11 clear water bay, hong kong, china - biorxiv · 11 clear water bay, hong kong, china 12 13 14...

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Title: 1

Descent of Bacteria and Eukarya from an archaeal root of life 2

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Authors: 5

Xi Long, Hong Xue and J. Tze-Fei Wong* 6

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Affiliation: 9

Division of Life Science, Hong Kong University of Science and Technology, 10

Clear Water Bay, Hong Kong, China 11

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*Corresponding author: 14

Email: [email protected] 15

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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 23, 2019. ; https://doi.org/10.1101/745372doi: bioRxiv preprint

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Abstract 17

The three biological domains delineated based on SSU rRNAs are confronted by 18

uncertainties regarding the relationship between Archaea and Bacteria, and the origin of 19

Eukarya. Herein the homologies between the paralogous valyl-tRNA and isoleucyl-tRNA 20

synthetases in a wide spectrum of species revealed vertical gene transmission from an 21

archaeal root of life through a Primitive Archaea Cluster to an Ancestral Bacteria Cluster of 22

species. The higher homologies of the ribosomal proteins (rProts) of eukaryotic Giardia 23

toward archaeal relative to bacterial rProts established that an archaeal-parent rather than a 24

bacterial-parent underwent genome merger with an alphaproteobacterium to generate 25

Eukarya. Moreover, based on the top-ranked homology of the proteins of Aciduliprofundum 26

among archaea toward the Giardia and Trichomonas proteomes and the pyruvate phosphate 27

dikinase of Giardia, together with their active acquisition of exogenous bacterial genes 28

plausibly through foodchain gene adoption, the Aciduliprofundum archaea were identified as 29

leading candidates for the archaeal-parent of Eukarya. 30

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Molecular evolution analysis of small subunit ribosomal RNA (SSU rRNA) yielded a 32

universal but unrooted tree of life (ToL) that comprises the three biological domains of 33

Archaea, Bacteria and Eukarya1. A ToL of transfer RNAs based on the genetic distances 34

between the 20 classes of tRNA acceptors for different amino acids located its root near the 35

hyperthermophilic archaeal methanogen Methanopyrus (Mka)2. Although this rooting is 36

supported by a wide range of evidence3-9, and the age of ~2.7 Gya for the Methanopyrus 37

lineage as the oldest among living organisms10, the phylogenies of the three biological 38

domains are beset by two fundamental problems: viz. the uncertain relationship between 39

Archaea and Bacteria, and the identity of the prokaryotic-parent that underwent genome 40

merger with an alphaproteobacterium to give rise to Eukarya. As long as these two problems 41

remain unresolved, the nature of the root of life is open to diverse formulations11-15. 42

Accordingly, the objective of the present study is to examine the pathways of descent of 43

Bacteria and Eukarya from an archaeal root of life, and the nature of the archaeal-parent of 44

Eukarya. 45

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The antiquity of proteins could be assessed based on the increasing divergence of paralogous 47

proteins in time16. Applying this approach, BLASTP was performed between the intraspecies 48

valyl-tRNA synthetase (VARS) and isoleucyl-tRNA synthetase (IARS) in the genomic 49

sequences for 5,398 species in NCBI Genbank. Arrangement of the BLASTP bitscores 50

obtained in descending order (Supplementary Table S1 and partly in Fig. 1) showed that the 51

119 highest scoring species were all archaeons, topped by Mka and including Mfe, Afu, Mnt 52

and Mja with bitscores of 473, 436, 387, 387 and 387 respectively. The top scoring bacterium 53

was the Clostridium Mau with a bit score of 378, and the top scoring eukaryote was the 54

filamentous brown alga Esi with a bit score of 240. These results established the foremost 55

antiquity of Mka among extant organisms. 56


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The positions of some of the species analyzed in Fig. 1 were indicated on the SSU rRNA tree, 58

with their intraspecies VARS-IARS bitscores expressed in circles colored according to the 59

thermal scale (Fig. 2a). There was a concentration of euryarchaeons with high VARS-IARS 60

homology in a ‘Primitive Archaea Cluster’ spread between Hal and Mfe. In the Bacteria 61

domain, there was likewise an ‘Ancestral Bacteria Cluster’ with high VARS-IARS homology 62

