial than to (1) Archaeal ORFs. Fig. 2a shows the hit number and Z i values at each E-value for yeast ORFs involved in DNA repair. According to the result of the primary t-test, the similarity of this yeast ORF group to Archaeal ORFs is revealed. On the other hand, yeast ORFs involved in cytoplasm organization show similarity to Bacterial ORFs (Fig. 2b). The results of t-tests for each yeast ORF group classified by category and sub-category are summarized in Table 1. According to the secondary t-test, shown in the last column, yeast ORF groups that show homology to Archaeal ORFs are indicated by A, while those that show homology to Bacterial ORFs are indicated by B. DISCUSSION In this analysis, we counted the numbers of orthologous ORFs between individual yeast functional categories and individual bacteria. By this method, it was possible to detect the correct origin of functional categories in

4 372 T. HORIIKE et al. Table 1. Results of the homology-hit analysis. Yeast ORFs in each group, classified by category and sub-category, were obtained from the MIPS Saccharomyces cerevisiae-functional Catalogue. The decision about origin as Archaeal (A) or Bacterial (B), indicated in the last column, was made as follows. A: Mean of Z (1) i values is positive and the ratio Z (2) /t n-1 (0.1) is greater than one. B: The mean of the Z (1) i values is negative and the ratio Z (2) /t n-1 (0.1) is greater than one. ---: The hit number of ORFs is not enough for the criterion. ORFs noted as mitochondrial were removed from the 44 categories (or sub-categories). A* and B* indicate categories newly determined by the present analysis. Categories Sub-categories Number of ORF (1) Mean of Z i Z (2) /t n-1 (0.1) Decision METABOLISM amino-acid metabolism B nitrogen and sulphur metabolism B nucleotide metabolism B C-compound and carbohydrate metabolism B lipid, fatty-acid and isoprenoid metabolism B* metabolism of vitamins, cofactors, and prosthetic groups B ENERGY B CELL GROWTH, CELL DIVISION cell growth AND DNA SYNTHESIS budding, cell polarity pheromone response, mating-type determination, sex-specific proteins sporulation and germination meiosis DNA synthesis and replication A recombination and DNA repair cell cycle control and mitosis A TRANSCRIPTION rrna transcription A trna transcription mrna transcrition A other transcription activities PROTEIN SYNTHESIS ribosomal proteins A translation (initiation, elongation and termination) A PROTEIN DESTINATION protein folding and stabilization B* protein targeting, sorting and translocation protein modification assembly of protein complexes A* proteolysis TRANSPORT FACILITATION INTRACELLULAR TRANSPORT vesicular transport vacuolar transport cellular import nuclear transport CELLULAR BIOGENESIS SIGNAL TRANSDUCTION B* CELL RESCUE, DEFENSE, stress response B CELL DEATH AND AGEING DNA repair A* detoxificaton B IONIC HOMEOSTASIS B CELLULAR ORGANIZATION organization of plasma membrane B* organization of cytoplasm B* organization of cytoskeleton organization of endoplasmic reticulum A organization of golgi nuclear organization A vacuolar and lysosomal organization MITOCHONDRIA-RELATED ORFS B

5 Origin of eukaryotic cell nuclei by the symbiosis 373 Fig. 2. Hit numbers of yeast ORFs to various bacterial ORFs and Z (1) i values at each threshold. Hit numbers of yeast ORF groups (sub-category) for DNA repair (a, upper panel), cytoplasm organization (b, upper panel) to the ORFs of each Archaea or Bacteria at each threshold (E-values) are plotted on the vertical axes. E-values as -loge scale are shown on the horizontal axes. Red lines correspond to Archaea: A. pernix (ape, open circles), Halobacterium sp. (Hsp, closed circles), M. thermoautotrophicum (mth, open squares), M. jannaschii (mja, closed squares), A. fulgidus (afu, open triangles), T. acidophilum (tac, closed triangles), P. abyssi (pab, inverse open triangles) and P. horikoshii (pho, inverse closed triangles). Blue lines correspond to Bacteria: A. aeolicus (aae, open circles), T. maritima (tma, closed circles), B. subtilis (bsu, open squares), M. tuberculosis (mtu, closed squares), Synechocystis sp. (ssp, open triangles), E. coli (eco, closed triangles), H. influenzae (hin, open inverse triangles), P. aeruginosa (pae, closed inverse triangles), V. cholerae (vch, open diamonds), X. fastidiosa (xfa, closed diamonds), H. pylori (hpy, open stars) and D. radiodurans (dra, closed stars). Z (1) i values at each E-value for the yeast ORF groups (sub-category) of DNA repair (a, lower panel) and cytoplasm organization (b, lower panel) are shown on the vertical axes. E-values as -loge are shown on the horizontal axes. Z (1) i values are shown in the range of E-value where the hit number was 5 or more in (at least one) bacterium. The double-headed arrow denotes the region in which this criterion was not satisfied. eukaryotic cells. Two categories, assembly of protein complexes and DNA repair, were newly judged to be of Archaeal origin, while five categories, lipid (fatty-acid and isoprenoid) metabolism, protein folding and stabilization, signal transduction, organization of plasma membrane and organization of cytoplasm, were newly judged to be of Bacterial origin. The origins of 18 other categories are the same as determined in the previous analysis. The origins of two categories (meiosis and cellular import, which were concluded to be of Bacterial origin in the previous analysis) were not decided. The reason for this can be explained as follows. Gene duplication produced various genes belonging to many functional categories with homologies to the original gene. The number of such genes originating from Archaea was larger than that originating from Bacteria. In the previous analysis, only one gene with the above history was counted as an orthologous gene. However, in the present analysis, those genes were also counted, resulting in a relative increase in the hit number to Archaea. In this study, the origins of 25 yeast ORF groups as Archaeal or Bacterial were clearly determined (Table 1). As in the previous analysis, categories judged as being of Archaeal origin are related to the nucleus, while those judged to be of Bacterial origin are related to the cytoplasm. Nuclear membrane is not composed of Archaeal lipids. Nuclear membrane has been changed, because environment around nucleus is quite different from that of cell. The membranes of mitochondria and chloroplast also have been specialized due to the role. Endoplasmic reticulum is connected continuously to the nuclear membrane; therefore it is possible to regard endoplasmic reticulum as nuclear related. Ribosomes are on the endoplasmic reticulum and in the cytoplasm. However translation is closely related to transcription. So it is possible to regard translation as nuclear related process. Especially, the organization of plasma membrane was newly judged to be the Bacterial origin, while the origins of two ORF groups, assembly of protein complexes and DNA repair, both of which lie on the information pathway from DNA to protein, were newly judged to be of Archaeal

