Possible Crystallization Process in the Origin of Bacteria, Archaea, Viruses, and Mobile Elements

1. Introduction

A phylogenetic analysis of ribosomal DNA (rDNA) revealed three domains of life, Bacteria, Archaea, and Eucarya (Figure 1A) [1,2]. The theory that symbioses of Bacteria and Archaea yielded Eucarya (Figure 1A(a)) and Plante (Figure 1A(b)) was proposed by Margulis [3]. Subsequently, Woese proposed an order of cellular evolution from an RNA world [4] to the last universal common ancestor (LUCA) of Bacteria and Archaea mediated by a progenote (Figure 1A) [5,6].

Koonin and Martin hypothesized that early cells evolved via 18 steps (Figure 1B(a)) at ancient submarine alkaline vents (Figure 1B(b)) [7,8]. Of note, the size of honeycomb-like chambers at both extant and fossil vents (Figure 1B(b)) [7] is nearly equivalent to the size of eukaryotic cells (Figure 1A). The LUCA was predicted to be non-free-living [5,7] with the extant genetic code, translation system, and hundreds of proteins [9,10].

Previously, we proposed a hypothesis to explain the origin of the genetic code and translation system [11]. The postulated scenario from an RNA world to the LUCA (Figure 1B(c)) [11] is quite different from that proposed by Koonin and Martin (Figure 1B(a)). The final step in the emergence of Bacteria and Archaea from the LUCA was called “crystallization”, as the stabilization of genetic systems, (Figure 1B(b)) by Woese [5].

In this study, we theoretically examined crystallization processes from the LUCA to extant cells. We hypothesize that crystallization was mainly driven by two physical laws (Eigen’s error catastrophe and Bejan’s constructal law) [12,13]. Remarkably, the order of steps (Figure 1B(c)) toward crystallization differs from that proposed by Koonin and Martin [7] (Figure 1B(a)). Complete scenarios for crystallization processes are presented.

2. The LUCA

2.1. Double-stranded U-DNA on a single operon

We have previously predicted that a pool of hundreds of single operons associated with both the translation system and extant standard genetic code existed before cells (Supplementary Figure S1) [11]. A small quantity of prebiotic dNTPs was likely essential for the RNA-to-DNA transition (Supplementary Figure S2), facilitating the cooperative emergence of ribonucleotide reductase (RNR) (Figure 2A) [14], primitive reverse transcriptase (RT) (Figure 2B), and primitive DNA-dependent RNA polymerase (DdRp) (Figure 2C). Since DNA is a more stable substance than RNA, antisense DNA is excellent template for multiple rounds of mRNA production (Figure 2D). This powerful driving force (Figure 2D) produced RT (Figure 2F) together with selective dNTP (Figure 2E), leading to T7 type DNA-dependent RNA polymerase (Figure 2G). Of note, RNA-dependent RNA polymerase (RdRp) (Supplementary Figure S1) has an RRM-palm domain (Supplementary Figure S3). Both RT (Figure 2F) and T7 phage DdRp (Figure 2G) also have the RRM-palm domain (Supplementary Figure S3). In addition to the RRM-palm fold, another DdRP derived from Bacteria or Archaea with a double psi-beta barrel domain (Supplementary Figure S3) emerged (Figure 2H) [15].

RNA digestion of RNA/DNA duplexes by RNaseH, an essential component of the retrovirus life cycle [16], could lead to single-stranded DNA (Figure 2I). To avoid the terminal replication problem, protein-primed type DNA synthesis could have emerged by utilizing a single-stranded DNA binding protein (SSB or RPA type) [17] and DNA-dependent DNA polymerases (DdDps) (Figure 2J(a)). The DdDp PolB (Figure 2J(a)) could be directly derived from T7 type DdRp (Figure 2G), primitive RdRp (Figure 2C), and RdRp (Figure S1). To simplify enzyme lineages in evolution, we marked these polymerases as PolB (Figure 2, Supplementary Figure S3). By contrast, DdDp (PolD) (Figure 2J(a)) could be derived from cellular DdRp (Figure 2H), marked PolD. Remarkably, DdDp with another fold (Supplementary Figure S3) could have emerged simultaneously, leading to PolA, PolC, and PolY (Figure 2J(a)). Then, dsDNA might emerge in the LUCA (Figure 2J(b)).

Purified Escherichia coli replicative Pol III (PolC) has a processivity of only 10 or 200 nucleotides in the absence or presence of SSB, respectively [18]. To complete the DNA replication of a single S10 operon (5.2 kbp) (Supplementary Figure S1B), DdDp should be loaded on ssDNA template, more than 26 (5,200/200) times. Since multiple DdDps, including lesion bypass-type PolY, could function cooperatively in the completion of DNA replication (Figure 2K), they share similar characteristics depending on the ssDNA template and primer for 5′ to 3′ directional DNA synthesis, as firstly described by Kornberg [18].

