The nature of the last universal common ancestor and its impact on the early Earth system.

The nature of the last universal common ancestor (LUCA), its age and its impact on the Earth system have been the subject of vigorous debate across diverse disciplines, often based on disparate data and methods. Age estimates for LUCA are usually based on the fossil record, varying with every reinterpretation. The nature of LUCA's metabolism has proven equally contentious, with some attributing all core metabolisms to LUCA, whereas others reconstruct a simpler life form dependent on geochemistry. Here we infer that LUCA lived ~4.2 Ga (4.09-4.33 Ga) through divergence time analysis of pre-LUCA gene duplicates, calibrated using microbial fossils and isotope records under a new cross-bracing implementation. Phylogenetic reconciliation suggests that LUCA had a genome of at least 2.5 Mb (2.49-2.99 Mb), encoding around 2,600 proteins, comparable to modern prokaryotes. Our results suggest LUCA was a prokaryote-grade anaerobic acetogen that possessed an early immune system. Although LUCA is sometimes perceived as living in isolation, we infer LUCA to have been part of an established ecological system. The metabolism of LUCA would have provided a niche for other microbial community members and hydrogen recycling by atmospheric photochemistry could have supported a modestly productive early ecosystem.

Biophysical Interactions Underpin the Emergence of Information in the Genetic Code.

The genetic code conceals a 'code within the codons', which hints at biophysical interactions between amino acids and their cognate nucleotides. Yet, research over decades has failed to corroborate systematic biophysical interactions across the code. Using molecular dynamics simulations and NMR, we have analysed interactions between the 20 standard proteinogenic amino acids and 4 RNA mononucleotides in 3 charge states. Our simulations show that 50% of amino acids bind best with their anticodonic middle base in the -1 charge state common to the backbone of RNA, while 95% of amino acids interact most strongly with at least 1 of their codonic or anticodonic bases. Preference for the cognate anticodonic middle base was greater than 99% of randomised assignments. We verify a selection of our results using NMR, and highlight challenges with both techniques for interrogating large numbers of weak interactions. Finally, we extend our simulations to a range of amino acids and dinucleotides, and corroborate similar preferences for cognate nucleotides. Despite some discrepancies between the predicted patterns and those observed in biology, the existence of weak stereochemical interactions means that random RNA sequences could template non-random peptides. This offers a compelling explanation for the emergence of genetic information in biology.

Prebiotic Synthesis of Aspartate Using Life's Metabolism as a Guide.

A protometabolic approach to the origins of life assumes that the conserved biochemistry of metabolism has direct continuity with prebiotic chemistry. One of the most important amino acids in modern biology is aspartic acid, serving as a nodal metabolite for the synthesis of many other essential biomolecules. Aspartate's prebiotic synthesis is complicated by the instability of its precursor, oxaloacetate. In this paper, we show that the use of the biologically relevant cofactor pyridoxamine, supported by metal ion catalysis, is sufficiently fast to offset oxaloacetate's degradation. Cu2+-catalysed transamination of oxaloacetate by pyridoxamine achieves around a 5% yield within 1 h, and can operate across a broad range of pH, temperature, and pressure. In addition, the synthesis of the downstream product ß-alanine may also take place in the same reaction system at very low yields, directly mimicking an archaeal synthesis route. Amino group transfer supported by pyridoxal is shown to take place from aspartate to alanine, but the reverse reaction (alanine to aspartate) shows a poor yield. Overall, our results show that the nodal metabolite aspartate and related amino acids can indeed be synthesised via protometabolic pathways that foreshadow modern metabolism in the presence of the simple cofactor pyridoxamine and metal ions.

Redox stress shortens lifespan through suppression of respiratory complex I in flies with mitonuclear incompatibilities.