spread between Det and Mau. The deepest branching species in the Bacteria domain were 63

two members of the Aquificae phylum, viz. the anaerobic Det with high VARS-IARS 64

homology, and the microaerobic, low-homology Aae. Since mutations could cause loss of 65

homology more easily than gain of homology, this suggests that Aae has evolved far from the 66

ancestral Aquificiae species possibly as part of the wave of tumultuous changes undergone by 67

former anaerobes in response to the appearance of atmospheric oxygen17, thereby sustaining 68

extensive evolutionary erosion of its VARS-IARS homology. The enhanced resistance of 69

paralogue homology to perturbation by horizontal gene transfer (HGT), due to the difficulty 70

of transfer of a pair of proteins compared to transfer of a single protein, was illustrated by the 71

preservation of low VARS-IARS bitscores in the proteobacterial region of the tree against 72

any shift toward elevated VARS-IARS homology on account of HGT events, despite the high 73

HGT-susceptibility of for example Eco, which acquired 18% of its genes through HGT 74

subsequent to its departure from Salmonella enterica about 100 million years ago18. 75

Previously, based on the intraspecies alloacceptor tRNA-distances of various species on the 76

tRNA tree, LUCA was positioned between the branches leading to Mka and Ape at a distance 77

ratio of 1.00 from the Mka branch versus 1.14 from the Ape branch2, and this position was 78

adopted in the SSU rRNA tree in Fig. 2. 79

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Given the relative paucity of HGT effects on VARS-IARS homology in a majority if not all 81


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of the species on the SSU rRNA tree, the parallel prominence of the high VARS-IARS 82

homology species in the Primitive Archaea Cluster and the Ancestral Bacteria Cluster was 83

explicable by vertical genetic transmission of the VARS and IARS genes from an 84

Mka-proximal root of life to the archaeal cluster, and in turn to the bacterial cluster. Since the 85

top ranked bacterial bitscore of Mau at 378 was between that of Mac at 382 and Pfu at 369, 86

the results suggest that the Ancestral Bacteria Cluster branched off from the Primitive 87

Archaea Cluster close to the Mka-proximal root of Archaea. The medium VARS-IARS 88

bitscores of Esi, Tps, Bpr and Cme among the Eukarya (Fig. 2a) were indicative of the 89

extension of the intraspecies VARS-IARS homology into this domain. The much higher 90

VARS homologies (colored squares) and IARS homologies (colored triangles) between 91

bacterial species and the eukaryote Gla compared to those between archaeal species and Gla 92

indicated that Eukarya received the VARS and IARS genes from the Bacteria instead of the 93

Archaea domain (Fig. 2b). 94

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The aligned segments of VARS and IARS from the archaeon Mka, the bacterium Mau and 96

the eukaryote Esi in Fig. 3 were portions of the six complete sequences (Supplementary Fig. 97

S1). These segments showed 42/207 columns where all six sequences carried the same amino 98

acid, thereby providing strong evidence for the vertical transmission of VARS and IARS 99

genes from Archaea to Bacteria and Eukarya. 100

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It has been suggested that an endosymbiotic event between an archaeal-parent and an 102

alphaproteobacterium acting as mitochondrion-parent led to the formation of the Last 103

Eukaryotic Common Ancestor (LECA) and ushered in the Eukarya domain19-21. Proposals 104

regarding the identity of the archaeal-parent have focused on a range of prokaryotes including 105

Thermoplasmatales where the lack of a rigid cell wall could facilitate its engulfment of the 106


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mitochondrion-parent to bring about endosymbiosis22-24; and various archaeons, especially 107

the Asgard archaeons Lok and Tho25,26, that are enriched in eukaryotic signature proteins 108

(ESPs)27. There is no consensus regarding a choice between these two groups of organisms28. 109

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Upon BLASTP comparisons of the fifty-six ribosomal proteins (rProts) of Gla, the lowest 111

branching eukaryote on the SSU rRNA tree, with corresponding prokaryotic rProts, 112

fifty-three of them yielded higher bitscores with archaeons relative to bacteria (Fig. 4a), 113

indicating that eukaryogenesis began with an archaeal-parent instead of a bacterial-parent. 114