2.2. Discrimination of sense and antisense DNA

Extant T7 RNA polymerase (PolB) and bacterial/archaeal RNA polymerase (PolD) cannot discriminate sense or antisense strands of duplex DNA without promoters (Figure 3A(a)(b)). T7 RNA polymerase directly recognizes the promoter. E. coli RNA polymerase recognizes the promoter via σ (Figure 3A(c)). TFIIB in both Achaea and Eucarya interacts with the surface of RNA polymerase, which is structurally equivalent to the σ-interacting surface of E. coli RNA polymerase [19,20]. TFIIB may have been the primary promoter recognition protein in the ancient world (Figure 3A(c)) [19,20,21]. Although the internal duplication of TFIIB yielded TFIIB′, there was no internal duplication in TBP at that time (Figure 3A(d)) [20]. Extant TBP, the primary site of promoter recognition, has an internal duplication (Figure 3A(e)) [20]. Importantly, various transcription systems produce mRNA equally (Figure 3A(c)(d)(e)), implicating that such weak selection allowed divergence of transcription initiations. Ohta proposed the nearly neutral theory explaining how weak selection shapes rates of evolution and patterns of genetic variation (allele frequency distributions) under different population sizes [22]. Similar to Ohta’s concept, the nearly neutral selection on mRNA may explain patterns of molecular variation in these systems.

2.3. Strand displacement and unidirectional replication

Duplex DNA (up to 30 kbp) could be replicated, as follows (Figure 3B). As in extant adenovirus DNA replication, protein-primed 5′ to 3′ directional replication of one template strand occurs and the other strand is displaced and stabilized by SSB [18,23]. After the replication of one strand is completed, the replication of the other strand occurs (Figure 3B). DNA polymerases and SSB (or RPA) are required for duplex DNA replication, suggesting that DdDp and SSB (RPA) described in Figure 2J were available. In addition to three types of RNA (Figure 3C (1)(2)(3), Supplementary Figure S1) in the progenote, the LUCA had four types of DNA (Figure 2F, 2J, 3B, 3C(4)(5)(6)(7)). Various nucleic acids could directly or indirectly produce mRNA, as predicted by the nearly neutral theory [22]. Nucleic acids (1)–(7) in Figure 3C correspond to types I–VI among VII types in a classification of viruses [24]. Considering the diversification of RNR (Figure 2A), multiple DdRps (Figure 2G, 2H), multiple DdDps and SSBs (RPA) (Figure 2J), multiple transcription initiation systems (Figure 3A), two-types of DNA ligases (Supplementary Figure S4A), and the variety of nucleic acids (Figure 3C), substantial diversification from the LUCA could occur. Thus, the LUCA can be considered the garden of Baltimore [24].

3. End of the LUCA

3.1. Beginning of genetic linkage and vertical inheritance

The balance among single operons encoding various products (Figure 3C) could be rapidly destabilized by an increase in dsDNA due to the gain of the homologous recombination (HR) system, which relies on common recombinases (Figure 3D(a)). Since the maximum genome length of extant RNA viruses is 30 kbp (Supplementary Figure S1C), the superior physical nature of dsDNA enabled the generation of genomes exceeding 30 kbp. The evolution of HR to repair errors during DNA replication (Figure 3D(b)) could have been a long process in the robust LUCA (Figure S4A, S4B). In addition, two independent DNA ligases [25] could have emerged to repair template ssDNA with single-stranded breaks (Figure S4A(a)). Thus, the basic tool kit for the completion of DNA replication on damaged templates could have been established in the LUCA (Supplementary Figure S4C).

In addition to its repair function, extant HR accelerates a variety of genomic rearrangements, including duplications (Supplementary Figure S5A) [26]. Furthermore, HR greatly accelerates protein evolution (Figure S5B, S5C, S5E). Eukaryotic HR-mediated meiotic recombination prompted species diversification [27]. Moreover, biotechnologies, such as knockouts and gene-editing by CRISPR/Cas9, rely on cellular HR [28,29]. Thus, HR has been a key event in Darwinian evolution since it arose in the LUCA.

