Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation.

Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in environmental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.

A CRISPR interference system for engineering biological nitrogen fixation.

A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, Azotobacter vinelandii (A. vinelandii). Here, we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in A. vinelandii that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by ~60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system fills an important gap for engineering microbial nitrogen fixation for desired purposes.IMPORTANCEAll life on Earth requires nitrogen to survive. About 78% of the atmosphere alone is nitrogen, yet humans cannot use it directly. Instead, we obtain the nitrogen we need for our survival through the food we eat. For more than 100 years, a substantial portion of agricultural productivity has relied on industrial methods for nitrogen fertilizer synthesis, which consumes significant amounts of nonrenewable energy resources and exacerbates environmental degradation and human-induced climate change. Promising alternatives to these industrial methods rely on engineering the only biological pathway for generating bioaccessible nitrogen: microbial nitrogen fixation. Bioengineering strategies require an extensive understanding of underlying genetics in nitrogen-fixing microbes, but genetic tools for this critical goal remain lacking. The CRISPRi gene silencing system that we report, developed in the broadly utilized nitrogen-fixing bacterial model, Azotobacter vinelandii, is an important step toward elucidating the complexity of nitrogen fixation genetics and enabling their manipulation.

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background.

Synonymous mutations are changes to DNA sequence, which occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation elongation factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels as well as global polysome abundance on RNA transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.IMPORTANCEThis study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein elongation factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway.

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron-sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron-sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways.

The modular biochemical reaction network structure of cellular translation.

Translation is an essential attribute of all living cells. At the heart of cellular operation, it is a chemical information decoding process that begins with an input string of nucleotides and ends with the synthesis of a specific output string of peptides. The translation process is interconnected with gene expression, physiological regulation, transcription, and responses to signaling molecules, among other cellular functions. Foundational efforts have uncovered a wealth of knowledge about the mechanistic functions of the components of translation and their many interactions between them, but the broader biochemical connections between translation, metabolism and polymer biosynthesis that enable translation to occur have not been comprehensively mapped. Here we present a multilayer graph of biochemical reactions describing the translation, polymer biosynthesis and metabolism networks of an Escherichia coli cell. Intriguingly, the compounds that compose these three layers are distinctly aggregated into three modes regardless of their layer categorization. Multimodal mass distributions are well-known in ecosystems, but this is the first such distribution reported at the biochemical level. The degree distributions of the translation and metabolic networks are each likely to be heavy-tailed, but the polymer biosynthesis network is not. A multimodal mass-degree distribution indicates that the translation and metabolism networks are each distinct, adaptive biochemical modules, and that the gaps between the modes reflect evolved responses to the functional use of metabolite, polypeptide and polynucleotide compounds. The chemical reaction network of cellular translation opens new avenues for exploring complex adaptive phenomena such as percolation and phase changes in biochemical contexts.

A beneficial synonymous substitution in EF-Tu is contingent on genetic background.

Synonymous mutations are changes to DNA sequence that occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation Elongation Factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels, as well as the polysome abundance on global transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.

Assessment of Stoichiometric Autocatalysis across Element Groups.

Autocatalysis has been proposed to play critical roles during abiogenesis. These proposals are at odds with a limited number of known examples of abiotic (and, in particular, inorganic) autocatalytic systems that might reasonably function in a prebiotic environment. In this study, we broadly assess the occurrence of stoichiometries that can support autocatalytic chemical systems through comproportionation. If the product of a comproportionation reaction can be coupled with an auxiliary oxidation or reduction pathway that furnishes a reactant, then a Comproportionation-based Autocatalytic Cycle (CompAC) can exist. Using this strategy, we surveyed the literature published in the past two centuries for reactions that can be organized into CompACs that consume some chemical species as food to synthesize more autocatalysts. 226 CompACs and 44 Broad-sense CompACs were documented, and we found that each of the 18 groups, lanthanoid series, and actinoid series in the periodic table has at least two CompACs. Our findings demonstrate that stoichiometric relationships underpinning abiotic autocatalysis could broadly exist across a range of geochemical and cosmochemical conditions, some of which are substantially different from the modern Earth. Meanwhile, the observation of some autocatalytic systems requires effective spatial or temporal separation between the food chemicals while allowing comproportionation and auxiliary reactions to proceed, which may explain why naturally occurring autocatalytic systems are not frequently observed. The collated CompACs and the conditions in which they might plausibly support complex, "life-like" chemical dynamics can directly aid an expansive assessment of life's origins and provide a compendium of alternative hypotheses concerning false-positive biosignatures.

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii.

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

Informatic Capabilities of Translation and Its Implications for the Origins of Life.