Incompatibilities between mitochondrial and nuclear genes can perturb respiration, biosynthesis, signaling and gene expression. Here we investigate whether mild mitonuclear incompatibilities alter the physiological response to redox stress induced by N-acetyl cysteine (NAC). We studied three Drosophila melanogaster lines with mitochondrial genomes that were either coevolved (WT) or mildly mismatched (BAR, COX) to an isogenic nuclear background. Responses to NAC varied substantially with mitonuclear genotype, sex, tissue and dose. NAC caused infertility and high mortality in some groups, but not others. Using tissue-specific high-resolution fluorespirometry, we show that NAC did not alter H2O2 flux but suppressed complex I-linked respiration in female flies, while maintaining a reduced glutathione pool. The high mortality in BAR females was associated with severe (>50 %) suppression of complex I-linked respiration, rising H2O2 flux in the ovaries, and significant oxidation of the glutathione pool. Our results suggest that redox stress is attenuated by the suppression of complex-I linked respiration, to the point of death in some mitonuclear lines. We propose that suppression of complex I-linked respiration is a general mechanism to maintain redox homeostasis in tissues, which could offset oxidative stress in ageing, producing a metabolic phenotype linked with epigenetic changes and age-related decline.

The limits of metabolic heredity in protocells.

The universal core of metabolism could have emerged from thermodynamically favoured prebiotic pathways at the origin of life. Starting with H2 and CO2, the synthesis of amino acids and mixed fatty acids, which self-assemble into protocells, is favoured under warm anoxic conditions. Here, we address whether it is possible for protocells to evolve greater metabolic complexity, through positive feedbacks involving nucleotide catalysis. Using mathematical simulations to model metabolic heredity in protocells, based on branch points in protometabolic flux, we show that nucleotide catalysis can indeed promote protocell growth. This outcome only occurs when nucleotides directly catalyse CO2 fixation. Strong nucleotide catalysis of other pathways (e.g. fatty acids and amino acids) generally unbalances metabolism and slows down protocell growth, and when there is competition between catalytic functions cell growth collapses. Autocatalysis of nucleotide synthesis can promote growth but only if nucleotides also catalyse CO2 fixation; autocatalysis alone leads to the accumulation of nucleotides at the expense of CO2 fixation and protocell growth rate. Our findings offer a new framework for the emergence of greater metabolic complexity, in which nucleotides catalyse broad-spectrum processes such as CO2 fixation, hydrogenation and phosphorylation important to the emergence of genetic heredity at the origin of life.

A prebiotic basis for ATP as the universal energy currency.

ATP is universally conserved as the principal energy currency in cells, driving metabolism through phosphorylation and condensation reactions. Such deep conservation suggests that ATP arose at an early stage of biochemical evolution. Yet purine synthesis requires 6 phosphorylation steps linked to ATP hydrolysis. This autocatalytic requirement for ATP to synthesize ATP implies the need for an earlier prebiotic ATP equivalent, which could drive protometabolism before purine synthesis. Why this early phosphorylating agent was replaced, and specifically with ATP rather than other nucleoside triphosphates, remains a mystery. Here, we show that the deep conservation of ATP might reflect its prebiotic chemistry in relation to another universally conserved intermediate, acetyl phosphate (AcP), which bridges between thioester and phosphate metabolism by linking acetyl CoA to the substrate-level phosphorylation of ADP. We confirm earlier results showing that AcP can phosphorylate ADP to ATP at nearly 20% yield in water in the presence of Fe3+ ions. We then show that Fe3+ and AcP are surprisingly favoured. A wide range of prebiotically relevant ions and minerals failed to catalyse ADP phosphorylation. From a panel of prebiotic phosphorylating agents, only AcP, and to a lesser extent carbamoyl phosphate, showed any significant phosphorylating potential. Critically, AcP did not phosphorylate any other nucleoside diphosphate. We use these data, reaction kinetics, and molecular dynamic simulations to infer a possible mechanism. Our findings might suggest that the reason ATP is universally conserved across life is that its formation is chemically favoured in aqueous solution under mild prebiotic conditions.