Although the yellow body phenotype is not biochemically related to the white eye phenotype in Drosophila melanogaster, the alleles are inherited together due to their linkage on the same chromosome (the same dsDNA molecule) (Figure 4A) [30]. Morgan’s genetic linkage could be the first step in the fusion of two single operons by HR. Transcription initiation could be divided into at least five systems (Figure 3A(e)), and homologous transcriptional system fusion could be beneficial over heterologous one (Figure 3B). In the LUCA, genes associated with different phenotypes could be genetically linked, as observed in the extant fruit fly. For instance, the σ genome (bacterial lineage) harbored genes encoding DNA polymerases (Figure 2J), including PolA, PolB, PolC, and PolY, and NAD-dependent DNA ligase (Supplementary Figure S4A(a)). By contrast, the TBP genome (archaeal lineage) encoded the DNA polymerases (Figure 2J) PolB, PolD, and PolY and ATP-dependent DNA ligase (Supplementary Figure S4A(a)). Thus, the difference in transcription initiation easily created the so-called DNA replication divide between Bacteria and Archaea [31]. A network of transcription factors evolved rapidly to regulate biological processes in both σ- and TBP genomes lineages (Supplementary Figure S6). Extinct genomes not found in free-living cells (Figure 4C(a)(c)(d)) could also acquire DNA polymerases and DNA ligases in different combinations. We speculated that initial replication mechanism evolved more than twice in the LUCA.

3.2. Beginning of horizontal gene transfer (HGT)

The integration of DNA fragments into the genome by HGT under different transcription initiation systems and subsequent mutations in promoter regions was an important process (Figure 4B(c)). Genomes incorporated genes encoding distinct proteins by vertical transmission (Figure 4B(b)), which was initially experimentally demonstrated by Mendel [32]. Over time, beneficial mutations could be transferred among genomes by HGT (Figure 4B(c)).

3.3. Evolution of two different DNA replication machineries

The replication of large DNA molecules took more time than that required for the replication of small DNA. Accordingly, Mullis-type DNA replication (i.e., the simultaneous replication of both strands) emerged (Figure 5A(a)). Furthermore, replication and transcription occurred simultaneously and collision between these processes should occur occasionally (Figure 5A(b)) [33,34,35,36]. Topological tortional stress has to be increased, as shown in Figure 5A(a) (b). After the publication of the DNA duplex model [37], Delbrück claimed that it is impossible to separate dsDNA into two ssDNAs due to a topological constraint [38]; however, the σ genome and TBP genome easily resolved this topological issue by the independent evolution of topoisomerases, TopoIIA and TopoIIB, respectively (Figure 5A(c)) [39,40]. TopoIA is common to both lineages (Figure 5A(c)), which may be explained by HGT. The TopoIB gene is distributed in various extant Bacteria and Archaea (Figure 5A(c)), suggesting that TopoIB arose in either Bacteria or Archaea, was transferred to other lineages by HGT, and was inherited by Eucarya.

Resolving topological issues enabled independent origins of dnaB helicase and MCM helicase in each genome (Figure 5A(d)). The dnaB helicase moves in the 5′ to 3′ direction [41]. MCM is translocated in the opposite direction [42]. Of note, dnaB could be derived from the duplication of the recA domain (Figure 5A(d), Figure S5C) [43]. Other helicases, including superfamily I and II helicases, may have arisen [44] and spread to both genomes by HGT (Figure 5A(e)). The rapid progression of the replication fork by topoisomerase and helicase enabled high-speed leading strand DNA synthesis, triggering the evolution of the clamp loader and clamp (Figure 5B(a)(b)) [45,46]. Since both the clamp loader and clamp are sequentially and structurally similar in both genomes [47,48], after they arose in either genome, they could be transferred to the other genome and thereby exert important functions.

High-speed replication fork opening and leading strand synthesis provided space for lagging strand synthesis, triggering a two-fold increase in the total replication speed. Under the same driving force, dnaG primase in the σ genome and PriS/PriL primase in the TBP genome could have arose independently (Figure 5C) [49]. Remarkably, dnaG could be derived from the previously established Topo domain [50] (Figure 5A(c)). The Okazaki fragment [51] is 1,000 or 100 nucleotides in Bacteria or Archaea, respectively (Figure 5C) [52,53].

The coordination of leading and lagging strand synthesis (Figure 5D(a)) [54] could be achieved by highly mutual interactions among replicative proteins (Figure 5D(b)). For instance, dnaB helicase and MCM helicase formed large protein complexes, the primosome and CMG (Cdc45-Mcm-GINS) helicase in Bacteria and Eucarya, respectively [55,56]. In addition to MCM, Cdc45 and GINS [57] are conserved in Eucarya and Archaea. T7 phage gene 4 encodes a primase and helicase fusion protein [58]. Mouse DNA polymerase α is a polymerase and primase complex [59]. Although E. coli PolIII has a processivity of only 10 nucleotides (Figure 2J), that of PolIII holoenzyme is more than 10⁵ nucleotides [18]. Thus, the equivalent replication components of the σ genome and TBP genome were no longer exchangeable, leading to a complete DNA replication divide between Bacteria and Archaea (Figure 5D(c)) [31]. Further evolution of DNA replication machineries in the σ genome and TBP genome, based on Jacob’s replicon theory [60], is summarized in Supplementary Figure S7.