S21e and L36e yielded no bitscore with any archeal or bacterial rProt, suggesting that they 115

were derived from a prokaryote not surveyed in the present study, altered beyond recognition 116

by BLASTP, or invented by the eukaryogenesis system. The S7e of eleven eukaryotes from 117

Table 1 although not that of Gla showed detectable homology toward the archaeon Alt. The 118

Sce, Esi and Hsa rProts differed from those of Gla in two aspects: about one-sixth of the 119

rProts that showed higher homology toward archaea relative to bacteria in Gla switched to 120

lower homology toward archaea relative to bacteria in Sce, Esi and Hsa; and there were also 121

some additional rProts in Sce, Esi and Hsa, mainly bacteria-derived ones, not found in Gla 122

(Supplementary Fig. S2). These findings pointed to a significant influx of bacterial rProts into 123

Sce, Esi and Hsa after their divergence from Gla, resulting in the replacement of some of the 124

archaea-derived rProts found in Gla by bacteria-derived ones. Acf, Abo and Mac displayed 125

the highest BLASTP bitscores among archaeons toward Gla pertaining to pyruvate phosphate 126

dikinase (EC2.7.9.1), which would be consistent with a possible role of these three archaeons 127

as archaeal-parent. Interestingly, the bitscores were high for Hei and Tho but low for Odi and 128

Lok among the Asgard archaea, and high for Tac and Tvo but low for Fac, Min and Mte 129

among the Thermoplasmatales (Fig. 4b). 130

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Figure 4c shows the prokaryotic distribution of homologues of some the 162 Gla proteins that 132

were ESPs or homologous toward a limited number of prokaryotes (Supplementary Table S2). 133

Tho, Odi, Xca and Lok, the four prokaryotes endowed with the largest numbers of the 134

Gla-homologous proteins, harbored only 26, 19, 17 and 16 of these Gla-homologous proteins 135

respectively (Supplementary Table S3), and the four Asgard archaeons also did not fully 136

share their Gla-like proteins with one another via HGT, thus underlining the difficulty for any 137

one archaeon or bacterium to accumulate a sufficient number of eukaryote-type proteins to 138

launch the Eukarya domain relying only on their own inventiveness and HGT. On the other 139

hand, one or more potential prokaryotic sources were found for each of the Gla-homologous 140

proteins despite the survey of only a small spectrum of prokaryotes, indicating that the 141

obstacle to eukaryogenesis posed by gene deficiency may be overcome if some dependable 142

mechanism were available to assemble the requisite genes from a broad spectrum of 143

prokaryotes. Addressing the inadequacy of ESP coverage by single archaeal species29,30, it 144

was suggested that HGTs, or development of phagocytosis by an ESP-rich archaeon might 145

provide a solution26,31. However, the frequencies of HGTs might be a limiting factor10,32, and 146

rProts could be particularly resistant to HGT33. 147

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During eukaryogenesis, the archaeal-parent might join up with the mitochondrion-parent to 149

develop directly into LECA in a mitochondria-early scenario, or it might serve as First 150

Eukaryotic Common Ancestor (FECA), and go through successive generations of genomic 151

expansion prior to merger with the mitochondrion-parent to form LECA in a 152

mitochondria-late scenario34. By measuring the phylogenetic distances between different 153

components of LECA and their closest prokaryotic relatives, evidence has been obtained in 154

support of a mitochondria-late time table, with the appearance of nucleolus preceding that of 155

nucleus, endomembrane system and finally mitochondria35. Previously, the proteins of the 156


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eukaryote Sce were observed to contain a substantial variety of bacterial proteins and also 157

some archaeal ones, and it was pointed out that the influx or bacterial genes into Sce could 158

not be explained by a merger between archaeal-parent with another bacterium besides the 159

mitochondrion-parent, or by Sce uptake of bacterial genes through ingestion of bacteria as 160

food. Instead, the mitochondrion-parent was a major source of the exogenous bacterial 161

proteins in Sce20. When the eukaryotic Gla and Trv proteomes were employed as homology 162

probes for BLASTP query against various prokaryotic proteomes, it gave rise to so many hits 163

with both archaea and bacteria (Supplementary Table S4) that the influx of both archaeal and 164