Repeat sequences limit the effectiveness of lateral gene transfer and favored the evolution of meiotic sex in early eukaryotes.

The transition from prokaryotic lateral gene transfer to eukaryotic meiotic sex is poorly understood. Phylogenetic evidence suggests that it was tightly linked to eukaryogenesis, which involved an unprecedented rise in both genome size and the density of genetic repeats. Expansion of genome size raised the severity of Muller's ratchet, while limiting the effectiveness of lateral gene transfer (LGT) at purging deleterious mutations. In principle, an increase in recombination length combined with higher rates of LGT could solve this problem. Here, we show using a computational model that this solution fails in the presence of genetic repeats prevalent in early eukaryotes. The model demonstrates that dispersed repeat sequences allow ectopic recombination, which leads to the loss of genetic information and curtails the capacity of LGT to prevent mutation accumulation. Increasing recombination length in the presence of repeat sequences exacerbates the problem. Mutational decay can only be resisted with homology along extended sequences of DNA. We conclude that the transition to homologous pairing along linear chromosomes was a key innovation in meiotic sex, which was instrumental in the expansion of eukaryotic genomes and morphological complexity.

A biophysical basis for the emergence of the genetic code in protocells.

The origin of the genetic code is an abiding mystery in biology. Hints of a 'code within the codons' suggest biophysical interactions, but these patterns have resisted interpretation. Here, we present a new framework, grounded in the autotrophic growth of protocells from CO2 and H2. Recent work suggests that the universal core of metabolism recapitulates a thermodynamically favoured protometabolism right up to nucleotide synthesis. Considering the genetic code in relation to an extended protometabolism allows us to predict most codon assignments. We show that the first letter of the codon corresponds to the distance from CO2 fixation, with amino acids encoded by the purines (G followed by A) being closest to CO2 fixation. These associations suggest a purine-rich early metabolism with a restricted pool of amino acids. The second position of the anticodon corresponds to the hydrophobicity of the amino acid encoded. We combine multiple measures of hydrophobicity to show that this correlation holds strongly for early amino acids but is weaker for later species. Finally, we demonstrate that redundancy at the third position is not randomly distributed around the code: non-redundant amino acids can be assigned based on size, specifically length. We attribute this to additional stereochemical interactions at the anticodon. These rules imply an iterative expansion of the genetic code over time with codon assignments depending on both distance from CO2 and biophysical interactions between nucleotide sequences and amino acids. In this way the earliest RNA polymers could produce non-random peptide sequences with selectable functions in autotrophic protocells.

Spontaneous assembly of redox-active iron-sulfur clusters at low concentrations of cysteine.

Iron-sulfur (FeS) proteins are ancient and fundamental to life, being involved in electron transfer and CO2 fixation. FeS clusters have structures similar to the unit-cell of FeS minerals such as greigite, found in hydrothermal systems linked with the origin of life. However, the prebiotic pathway from mineral surfaces to biological clusters is unknown. Here we show that FeS clusters form spontaneously through interactions of inorganic Fe2+/Fe3+ and S2- with micromolar concentrations of the amino acid cysteine in water at alkaline pH. Bicarbonate ions stabilize the clusters and even promote cluster formation alone at concentrations >10 mM, probably through salting-out effects. We demonstrate robust, concentration-dependent formation of [4Fe4S], [2Fe2S] and mononuclear iron clusters using UV-Vis spectroscopy, 57Fe-Mössbauer spectroscopy and 1H-NMR. Cyclic voltammetry shows that the clusters are redox-active. Our findings reveal that the structures responsible for biological electron transfer and CO2 reduction could have formed spontaneously from monomers at the origin of life.

Mitonuclear Interactions Produce Diverging Responses to Mild Stress in Drosophila Larvae.