3.4. Necessity of DNA repair for the increase in DNA genome size

According to Eigen’s error-catastrophe theory (Figure 6A) [12], it is impossible for the DNA genome to increase in size without the evolution of a DNA repair system. Translesion polymerase (PolY) (Figure 2K, 6B(a)) [61], SSB repair by DNA ligases (Supplementary Figure S4A), and recombinases (Figure 3D, 6B(a)) may have been established in the LUCA (before genome fusion). During genome enlargement, proof-reading of DNA polymerases [18] and a mismatch repair system could have arisen independently in each genome (Figure 6B(a)) [62,63,64]. Enzymes involved in base excision repair (BER) [65,66,67] are largely shared between Bacteria and Archaea, suggesting that genes encoding the enzymes involved in BER in either the σ genome or TBP genome are easily transferred into the other genome by HGT (Figure 4B(c)) [68,69,70].

Although E. coli has an efficient nucleotide excision repair (NER) system mediated by UvrABC [71], an NER enzyme is lacking in most species of Archaea. This point will be discussed later (see Figure 6C). Among 138 human DNA repair genes, 70 genes are associated with hereditary diseases [72]. Among these 70 genes, 26 representative genes are listed in Figure 6B(b), such as XP-V [73], NBS [74], and XP-A [75]. Thus, Eigen’s error-catastrophe, a physical law, has dominantly governed biological processes in all organisms, including humans, since the LUCA era.

3.5. Transition from U-DNA to T-DNA

Cytosine often changes into uracil via deamination, leading to mutations during subsequent DNA replication [18]. Extant living cells resolve this problem using T-DNA, rather than U-DNA. This transition simultaneously requires the invention of thymidylate synthetase (TS), uracil elimination by dUMPase, and removal of mis-incorporated uracil by glycosylase (Figure 6B(a)) [18,76]. One scenario for the transition from U-DNA to T-DNA is described in Supplementary Figure S8.

3.6. Transcription-coupled NER

Hanawalt’s group first proposed transcription-coupled repair [77]. RNA polymerase in the elongation step is an excellent sensor of damaged antisense DNA (Figure 6C(a)), and the preferential repair of antisense DNA is highly beneficial for restoring normal transcription. In Eucarya, transcription-coupled NER [78] is operated by various proteins, including XP-B, XP-D, XP-F, and XP-G, all of which are conserved in Archaea (Figure 6C(b)). Thus, TCR-NER might mainly function in Archaea to repair DNA damage. It is possible that global genome (GG)-NER in Eucarya (Figure 6C(c)) [79] evolved from TCR-NER via the Eucarya-specific evolution of XP-A, XP-C, and XP-E.

3.7. Necessity of DNA compaction

After the evolution of DNA replication (Figure 5, Supplemental Figure S7) and DNA repair systems (Figure 6B,C), the DNA genome can increase 8.5 cm in length (Figure 6B(a)). Venter’s minimal synthetic cell JCVI-syn3 has a 543 kbp DNA genome [80], with an estimated length of 185 µm. Since the average size of each chamber in a hydrothermal vent (Figure 1B(b), 6D) is 50 µm, a 185-µm-long DNA genome should be compacted 3.7-fold in the chamber. HU and histone evolution in the σ genome and TBP genome, respectively [81,82], easily solved the compaction problem (Figure 6D).

4. Toward proto-cells

4.1. A lack of essential genes in lethal in extant cells

Giant viruses carrying a 1,259 kb genome (lager than the genome of free-living JCVI-syn3) (Figure 7B(a)) [83,84] are not free-living but are very active in host cells (Figure 7A). The virus factory in Eucarya freely imports and/or exports necessary biomolecules. Thus, an open system is required for the survival of taxa with incomplete genomes, like giant viruses. If extant cells lose a single essential gene, they cannot survive (Figure 7B(b)). For example, the lack of arginine biosynthesis in Neurospora crassa leads to cell death [85]. The lethality of cdc2 mutations in Schizosaccharomyces pombe was rescued by the Homo sapiens homologue of the cdc2 gene [86]. As previously reported [87,88], we hypothesized that intermediate proto-cells with an incomplete genome before reaching the free-living state had a permeable membrane to freely exchange necessary biomolecules within the chamber (Figure 7C). A variety of membrane-bound proteins could have evolved. Since the cell size of JCVI-syn3A is 1 µm, the 185 µm DNA genome of JCVI-syn3A should be compacted more than 185-fold (Figure 7B(a), 7D). Such compaction could be achieved by the SMC protein [89], which could have arisen in either the σ genome or TBP genome, followed by HGT into the other genome (Figure 7D). In Eucarya, condensation compacts metaphase chromosomes [90] and cohesion attaches two sister chromatids [91]. The consideration on Figure 7C,D, prompts the question of what type of Darwinian driving force creates proto-cells with permeable membrane and highly compacted DNA genomes.