bacterial genes into the eukaryogenesis system had to be mediated by some highly efficient 165

mechanism; and the similar prokaryotic-homology spectra for Gla and Trv (Fig. 5a) suggest 166

that a majority of the prokaryotic genes in these two eukaryotic genomes entered the 167

eukaryogenic lineage prior to the divergence between Gla and Trv. In fact, archaea have long 168

relied on bacteria as a source of genetic diversity, and there was precedent of influx of 169

bacterial genes being a determinant of archaeal phylogenies: a large number of bacterial 170

genes entered into the methanogen that begot the haloarchael archaeons36,37. In view of this, 171

an influx of beneficial prokaryotic genes into the archaeal-parent lineage likely began prior to 172

the FECA stage and continued through LECA to the early eukaryotes as illustrated by the 173

entry of bacterial rProt genes into Sce, Esi and Hsa (Supplementary Fig. S2). 174

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Among 46 archael proteomes analyzed, the Abo and Acf proteomes displayed the highest 176

average homology bitscores toward the eukaryotic proteomes of both Gla and Trv (Fig. 5b, 177

Supplementary Fig. S3 and Supplementary Table S4), which suggests that these archaeons 178

could be candidate archaeal-parents. Once a bacterial protein entered into the 179

FECA-eukaryote lineage, its bacterial and eukaryotic versions became segregated 180

locationwise, and evolved independently. The divergence between the two versions would 181


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thus increase with time, as in the case of functional paralogues such as VARS and IARS. 182

Accordingly, the bitscores of Ctr and Tpa proteins were high toward Gla and Trv likely 183

because they were taken up late by the eukaryogenic lineage, whereas the bitscores of Mpn 184

and Aae were low likely because they were taken up early (Fig. 5b and Supplementary Fig. 185

S3). Moreover, when the 46 archaeons were compared regarding their ability to import 186

bacterial genes into their own genomes, several archaeons with relatively large proteomes, 187

viz. Hla, Hgi and Mac (with 3,704 to 4,469 protein genes), as well as Lok (5,378 protein 188

genes) and Pfu (2,053 protein genes), displayed distinctive homologies toward bacteria (Fig. 189

5c left panel). However, when the individual archaeal bitscores were normalized with respect 190

to the protein gene numbers of the archaeons, the normalized bitscores of the small-proteome 191

Abo-group of seven euryarchaeons comprising Abo, Acf, Mte, Tvo and Tac (each with 192

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they took as food41. To perform FGA, Abo would employ its array of thirty proteases to 207

digest away the proteins of dead prokaryotes to prepare their naked DNA, import it through 208

the Beveridge bridal S-layer of its cell surface (S-layer lattices are known to house regular 209

channels of 2-6 nm diameter42) for implantation into its own genome. In using proteases to 210

purify DNA for cloning, Abo predates by eons the same usage by modern genetic 211

engineering. The S-layer of Abo is highly flexible and can be bent to form small blebbing 212

vesicles with sharp curvature, indicating that the bonding forces between the S-layer subunits 213

are unusually weak or transient. These vesicles can bud off and anneal with other cells43. 214

Pseudomonas aeruginosa also releases comparable membrane vesicles that contain the 215

pseudomonas quinoline signal for cell-cell communication and group behavior44; and 216

Sulfolobus islandicus produces cell-derived S-layer coated spherical membrane vesicles of 217

90-180 nm diameter45. Importantly, such flexibility of the Abo cell surface could facilitate the 218

formation of eukaryotic endomembrane and the prerequisite phagocytosis machinery for 219

eukaryogenesis31,46,47. Furthermore, while all prokaryotic cells evolve on the basis of 220

nucleotidyl mutations through the replacement, addition and subtraction of nucleotides, FGA 221

gene uptake would enable the cells to evolve also on the basis of gene-content mutations 222

through the replacement, addition and subtraction of genes, or gene clusters, expediting 223

eukaryogenesis by orders of magnitude. In the example of Tac, it has succeeded in acquiring 224

gene clusters from other organisms for rProts, NADH dehydrogenase, precorrin biosynthesis, 225

flagellar proteins and a protein degradation pathway, amounting to 32% of its total open 226

reading frames plausibly via FGA40. Besides, the blebbing vesicles of Abo and Acf could 227

mediate gene exchanges between cells engaged in eukaryogenesis, thus further facilitating the 228

process. Therefore, based on the highest archaeal BLASTP bitscores of Abo and Acf toward 229