Mitochondrial function depends on direct interactions between respiratory proteins encoded by genes in two genomes, mitochondrial and nuclear, which evolve in very different ways. Serious incompatibilities between these genomes can have severe effects on development, fitness and viability. The effect of subtle mitonuclear mismatches has received less attention, especially when subject to mild physiological stress. Here, we investigate how two distinct physiological stresses, metabolic stress (high-protein diet) and redox stress [the glutathione precursor N-acetyl cysteine (NAC)], affect development time, egg-to-adult viability, and the mitochondrial physiology of Drosophila larvae with an isogenic nuclear background set against three mitochondrial DNA (mtDNA) haplotypes: one coevolved (WT) and two slightly mismatched (COX and BAR). Larvae fed the high-protein diet developed faster and had greater viability in all haplotypes. The opposite was true of NAC-fed flies, especially those with the COX haplotype. Unexpectedly, the slightly mismatched BAR larvae developed fastest and were the most viable on both treatments, as well as control diets. These changes in larval development were linked to a shift to complex I-driven mitochondrial respiration in all haplotypes on the high-protein diet. In contrast, NAC increased respiration in COX larvae but drove a shift toward oxidation of proline and succinate. The flux of reactive oxygen species was increased in COX larvae treated with NAC and was associated with an increase in mtDNA copy number. Our results support the notion that subtle mitonuclear mismatches can lead to diverging responses to mild physiological stress, undermining fitness in some cases, but surprisingly improving outcomes in other ostensibly mismatched fly lines.

The need for high-quality oocyte mitochondria at extreme ploidy dictates mammalian germline development.

Selection against deleterious mitochondrial mutations is facilitated by germline processes, lowering the risk of genetic diseases. How selection works is disputed: experimental data are conflicting and previous modeling work has not clarified the issues; here, we develop computational and evolutionary models that compare the outcome of selection at the level of individuals, cells and mitochondria. Using realistic de novo mutation rates and germline development parameters from mouse and humans, the evolutionary model predicts the observed prevalence of mitochondrial mutations and diseases in human populations. We show the importance of organelle-level selection, seen in the selective pooling of mitochondria into the Balbiani body, in achieving high-quality mitochondria at extreme ploidy in mature oocytes. Alternative mechanisms debated in the literature, bottlenecks and follicular atresia, are unlikely to account for the clinical data, because neither process effectively eliminates mitochondrial mutations under realistic conditions. Our findings explain the major features of female germline architecture, notably the longstanding paradox of over-proliferation of primordial germ cells followed by massive loss. The near-universality of these processes across animal taxa makes sense in light of the need to maintain mitochondrial quality at extreme ploidy in mature oocytes, in the absence of sex and recombination.

Mother's curse is pervasive across a large mitonuclear Drosophila panel.

The maternal inheritance of mitochondrial genomes entails a sex-specific selective sieve, whereby mutations in mitochondrial DNA can only respond to selection acting on females. In theory, this enables male-harming mutations to accumulate in mitochondrial genomes as long as they are neutral, beneficial, or only slightly deleterious to females. Ultimately, this bias could drive the evolution of male-specific mitochondrial mutation loads, an idea known as mother's curse. Earlier work on this hypothesis has mainly used small Drosophila panels, in which naturally sourced mitochondrial genomes were coupled to an isogenic nuclear background. The lack of nuclear genetic variation in these designs has precluded robust generalization. Here, we test the predictions of mother's curse using a large Drosophila mitonuclear genetic panel, comprising nine isogenic nuclear genomes coupled to nine mitochondrial haplotypes, giving a total of 81 different mitonuclear genotypes. Following a predictive framework, we tested the mother's curse hypothesis by screening our panel for wing size. This trait is tightly correlated with overall body size and is sexually dimorphic in Drosophila. Moreover, growth is heavily reliant on metabolism and mitochondrial function, making wing size an ideal trait for the study of the impact of mitochondrial variation. We detect high levels of mitonuclear epistasis, and more importantly, we report that mitochondrial genetic variance is larger in male than female Drosophila for eight out of the nine nuclear genetic backgrounds used. These results demonstrate that the maternal inheritance of mitochondrial DNA does indeed modulate male life history traits in a more generalisable way than previously demonstrated.