4.2. Coacervate formation triggering lipid divide

The reconstructed metabolism of the LUCA is quite similar to that of extant cells [9,10]. A symbolic metabolic map of the LUCA is illustrated in Figure 8A(a). DNA rearrangement and DNA compaction via SMC and transcription factors enabled operons encoding components of metabolic pathways (1), (2), and (3) in Figure 8A(a)(b) to become closer in limited space (Figure 8A(b)), as observed in Eukarya [92]. High concentrations of ribosomes, enzymes, and metabolites could trigger the formation of coacervate [93], also called liquid droplets (Figure 8A(c)) [94,95,96,97,98], leading to efficient biochemistry.

The lipid divide between Bacteria (σ genome) and Archaea (TBP genome) [99] can be explained by equal contributions of fatty acids and fatty alcohols to the biochemistry of coacervate (Figure 8B(a)). Moreover, if di-acyl-glycerol is a better biochemical facilitator than is mono-acyl-glycerol, which is a better biochemical facilitator than either glycerol or fatty acids (Figure 8B(b)), then the σ genome could obtain all biochemical steps one-by-one (Figure 8B(c) upper column). A similar scenario (Figure 8B(b)) could occur independently in the TBP genome (Figure 8B(c) lower column).

Simultaneously and independently, a phase transition could happen in both σ and TBP incomplete genomes, leading to the development of a permeable membrane (Figure 8B(d)). Importantly, if the hypotheses described in Figure 8 are correct, the enlargement, rearrangement, and proper compaction of the DNA genome (Figure 5, 6), enabling better biochemistry, will inevitably produce proto-cells in the chamber. Then, both the replication divide and lipid divide between Bacteria and Archaea could be fully completed at this stage (Figure 8B(d)).

4.3. Bejan’s “constructal law” in transporter and channel creation

The coevolution of the membrane and membrane-bound proteins was proposed [48]. We hypothesize that such coevolution occurs under Bejan’s “constructal law,” a law of thermodynamics [13]. Bejan stated that “For a finite-size system to persist in time (to live), it must evolve in such a way that it provides easier access to the imposed (global) currents that flow through it.” [100]. Under the constructal law, a high concentration of substance A inside a proto-cell moves outward through a permeable membrane; then, to improve this flow, transporter A from the inner to outer space evolved (Figure 9A(a)). Similarly, transporter B and dual transporters C/D emerged. Since the proto-cell produces substance A and consumes substance B, such transporters could facilitate metabolism in proto-cells substantially.

4.4. ATPase and the electron transport system

Protons and electrons are produced from H₂S in the chambers of extant alkaline vents [7,8,101,102,103,104,105,106]. External protons move into proto-cells through the permeable membrane, accumulate in proto-cells, and move outside, reaching maximum entropy by the second law of thermodynamics (Figure 9B). Under the constructal law, both inward and outward transporters (channel) of protons inevitably emerged (Figure 9B). A variety of inward and outward proton transporters likely arose (Figure 9C, Supplementary Figure S9). Importantly, all proton transporters contributed equally to the improved current under constructal law, as predicted by the nearly neutral theory [22]. However, among these, proton current-driven ATPase (Figure 9C(d)) and outward proton pumps coupled with electron transport (Figure S9B(e)) were positively selected due to the benefits with respect to energy and biochemistry for proto-cells. After the ATPase or electron transport system arose in either the σ or TBP genome, the corresponding gene could be transferred into the other genome and retained due to its important function. In addition to the transport of small molecules, incomplete genomes also require macromolecule transport mechanisms. As hypothesized previously [107,108], a proposed ancient membrane-based macromolecule exchange system is summarized in Supplemental Figure S10.

4.5. Omnis cellula a cellula

The continuous supply of lipids to membrane vesicles in vitro inevitably leads to vesicle division due to the physical nature of the lipid bilayer [96]. Thus, under constructal law, FtsZ-mediated cytokinesis [109,110] arose (Figure 10A(a)(b)). Since the concentration of all molecules on one side after cytokinesis is counter to the second law of thermodynamics (Figure 10A(c)), after proto-cell division, equal segregation of chromosomes yields maximum entropy in the production of two daughter proto-cells (Figure 10A(d)(e)). Since pathways for the unlinking of replicated DNA were already established (Supplementary Figure S7C) and membrane-vesicle division is an intrinsic property of a lipid bilayer [96], the development of anchoring DNA on the membrane enabled the equal segregation of duplicated DNA genomes into daughter cells (Figure 10B); subsequently, ParA, ParB, and ParS in the σ genome and SegA and SegB in the TBP genome could emerge (Figure 10B) [111].