Gla pertaining to pyruvate phosphate dikinase (Fig. 4b), their highest average archaeal 230

bitscores toward Gla and Trv proteomes (Fig. 5b and Supplementary Fig. S3) and highest 231


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archaeal normalized bitscores toward bacteria (Fig. 5c), blebbing membrane vesicles, and 232

possession of nine out of ten glycolytic enzymes needed for metabolic cooperation with 233

mitochondrial respiration, these Aciduliprofundum archaea represent exceptionally 234

advantaged candidates for the archaeal-parent role. They even share with the deep branching 235

eukaryote Gla, the rProts of which have remained more archaeal than those of Sce, Esi and 236

Hsa, the scavenger life style. Between Acf and Abo, the catalase/peroxidase HP1-encoding 237

facultatively anaerobic Acf might be more resistant to oxidative damage than Abo during 238

merger with an alphaproteobacterium, and could hunt a wide range of ecological niches for 239

beneficial food species. The bitscore 936 of Acf toward Gla with respect to pyruvate 240

phosphate dikinase was also slightly higher than the 928 bitscore of Abo. The Asgard 241

archaeons have been highly regarded as candidate archaeal-parents on the strength of their 242

important ESP genes, which could render any one of them a valuable food species as well for 243

the archaeal-parent. Moreover, the new cultivatable Asgard Candidatus Prometheoarchaeum 244

syntrophicum MK-D148,49 can degrade amino acids through syntrophy, and display wisp-like 245

membrane protrusions indicative of flexible cell surface and possible FGA activity in keeping 246

with the significant albeit modest bitscores between Lok and the bacterial species Bja and Tht, 247

and between Odi and several bacterial species, in Fig. 5c left and right panels respectively. 248

249

Based on the BLASTP bitscores between the proteins of various prokaryotes and the 250

mitochondrial gene-encoded proteins in four eukaryotes (Supplementary Table S5), the 251

alphaproteobacteria Haematobacter, Chelativorans and Tateyamaria were closest to the 252

lineage of the mitochondrion-parent (Fig. 5d). 253

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In conclusion, in the present study, Methanopyrus kandleri was found to be the top-ranked 255

organism in VARS-IARS homology among 5,398 species from all three biological domains, 256


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and therefore close to the root of life. Moreover, the high VARS-IARS homologies in the 257

Primitive Archaea Cluster and in the Ancestral Bacteria Cluster delineated a pathway of 258

descent of Bacteria from Archaea that diverged early from Archaea to form the Bacteria 259

domain. The preeminent homology between the Gla rProts and archaeal rProts established 260

that the prokaryote-parent of Eukarya that entered into genome merger with an 261

alphaproteobacterial mitochondrion-parent was an archaeal-parent. The archaeal-parent was 262

suggested to a scavenger archaeon such as the Aciduliprofundum archaea, capable of 263

generating a chimeric eukaryote through large scale foodchain gene adoption. 264

Notwithstanding such elaborate phylogenetic developments, the asterisked columns in Fig. 3, 265

where all six aligned protein sequences showed the same Val or Leu residue despite the ease 266

with which Val, Leu and Ile can be interchanged in evolution, represented a level of protein 267

sequence conservation across two different proteins, three biological domains, and two giga 268

year-plus time span that required the vertical genetic descent of Bacteria and Eukarya from an 269

archaeal root of life. 270

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43. Reysenbach, A. L. & Flores, G. E. Electron microscopy encounters with unusual 372


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thermophiles helps direct genomic analysis of Aciduliprofundum boonei. Geobiology 6, 373

331-336 (2008). 374

44. Mashburn, L. M. & Whiteley, M. Membrane vesicles traffic signals and facilitate group 375

activities in a prokaryote. Nature 437, 422-425 (2005). 376

45. Prangishvili, D. et al. Sulfolobicins, specific proteinaceous toxins produced by strains of 377

the extremely thermophilic archaeal genus Sulfolobus. J. Bacteriol. 182, 2985-2988 378