6 374 T. HORIIKE et al. origin. A mosaic structure of the yeast genome constructed by the ORFs similar to archaeal ORFs or bacterial ORFs was confirmed. There are three possibilities for the appearance of this structure: gene transfer by mitochondrial symbiosis (Rivera et al., 1998; Ribeiro and Golding, 1998; Vellai T and Vida G, 1999), the accumulation of smallscale lateral gene transfer (Jain et al., 1998) and massive gene transfer by nuclear symbiosis (Lake, 1988; Lake and Rivera, 1994; Gupta et al., 1994; Moreira and Lopez-Garcia, 1998; Horiike et al., 2001). The ORFs related to the mitochondria were removed and analyzed separately. Although, hidden mitochondrial genes may remain (could not be excluded), their influence on the analysis may be negligible, because the proportion of mitochondrial genes to other genes is small. If only lateral gene transfer was responsible for the mosaic structure, then many ORFs must have been transferred from a specific bacterium (Archaea or Bacteria) to a specific functional category of yeast. However, the replacement of many genes in specific gene categories by lateral transfer is unlikely, because lateral gene transfer to genomic sequence of Eukaryote is very rare (Kidwell, 1993; Andrade et al., 1999). If the symbiosis of Archaea into Bacteria ocurred, many genes with same functions should be duplicated (derived from Archaea and Bacteria). This lead to the following three considerations: 1) It is difficult to replace one gene for a protein that is involved in a multi-complex structure with other proteins. 2) The syntheses of many proteins are regulated as a unit of each functional group comprising many proteins. 3) Many genes form an operon for several enzymes in a metabolic pathway and are expressed in a cascade fashion. These situations, formation of complex protein structures and the regulation of protein synthesis may have resulted in the discarding of genes in a category originating from Archaea or Bacteria as a unit, leading to the formation of a mosaic genome structure in the yeast. In addition to the judgment made for the functional category organization of plasma membrane as Bacterial origin to the above background, the possibility of symbiosis is the highest. Furthermore, translation within nuclei of mammalian cell was discovered (Iborra et al., 2001). It also supports the possibility Fig. 3. Schematic figure of a eukaryotic cell showing the origin of each sub-cellular organ and function as determined by Homology- Hit Analysis. Yeast ORF groups originating from Archaeal ORFs or Bacterial ORFs are shown in red or blue, respectively. Mitochondria-related ORFs (originating from Bacterial ORFs) are shown in green.

7 Origin of eukaryotic cell nuclei by the symbiosis 375 of symbiosis. Archaea might become Bacterial parasites and utilized Bacterial metabolites. In the meantime, Archaea discarded their original genes related to metabolism and became cell nuclei. This scheme is outlined in Fig. 3. The analysis was carried out on the assumption that the evolutionary rate of genes for time is constant. If many genes have serious variation in the rate of evolution, then our interpretation of the results is mistaken. However, the results are consistent with the phylogenic trees of many genes. For example, the genes for DNA replication, transcription and translation are shown to have homology to Archaea, while those for cytoplasm (homeostasis) have homology to Bacteria. Therefore, the influence of variation of evolutionary rate is small and negligible. 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