Coordination among DNA replication/segregation, membrane growth, anchoring DNA to the membrane, and cytokinesis could establish primitive proto-cell division (Figure 10C). Importantly, the equal division of proto-cells into daughter cells maximizes entropy within the proto-cell (Figure 10C). Moreover, the proliferation of proto-cells in a manner consistent with “omnis cellula a cellula” [112] maximizes total entropy in the chamber as well as entropy in each proto-cell (Figure 10D). Thus, the second law of thermodynamics could promote the establishment of extant living cells, following “omnis cellula a cellula.”

4.6. Punctuated equilibrium by the fusion of different systems

Gould proposed the concept of punctuated equilibrium based on fossil data [113]. In the history of life on our planet, the symbiosis of Archaea and Bacteria (Figure 1A(a), Supplementary Figure S11A(a)(2)) and the symbiosis of Eucarya and Cyanobacteria (Figure 1A(b), Supplementary Figure S11A(a)(3)) can be described as examples of punctuated equilibrium at the cellular level. Moreover, another example of punctuated equilibrium, the fusion of photosystem I and II, could yield Cyanobacteria (Supplementary Figure S11A(a)(1)), which produced oxygen and changed the environment of the whole planet.

Considering that free-living cells derived from giant viruses did not arise for 2 billion years (Supplementary Figure S11(b)) [83], punctuated equilibrium by system fusion likely occurred in the chamber of the original alkaline vent (Figure 11) during a relatively narrow time window (Supplementary Figure S11A(a)). Since we hypothesize that membrane-vesicle fusion and the split system were present at the chamber (Supplementary Figure S10), a cascade of proto-cell fusion could rapidly create new and complicated proto-cells in a pattern of punctuated equilibrium (Figure 11A). During proto-cell fusion, DNA rearrangement by HR could create new genomes (Figure 11B). Once useful protein machinery arose in either the σ genome or TBP genome, DNA encoding such machinery could be transferred into the other genome by HGT (Figure 11C). For instance, ATPase and SRP are common in Bacteria and Archaea [9,48,114]. By a cascade of proto-cell fusion (Supplementary Figure S12A) associated with genomic rearrangement (Supplementary Figure S12B(a)(b)), proto-cells carrying a nearly complete σ genome or TBP genome may have arisen, after which the permeable membrane could be transformed into a non-permeable membrane (Supplementary Figure S12B(c)).

4.7. Selfish cells trigger the emergence of selfish genes

Primitive living cells could develop active transporters using ATP created by ATPase (Figure 12A) and start to use all energy and resources at the chamber in a selfish manner, as do extant Bacteria and Archaea. Thus, all intermediates described in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figure 11, including proto-cells with incomplete genomes, would go extinct (Supplementary Figure S13A, see Figure 13A). Small genes encoding replicases, such as RdRp, RT, and DdDp, could become selfish DNA as in primitive viruses, and an arms race between selfish cells and selfish replicators is possible (Figure 12A,B). Most deadly viruses would be enveloped because the membrane-vesicle-mediated macromolecule transport system supported metabolism at the chamber (Supplementary Figure S10E). Furthermore, primitive viruses could infect proto-cells as well as primitive living cells, just like mimivirus (giant virus) infection by virophage (Supplementary Figure S13B) [115]. Thus, proto-cells should be affected by a shortage of energy and resources as well as viral infection, leading to rapid extinction at the original vent.

5. Independent time

5.1. Independent cell wall formation

To prevent the entry of enveloped viruses into primitive cells (Figure 12A), a cell wall may have evolved independently in primitive cells containing the σ or TBP genome (Figure 12B) [116]. The cell wall would be an intrinsically double-edged sword for Bacteria and Archaea. The endless supply of protons from the vent (Figure 12A) would not be accessible. However, the coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic mechanism [117] could be retained in cells with a cell wall (Figure 12B). Additionally, cell fusion (Figure 11) and membrane-mediated transport (Supplementary Figure S10E) would no longer be possible.

By contrast, primitive cells (Figure 12A) would no longer depend on the inorganic chamber of the original vent (Figure 12B). The cell wall could eliminate enveloped viruses (Figure 12C); however, capsid-based viruses would remain [118]. A flagellin motor could be anchored to the solid cell wall [119,120], leading to free-swimming Bacteria and Archaea (Figure 12D).

