Bacteria are the most abundant cells on Earth. They are generally regarded as ancient, but due to striking diversity in their metabolic capacities and widespread lateral gene transfer, the physiology of the first bacteria is unknown. From 1089 reference genomes of bacterial anaerobes, we identified 146 protein families that trace to the last bacterial common ancestor, LBCA, and form the conserved predicted core of its metabolic network, which requires only nine genes to encompass all universal metabolites. Our results indicate that LBCA performed gluconeogenesis towards cell wall synthesis, and had numerous RNA modifications and multifunctional enzymes that permitted life with low gene content. In accordance with recent findings for LUCA and LACA, analyses of thousands of individual gene trees indicate that LBCA was rod-shaped and the first lineage to diverge from the ancestral bacterial stem was most similar to modern Clostridia, followed by other autotrophs that harbor the acetyl-CoA pathway.

The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA's bacterial derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal derived and eukaryote specific genes. The surplus of bacterial derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host's chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggest that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin.

The metabolism of cells contains evidence reflecting the process by which they arose. Here, we have identified the ancient core of autotrophic metabolism encompassing 404 reactions that comprise the reaction network from H2, CO2, and ammonia (NH3) to amino acids, nucleic acid monomers, and the 19 cofactors required for their synthesis. Water is the most common reactant in the autotrophic core, indicating that the core arose in an aqueous environment. Seventy-seven core reactions involve the hydrolysis of high-energy phosphate bonds, furthermore suggesting the presence of a non-enzymatic and highly exergonic chemical reaction capable of continuously synthesizing activated phosphate bonds. CO2 is the most common carbon-containing compound in the core. An abundance of NADH and NADPH-dependent redox reactions in the autotrophic core, the central role of CO2, and the circumstance that the core’s main products are far more reduced than CO2 indicate that the core arose in a highly reducing environment. The chemical reactions of the autotrophic core suggest that it arose from H2, inorganic carbon, and NH3 in an aqueous environment marked by highly reducing and continuously far from equilibrium conditions. Such conditions are very similar to those found in serpentinizing hydrothermal systems.

Though all theories for the origin of life require a source of energy to promote primordial chemical reactions, the nature of energy that drove the emergence of metabolism at origins is still debated. We reasoned that evidence for the nature of energy at origins should be preserved in the biochemical reactions of life itself, whereby changes in free energy, ΔG, which determine whether a reaction can go forward or not, should help specify the source. By calculating values of ΔG across the conserved and universal core of 402 individual reactions that synthesize amino acids, nucleotides and cofactors from H2, CO2, NH3, H2S and phosphate in modern cells, we find that 95–97% of these reactions are exergonic (ΔG ≤ 0 kJ⋅mol−1) at pH 7-10 and 80-100°C under nonequilibrium conditions with H2 replacing biochemical reductants. While 23% of the core’s reactions involve ATP hydrolysis, 77% are ATP-independent, thermodynamically driven by ΔG of reactions involving carbon bonds. We identified 174 reactions that are exergonic by –20 to –300 kJ⋅mol−1 at pH 9 and 80°C and that fall into ten reaction types: six pterin dependent alkyl or acyl transfers, ten S-adenosylmethionine dependent alkyl transfers, four acyl phosphate hydrolyses, 14 thioester hydrolyses, 30 decarboxylations, 35 ring closure reactions, 31 aromatic ring formations, and 44 carbon reductions by reduced nicotinamide, flavins, ferredoxin, or formate. The 402 reactions of the biosynthetic core trace to the last universal common ancestor (LUCA), and reveal that synthesis of LUCA’s chemical constituents required no external energy inputs such as electric discharge, UV-light or phosphide minerals. The biosynthetic reactions of LUCA uncover a natural thermodynamic tendency of metabolism to unfold from energy released by reactions of H2, CO2, NH3, H2S, and phosphate.

The possible evolutionary significance of pyrophosphate (PPi) has been discussed since the early 1960s. Lipmann suggested that PPi could have been an ancient currency or a possible environmental source of metabolic energy at origins, while Kornberg proposed that PPi vectorializes metabolism because ubiquitous pyrophosphatases render PPi forming reactions kinetically irreversible. To test those ideas, we investigated the reactions that consume phosphoanhydride bonds among the 402 reactions of the universal biosynthetic core that generates amino acids, nucleotides, and cofactors from H2, CO2, and NH3. We find that 36% of the core’s phosphoanhydride hydrolyzing reactions generate PPi, while no reactions use PPi as an energy currency. The polymerization reactions that generate ~80% of cell mass – protein, RNA, and DNA synthesis – all generate PPi, while none use PPi as an energy source. In typical prokaryotic cells, aminoacyl tRNA synthetases (AARS) underlie ~80% of PPi production. We show that the irreversibility of the AARS reaction is a kinetic, not a thermodynamic effect. The data indicate that PPi is not an ancient energy currency and probably never was. Instead, PPi hydrolysis is an ancient mechanism that imparts irreversibility, as Kornberg suggested, functioning like a ratchet’s pawl to vectorialize the life process toward growth. The two anhydride bonds in nucleoside triphosphates offer ATP-cleaving enzymes an option to impart either thermodynamic control (Pi formation) or kinetic control (PPi formation) upon reactions. This dual capacity explains why nature chose the triphosphate moiety of ATP as biochemistry’s universal energy currency.