(2000). 379

46. Poole, A. M. & Gribaldo, S. Eukaryotic origins: how and when was the mitochondrion 380

acquired? Cold Spring Harb. Perspect. Biol. 6, a015990; 10.1101/cshperspect.a015990 381

(2014). 382

47. McInerney, J., Pisani, D. & O'Connell, M. J. The ring of life hypothesis for eukaryote 383

origins is supported by multiple kinds of data. Philos. Trans. Royal Soc. B 370, 384

20140323; 10.1098/rstb.2014.0323 (2015). 385

48. Imachi, H. et al. Isolation of an archaeon at the prokaryote-eukaryote interface. 386

Preprint at https://doi.org/10.1101/726976 (2019). 387

49. Lambert, J. Scientists glimpse oddball microbe that could explain rise of complex life. 388

Nature 572, 294 (2019). 389

50. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence 390

alignments using Clustal Omega. Mol. Syst. Biol. 7, 539; 10.1038/msb.2011.75 (2011). 391

392


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Supplementary Information is available online. 393

394

Author Contribution 395

J.T.W. and H.X. conceived the study; X.L. collected the data and performed computational analysis; 396

and J.T.W., H.X. and X.L. wrote the paper. All authors read and approved the final manuscript. 397

398

Acknowledgments 399

This study was supported by University Grants Council of Hong Kong SAR (ITS/113/15FP), 400

and X.L. was recipient of a Hong Kong Government Ph. D. Fellowship. 401

402

Author information 403

The authors declare that they have no competing interests. Correspondence and material 404

requests should be addressed to T.F.W. ([email protected]). 405

406

Data availability 407

All data supporting the findings of this study are available within the paper and its 408

Supplementary Information files. 409

410

Methods 411

Source of data and materials 412

The protein and SSU rRNA sequences analyzed in the present study were retrieved from the 413

NCBI GenBank release 231 (ftp://ftp.ncbi.nlm.nih.gov/genomes/)51,52. For species without 414

available SSU rRNA information in NCBI, quality checked SSU rRNA sequences were 415

downloaded from the SILVA database release 132 (https://www.arb-silva.de/)53. For species 416

with multiple SSU rRNA sequences, the one yielding the highest total bitscore (using 417


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BLASTN54 with ‘-word_size’ flag set to 4) with SSU rRNAs of other species from the same 418

domain was employed for analysis. The alignment of the 83 SSU rRNA sequences used to 419

build the SSU rRNA tree was given in Supplementary File S1. Eukaryotic mitochondrial 420

gene-encoded protein sequences were retrieved from NCBI Protein database 421

(https://www.ncbi.nlm.nih.gov/protein) by searching species name of the eukaryote of 422

interest and setting the ‘Genetic compartments’ filter as ‘Mitochondrion’. 423

424

Estimation of nuclear or mitochondrial proteome homology 425

When the proteomes of two species were compared using BLASTP54 (with ‘-evalue’ flag 426

setting to 0.05), query and subject sequences that were the only best match of each other, viz. 427

query n has the highest bitscore with subject m, and subject m has the highest bitscore with 428

query n at the same time, were considered in calculating proteome similarity. 429

430

Estimation of protein family homology 431

Protein sequence similarity was estimated by the maximum bitscore of each pair of sequences 432

yielded by BLASTP54 with all default parameters. To estimate ribosomal protein similarities 433

among eukaryotes and prokaryotes, all seed sequences of 80 ribosomal protein families 434

(Supplementary Table S6) retrieved from Pfam database55 were blasted (with ‘-evalue’ flag 435

set to 0.05) against the proteomes of 21 eukaryotes, 46 archaea and 36 bacteria respectively. 436

For every ribosomal protein family, only one protein sequence of each species yielding the 437

highest bitscore with the seed sequences were selected for further analysis. To remove 438

false-positive sequences, the selected sequences were submitted to NCBI Batch CD-search 439

Tool56 to search against the Pfam database, and sequences that failed to map to the same 440

ribosomal protein family were removed. Finally, the eukaryotic ribosomal protein sequences 441

were blasted to all the prokaryotic ribosomal protein sequences, and the maximum bitscores 442