Genes encoding the postulated ESCRT-mediated membrane-vesicle transport system (Supplementary Figure S10E) would be lost in all Bacteria and most Archaea (Figure 12D) due to lack of necessity. Similarly, an archaeal lineage, Crenarchaeote, has two copies of PolB and could discard PolD [121] due to the redundancy of DNA polymerases (Figure 12D, Supplementary Figure S3). Extant slow-growing Archaea (with a doubling time of one month) with an elongated, branched form [122] maintains the ESCRT system for internal vesicle trafficking. Thus, ESCRT in some Archaea might be a molecular fossil remain of an ancient extinct membrane-vesicle transporting system at the chamber of the original vent (Supplementary Figure S10E). Restriction enzymes [123] and the CRISPR/Cas9 system [29] could have evolved during an arms race between capsid-based viruses and their hosts (Figure 12A, 12B, Supplementary Figure S14).

5.2. Why is the genetic code universal?

Free-living Bacteria and Archaea at the original vent would lead to the mass extinction of intermediates other than free-living cells (Figure 13A(a)(b)(c), Supplementary Figure S13). During the depletion of energy and resources at the original vent, viroid-type RNA [124] (Wang 2021) would survive (Figure 13B(a)). Small nucleic acids of any of the seven types summarized in Figure 3C could be synthesized and protected by the single jellyroll (SHR) protein derived from the proto-cell [118] or the membrane (Supplementary Figure S10E). The evolutionary process described in Figure 13B could rapidly create a variety of viruses. Since cell wall formation in primitive cells (Figure 12) could eliminate enveloped viruses, only capsid-based viruses would remain at the original vent.

Figure 13. Explanation for the universal standard genetic code. (A) Mass extinction at the original vent. (a) The original alkaline vent might have been occupied by free-living Bacteria and Archaea. (b) Event (a) might exhaust all energy and resources at the vent. (c) Events (a) and (b) might lead to the mass extinction of most of intermediates described in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figure 11. This would reduce the activity of the proto-cell, which is completely dependent on such energy and resources at the vent. (B) Purification of survivors. (a) Extant viroid RNAs can be copied by host DNA-dependent RNA polymerase. Thus, ancient viroid-type RNAs could survive a mass extinction (A). (b) During the exhaustion of energy and resources at the vent, small nucleic acids might be replicated to avoid extinction. The remaining nucleic acids could be protected by single jellyroll protein (SJR), which is derived from the proto-cell and later became a capsid protein, or by a lipid bilayer to prevent extinction. Nucleic acids covered with SJR or a membrane might enter living cells. Nucleic acids in cells could replicate. Thus, nucleic acids should encode replicase. Replicated molecules might remain in cells, leading to mobile elements. The extant mobile elements encode RCRE or RT in Bacteria and AEP in Archaea (Supplementary Figure S3), as observed in mitovirus, found in some extant mitochondria. Nucleic acids covered with SJR or lipids could escape from cells, leading to proto-viruses. During many infection cycles described in (A)(B), proto-viruses could rapidly evolve to become deadly viruses. Since cell wall formation (Figure 12B), enveloped viruses might be lost due to an inability of enter cells. Systems in ancestors of extant viruses could be crystalized at the original vent. (C) Mass extinction on the planet. Bacteria and Archaea with their corresponding viruses might leave the original vent and spread widely, eventually inhabiting all alkaline vents similar to the original one. Since other vents might use different genetic codes, process (B) might not produce viruses at these other vents. Thus, the genetic code at the original vent could become universal. (D) Against panspermia. Living cells with viruses were brought to the Earth 3.8 billion years ago according to the panspermia hypothesis (red arrow) (lower column), raising the question: why has other life (many green arrows) not been brought to the planet? Thus, it is likely that life originated on the Earth through the process described in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12 and Figure 13, 3.8 billion years ago (upper column).