CO2 reduction driven by a pH gradient.

All life on Earth is built of organic molecules, so the primordial sources of reduced carbon remain a major open question in studies of the origin of life. A variant of the alkaline-hydrothermal-vent theory for life's emergence suggests that organics could have been produced by the reduction of CO2 via H2 oxidation, facilitated by geologically sustained pH gradients. The process would be an abiotic analog-and proposed evolutionary predecessor-of the Wood-Ljungdahl acetyl-CoA pathway of modern archaea and bacteria. The first energetic bottleneck of the pathway involves the endergonic reduction of CO2 with H2 to formate (HCOO-), which has proven elusive in mild abiotic settings. Here we show the reduction of CO2 with H2 at room temperature under moderate pressures (1.5 bar), driven by microfluidic pH gradients across inorganic Fe(Ni)S precipitates. Isotopic labeling with 13C confirmed formate production. Separately, deuterium (2H) labeling indicated that electron transfer to CO2 does not occur via direct hydrogenation with H2 but instead, freshly deposited Fe(Ni)S precipitates appear to facilitate electron transfer in an electrochemical-cell mechanism with two distinct half-reactions. Decreasing the pH gradient significantly, removing H2, or eliminating the precipitate yielded no detectable product. Our work demonstrates the feasibility of spatially separated yet electrically coupled geochemical reactions as drivers of otherwise endergonic processes. Beyond corroborating the ability of early-Earth alkaline hydrothermal systems to couple carbon reduction to hydrogen oxidation through biologically relevant mechanisms, these results may also be of significance for industrial and environmental applications, where other redox reactions could be facilitated using similarly mild approaches.

Genome expansion in early eukaryotes drove the transition from lateral gene transfer to meiotic sex.

Prokaryotes acquire genes from the environment via lateral gene transfer (LGT). Recombination of environmental DNA can prevent the accumulation of deleterious mutations, but LGT was abandoned by the first eukaryotes in favour of sexual reproduction. Here we develop a theoretical model of a haploid population undergoing LGT which includes two new parameters, genome size and recombination length, neglected by previous theoretical models. The greater complexity of eukaryotes is linked with larger genomes and we demonstrate that the benefit of LGT declines rapidly with genome size. The degeneration of larger genomes can only be resisted by increases in recombination length, to the same order as genome size - as occurs in meiosis. Our results can explain the strong selective pressure towards the evolution of sexual cell fusion and reciprocal recombination during early eukaryotic evolution - the origin of meiotic sex.

How energy flow shapes cell evolution.

How mitochondria shaped the evolution of eukaryotic complexity has been controversial for decades. The discovery of the Asgard archaea, which harbor close phylogenetic ties to the eukaryotes, supports the idea that a critical endosymbiosis between an archaeal host and a bacterial endosymbiont transformed the selective constraints present at the origin of eukaryotes. Cultured Asgard archaea are typically prokaryotic in both size and internal morphology, albeit featuring extensive protrusions. The acquisition of the mitochondrial predecessor by an archaeal host cell fundamentally altered the topology of genes in relation to bioenergetic membranes. Mitochondria internalised not only the bioenergetic membranes but also the genetic machinery needed for local control of oxidative phosphorylation. Gene loss from mitochondria enabled expansion of the nuclear genome, giving rise to an extreme genomic asymmetry that is ancestral to all extant eukaryotes. This genomic restructuring gave eukaryotes thousands of fold more energy availability per gene. In principle, that difference can support more and larger genes, far more non-coding DNA, greater regulatory complexity, and thousands of fold more protein synthesis per gene. These changes released eukaryotes from the bioenergetic constraints on prokaryotes, facilitating the evolution of morphological complexity.