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are shown in Fig. 4a. 443

444

To determine non-rProt families with sequence homology between Gla and prokaryotes, the 445

Gla protein sequence in each instance was blasted against each of the 82 prokaryotic 446

proteomes, and the best matches were submitted to NCBI Batch CD-search Tool to search 447

against the Pfam database. Only cases where both query and subject sequences belonged to 448

the same protein family were kept, and 162 of these cases were shown in Supplementary 449

Table S2, and in part in Fig. 4c. 450

451

51. Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Sayers, E. W. GenBank. 452

Nucleic Acids Res. 44, D67-72 (2016). 453

52. O'Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, 454

taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733-745 455

(2016). 456

53. Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data 457

processing and web-based tools. Nucleic Acids Res. 41, D590-596 (2013). 458

54. Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinform. 10, 421; 459

10.1186/1471-2105-10-421 (2009). 460

55. El-Gebali, S. et al. The Pfam protein families database in 2019. Nucleic Acids Res. 47, 461

D427-D432 (2019). 462

56. Marchler-Bauer, A. & Bryant, S. H. CD-Search: protein domain annotations on the fly. 463

Nucleic Acids Res. 32, W327-331 (2004). 464

465

466


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Table 1. Partial list of species analyzed. 467Abbr. Species name Abbr. Species name Archaea Bacteria (Continued) Abo Aciduliprofundum boonei Cex Caldisericum exile Acf Aciduliprofundum sp. MAR08-339 Cje Campylobacter jejuni Aen C.Aenigmarchaeota archaeon Cpo Cloacibacillus porcorum Afu Archaeoglobus fulgidus Ctr Chlamydia trachomatis Aia Acidilobus sp. 7A Cvi Caulobacter vibrioides Alt C.Altiarchaeales archaeon Cvo Chelativorans sp. BNC1 Ape Aeropyrum pernix Det Desulfurobacterium thermolithotrophum Bat C.Bathyarchaeota archaeon Dra Deinococcus radiodurans Csu C.Caldiarchaeum subterraneum Dth Dictyoglomus thermophilum Csy Cenarchaeum symbiosum Eco Escherichia coli Dia C.Diapherotrites archaeon Hth Hungateiclostridium thermocellum Fac Ferroplasma acidiphilum Kol Kosmotoga olearia Ffo Fervidicoccus fontis Mau Mahella australiensis Hal Halobacterium salinarum Mhy Megamonas hypermegale Hei C.Heimdallarchaeota archaeon Mpn Mycoplasma pneumoniae Hgi Haloferax gibbonsii Mtu Mycobacterium tuberculosis Hla Halobiforma lacisalsi Pel Pelobacter sp. SFB93 Kcr C.Korarchaeum cryptofilum Pmo Petrotoga mobilis Lok C.Lokiarchaeota archaeon Rpr Rickettsia prowazekii Mac Methanosarcina acetivorans Rru Rhodospirillum rubrum Man C.Mancarchaeum acidiphilum Rso Ralstonia solanacearum Mar C.Marsarchaeota G2 archaeon Spn Streptococcus pneumoniae Mbo Methanoregula boonei Ssp Sporanaerobacter sp. NJN-17 Mco Methanocella conradii Syn Synechocystis sp. PCC 6803 Met C.Methanosuratus sp. Tht Thermobaculum terrenum Mfe Methanothermus fervidus Tis Tistrella mobilis Mic C.Micrarchaeota archaeon Tma Thermotoga maritima Min C.Methanomassiliicoccus intestinalis Tpa Treponema pallidum Mja Methanocaldococcus jannaschii Tte Caldanaerobacter subterraneus Mka Methanopyrus kandleri Xca Xanthomonas campestris Mlt C.Methanoliparum thermophilum Eukarya Mnt Methanonatronarchaeum thermophilum Aca Acanthamoeba castellanii Mph Methanophagales archaeon Bbo Babesia bovis Mte C.Methanoplasma termitum Bho Blastocystis hominis Nca C.Nitrosocaldus cavascurensis Bpr Bathycoccus prasinos Nga C.Nitrososphaera gargensis Cel Caenorhabditis elegans Nko C.Nitrosopumilus koreensis Cme Cyanidioschyzon merolae Nst C.Nanobsidianus stetteri Dme Drosophila melanogaster Odi C.Odinarchaeota archaeon Dre Danio rerio Pae Pyrobaculum aerophilum Esi Ectocarpus siliculosus Pfu Pyrococcus furiosus Gla Giardia intestinalis Sso Saccharolobus solfataricus Hsa Homo sapiens Tac Thermoplasma acidophilum Lma Leishmania major Tho C.Thorarchaeota archaeon Pfa Plasmodium falciparum Tvo Thermoplasma volcanium Pma Perkinsus marinus Woa C.Woesearchaeota archaeon Sce Saccharomyces cerevisiae Bacteria Spa Saprolegnia parasitica Aae Aquifex aeolicus Spo Schizosaccharomyces pombe Atu Agrobacterium tumefaciens Sra Strongyloides ratti Bap Buchnera aphidicola Tps Thalassiosira pseudonana Bja Bradyrhizobium japonicum Ttr Thecamonas trahens Blo Bifidobacterium longum Trv Trichomonas vaginalis Bsu Bacillus subtilis