Figure 13. Explanation for the universal standard genetic code. (A) Mass extinction at the original vent. (a) The original alkaline vent might have been occupied by free-living Bacteria and Archaea. (b) Event (a) might exhaust all energy and resources at the vent. (c) Events (a) and (b) might lead to the mass extinction of most of intermediates described in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figure 11. This would reduce the activity of the proto-cell, which is completely dependent on such energy and resources at the vent. (B) Purification of survivors. (a) Extant viroid RNAs can be copied by host DNA-dependent RNA polymerase. Thus, ancient viroid-type RNAs could survive a mass extinction (A). (b) During the exhaustion of energy and resources at the vent, small nucleic acids might be replicated to avoid extinction. The remaining nucleic acids could be protected by single jellyroll protein (SJR), which is derived from the proto-cell and later became a capsid protein, or by a lipid bilayer to prevent extinction. Nucleic acids covered with SJR or a membrane might enter living cells. Nucleic acids in cells could replicate. Thus, nucleic acids should encode replicase. Replicated molecules might remain in cells, leading to mobile elements. The extant mobile elements encode RCRE or RT in Bacteria and AEP in Archaea (Supplementary Figure S3), as observed in mitovirus, found in some extant mitochondria. Nucleic acids covered with SJR or lipids could escape from cells, leading to proto-viruses. During many infection cycles described in (A)(B), proto-viruses could rapidly evolve to become deadly viruses. Since cell wall formation (Figure 12B), enveloped viruses might be lost due to an inability of enter cells. Systems in ancestors of extant viruses could be crystalized at the original vent. (C) Mass extinction on the planet. Bacteria and Archaea with their corresponding viruses might leave the original vent and spread widely, eventually inhabiting all alkaline vents similar to the original one. Since other vents might use different genetic codes, process (B) might not produce viruses at these other vents. Thus, the genetic code at the original vent could become universal. (D) Against panspermia. Living cells with viruses were brought to the Earth 3.8 billion years ago according to the panspermia hypothesis (red arrow) (lower column), raising the question: why has other life (many green arrows) not been brought to the planet? Thus, it is likely that life originated on the Earth through the process described in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12 and Figure 13, 3.8 billion years ago (upper column).

Plasmids encoding RCRE (PolB) or AEP (PolB) and mobile elements encoding RT (PolB) (Supplementary Figure S3) cannot escape from host cells, just like Mitovirus [125]. We speculated that these plasmids and mobile elements lost capsids or were introduced from a primitive enveloped virus. In the latter case, cell wall formation in host cells could permanently trap RCRE, AEP, and RT (Figure 12D, 13B). The remaining nucleic acids not existing at the time of the LUCA (Baltimore definition class VII) (Figure 3C) [24] appeared in the σ genome as mobile elements (i.e., group II introns) (Figure 12D, 13B).

When Bacteria and Archaea with their viruses migrated from the original vent to the ocean, cells with viruses occupied all alkaline vents on the planet. Although intermediates described in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figure 11 could be present at some vents, they would eventually go extinct (Figure 13C), as in the original vent (Figure 13A). Gene flow between taxa and populations at different vents was not possible (Figure 13B), because the genetic code likely varied among vents [11], leading to the standard genetic code of the progenote (Supplementary Figure S1) and the LUCA (Figure 3) from the original vent and its global spread (Figure 13C).

According to Darwin’s statement “At the present day such matter (Events in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11 and Figure 12) be instantly devoured, or absorbed, which would not have been the case before creatures were formed” [126] (Figure 13D), we predict that the crystallization event only occurred once at the original vent 3.8 billion years ago (Figure 13D, upper column). Although an alternative evolutionary theory, panspermia [127] (Crick and Orgel 1973), has been proposed repeatedly [128], it does not effectively explain why Bacteria and Archaea reaching the Earth 3.8 billion years ago grew, while other taxa did not survive (Figure 13D, lower column).

6. Conclusion

Although viruses are believed to be parasites of host cells (Figure 14A(a)), the first free-living cells would likely be selfish parasites of the original vent (Figure 14(b)). Massive numbers of viruses may have been produced from the LUCA [129] at the original vent (Figure 13A(a), 14A(c)). Moreover, there are far more viruses than taxa belonging to Bacteria, Archaea, and Eucarya in the virosphere (Figure 14A(c)) [130] or cells on the human body (Figure 14A(d)) [131]. Thus, viruses should be included in the definition of life [132,133].

Key innovations for evolution from the progenote to free-living cells with viruses (Figure 14B, right cartoon of the vent) are summarized in Supplementary Figure S14. Some evolutionary steps described in Figure 7, Figure 8, Figure 9, Figure 10 and Figure 11 and S10–S12 can be tested in vitro and/or in silico (Supplementary Figure S15). Kornberg stated “I have never met a dull enzyme” [134], and key enzymes and proteins forming the extant biological world have been never dull since they emerged at the LUCA (Figure S14).

Although the progenote (Figure S1), the LUCA (Figure 3), and intermediates were not free-living (Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figure 11) (Figure 14B), all extant life including cells, viruses, and mobile elements are clearly descendants of these non-free-living materials. Cells, viruses, the LUCA, and progenote commonly replicate (Figure 14(a)). Although Darwin articulately described life as “descent with modification” (Figure 14(b)) [135], this statement could apply to man-made artifacts, such as smartphones, personal computers, televisions, and cars. Thus, we propose that life, including cells, viruses, mobile elements, the LUCA, and progenote, can be defined as “a natural system that self-replicates, yielding a nearly identical copy” (Figure 14B(c)).