Impact of mitonuclear interactions on life-history responses to diet.

Mitochondria are central to both energy metabolism and biosynthesis. Mitochondrial function could therefore influence resource allocation. Critically, mitochondrial function depends on interactions between proteins encoded by the mitochondrial and nuclear genomes. Severe incompatibilities between these genomes can have pervasive effects on both fitness and longevity. How milder deficits in mitochondrial function affect life-history trade-offs is less well understood. Here, we analyse how mitonuclear interactions affect the trade-off between fecundity and longevity in Drosophila melanogaster. We consider a panel of 10 different mitochondrial DNA haplotypes against two contrasting nuclear backgrounds (w1118 (WE) and Zim53 (ZIM)) in response to high-protein versus standard diet. We report strikingly different responses between the two nuclear backgrounds. WE females have higher fecundity and decreased longevity on high protein. ZIM females have much greater fecundity and shorter lifespan than WE flies on standard diet. High protein doubled their fecundity with no effect on longevity. Mitochondrial haplotype reflected nuclear life-history trade-offs, with a negative correlation between longevity and fecundity in WE flies and no correlation in ZIM flies. Mitonuclear interactions had substantial effects but did not reflect genetic distance between mitochondrial haplotypes. We conclude that mitonuclear interactions can have significant impact on life-history trade-offs, but their effects are not predictable by relatedness. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Promotion of protocell self-assembly from mixed amphiphiles at the origin of life.

Vesicles formed from single-chain amphiphiles (SCAs) such as fatty acids probably played an important role in the origin of life. A major criticism of the hypothesis that life arose in an early ocean hydrothermal environment is that hot temperatures, large pH gradients, high salinity and abundant divalent cations should preclude vesicle formation. However, these arguments are based on model vesicles using 1-3 SCAs, even though Fischer-Tropsch-type synthesis under hydrothermal conditions produces a wide array of fatty acids and 1-alkanols, including abundant C10-C15 compounds. Here, we show that mixtures of these C10-C15 SCAs form vesicles in aqueous solutions between pH ~6.5 and >12 at modern seawater concentrations of NaCl, Mg2+ and Ca2+. Adding C10 isoprenoids improves vesicle stability even further. Vesicles form most readily at temperatures of ~70 °C and require salinity and strongly alkaline conditions to self-assemble. Thus, alkaline hydrothermal conditions not only permit protocell formation at the origin of life but actively favour it.

Isoprenoids enhance the stability of fatty acid membranes at the emergence of life potentially leading to an early lipid divide.

Two key problems concern cell membranes during the emergence and early evolution of life: what was their initial composition, and why did the membranes of archaea and bacteria diverge? The composition of the first cell membranes could shed light on the most likely environment for the emergence of life. The opposing stereochemistry of modern lipid glycerol-phosphate headgroups in bacteria and archaea suggests that early membranes were composed of single chain amphiphiles, perhaps both fatty acids and isoprenoids. We investigated the effect of adding isoprenoids to fatty acid membranes using a combination of UV-visible spectroscopy, confocal microscopy and transmission electron microscopy. We tested the stability of these membranes across a pH range and under different concentrations of ionic species relevant to oceanic hydrothermal environments, including Na2+, Cl-, Mg2+, Ca2+, HC O 3 - , Fe3+, Fe2+ and S2-. We also tested the assembly of vesicles in the presence of Fe particles and FeS precipitates. We found that isoprenoids enhance the stability of membranes in the presence of salts but require 30-fold higher concentrations for membrane formation. Intriguingly, isoprenoids strongly inhibit the tendency of vesicles to aggregate together in the presence of either Fe particles or FeS precipitates. These striking physical differences in the stability and aggregation of protocells may have shaped the divergence of bacteria and archaea in early hydrothermal environments.