Note: C. in front of species name stands for Candidatus. Detailed species information was 468

given in Supplementary Table S1. 469


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470

471

Figure 1. Ranking of bitcores for the VARS-IARS homology of species of organisms in 472

descending order (from left to right). Bitscores were obtained using BLASTP for a 473

selection of archaeal (red), bacterial (green) and eukaryotic (blue) species. The complete list 474

of 1,185 archaeal, 3,621 bacterial and 592 eukaryotic species from NCBI Genbank release 475

231 and their bitscores are given in Supplementary Table S1. The full names and three-letter 476

abbreviations for a number of the species are indicated in Table 1. 477

478


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479

480

Figure 2. The rooted universal SSU rRNA tree. The tree was constructed using the 481

Fitch-Margoliash method from the alignment of SSU rRNAs from 33 archaeal, 31 bacterial, 482

and 19 eukaryotic species. Pairwise distances between the aligned sequences were estimated 483

using DNADIST in PHYLIP with Jukes and Cantor model. The thermal scale in (a) expresses 484

the bitscore for the VARS-IARS pair of each species (Supplementary Table S1), and that in 485

(b) expresses the genetic distances of VARS (squares), or IARS (triangles) between Gla and 486

other organisms on the tree. 487

488


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489

490

Figure 3. Segments of the aligned VARS and IARS sequences of Mka, Mau and Esi. The 491

six sequences were aligned using ClustalOmega50. Tick marks indicate the positions of the 492

sequence segments on the complete alignment (Supplementary Fig. S1). Similar amino acids 493

are colored in orange, and ≥ 50% conserved ones in blue. Asterisks indicate the six positions 494

displaying conservation of the same V or L amino acid in all six sequences. 495

496


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497

498

Figure 4. Proteins sequence homology between eukaryotic and prokaryotic species. (a) 499

BLASTP bitscores between Gla rProts and prokaryotic rProts. (b) Bitscores between Gla 500

pyruvate phosphate dikinase and prokaryotic proteins. Among all Gla proteins, this protein 501

elicited the highest total BLASTP bitscore from all the prokaryotes. (c) Bitscores displayed 502

by some of the 162-Gla proteins from Supplementary Table S2 towards prokaryotes. The 503

color coding and order of different prokaryotic species on the x-axis in (b) and (c) are the 504

same as those in (a). Bitscores with E-value < 0.05 are shown, except for asterisked entries in 505

(c) where E-value < 0.1. 506


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507

508

Figure 5. Interspecies protein homologies. (a) Total homology bitscores of Gla and Trv 509

proteomes towards prokaryotic proteomes. (b) Relationships between the number of 510

homologous hits (x-axis) and average bitscore per hit (y-axis) between prokaryotic and Gla 511

proteomes. (c) Homologous hits between archaeal proteomes (y-axis) and bacterial 512

proteomes (x-axis) with (left) or without (right) normalization based on the number of 513

proteins in the archaeon. Complete heat map is given in Supplementary Fig. S4. (d) Bitscores 514

of homologous hits of two eukaryotic species of mitochondrial gene-encoded proteins 515

towards prokaryotes. Total bitscores in each instance are shown in parentheses. 516

517

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