The ability to encode and convert heritable information into molecular function is a defining feature of life as we know it. The conversion of information into molecular function is performed by the translation process, in which triplets of nucleotides in a nucleic acid polymer (mRNA) encode specific amino acids in a protein polymer that folds into a three-dimensional structure. The folded protein then performs one or more molecular activities, often as one part of a complex and coordinated physiological network. Prebiotic systems, lacking the ability to explicitly translate information between genotype and phenotype, would have depended upon either chemosynthetic pathways to generate its components-constraining its complexity and evolvability- or on the ambivalence of RNA as both carrier of information and of catalytic functions-a possibility which is still supported by a very limited set of catalytic RNAs. Thus, the emergence of translation during early evolutionary history may have allowed life to unmoor from the setting of its origin. The origin of translation machinery also represents an entirely novel and distinct threshold of behavior for which there is no abiotic counterpart-it could be the only known example of computing that emerged naturally at the chemical level. Here we describe translation machinery's decoding system as the basis of cellular translation's information-processing capabilities, and the four operation types that find parallels in computer systems engineering that this biological machinery exhibits.

Enigmatic evolution of microbial nitrogen fixation: insights from Earth's past.

The evolution of nitrogen fixation undoubtedly altered nearly all corners of the biosphere, given the essential role of nitrogen in the synthesis of biomass. To date, there is no unified view on what planetary conditions gave rise to nitrogen fixation or how these conditions have sustained it evolutionarily. Intriguingly, the concentrations of metals that nitrogenases require to function have changed throughout Earth's history. In this review, we describe the interconnection of the metal and nitrogen cycles with nitrogenase evolution and the importance of ancient ecology in the formation of the modern nitrogen cycle. We argue that exploration of the nitrogen cycle's deep past will provide insights into humanity's immediate environmental challenges centered on nitrogen availability.

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism.

The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, which is also exhibited by extant A. vinelandii nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism.

Effects of RuBisCO and CO2 concentration on cyanobacterial growth and carbon isotope fractionation.

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a specific range of genetic and environmental factors that may impact ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO across varying atmospheric CO2 concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO2 fixation versus CO2 supply, and thus whole-cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO2 concentrations on both carbon isotope biosignatures and cyanobacterial physiology, including cell growth and oxygen evolution rates. We found that an increased pool of active RuBisCO does not significantly affect the 13 C/12 C isotopic discrimination (εp ) at all tested CO2 concentrations, yielding εp of ≈ 23‰ for both wild-type and mutant strains at elevated CO2 . We therefore suggest that expected variation in cyanobacterial RuBisCO expression patterns should not confound carbon isotope biosignature interpretation. A deeper understanding of environmental, evolutionary, and intracellular factors that impact cyanobacterial physiology and isotope discrimination is crucial for reconciling microbially driven carbon biosignatures with those preserved in the geologic record.

Very early evolution from the perspective of microbial ecology.

The universal ancestor at the root of the species tree of life depicts a population of organisms with a surprising degree of complexity, posessing genomes and translation systems much like that of microbial life today. As the first life forms were most likely to have been simple replicators, considerable evolutionary change must have taken place prior to the last universal common ancestor. It is often assumed that the lack of earlier branches on the tree of life is due to a prevalence of random horizontal gene transfer that obscured the delineations between lineages and hindered their divergence. Therefore, principles of microbial evolution and ecology may give us some insight into these early stages in the history of life. Here, we synthesize the current understanding of organismal and genome evolution from the perspective of microbial ecology and apply these evolutionary principles to the earliest stages of life on Earth. We focus especially on broad evolutionary modes pertaining to horizontal gene transfer, pangenome structure, and microbial mat communities.

Early Nitrogenase Ancestors Encompassed Novel Active Site Diversity.

Ancestral sequence reconstruction (ASR) infers predicted ancestral states for sites within sequences and can constrain the functions and properties of ancestors of extant protein families. Here, we compare the likely sequences of inferred nitrogenase ancestors to extant nitrogenase sequence diversity. We show that the most-likely combinations of ancestral states for key substrate channel residues are not represented in extant sequence space, and rarely found within a more broadly defined physiochemical space-supporting that the earliest ancestors of extant nitrogenases likely had alternative substrate channel composition. These differences may indicate differing environmental selection pressures acting on nitrogenase substrate specificity in ancient environments. These results highlight ASR's potential as an in silico tool for developing hypotheses about ancestral enzyme functions, as well as improving hypothesis testing through more targeted in vitro and in vivo experiments.

Early divergence of translation initiation and elongation factors.

Protein translation is a foundational attribute of all living cells. The translation function carried out by the ribosome critically depends on an assortment of protein interaction partners, collectively referred to as the translation machinery. Various studies suggest that the diversification of the translation machinery occurred prior to the last universal common ancestor, yet it is unclear whether the predecessors of the extant translation machinery factors were functionally distinct from their modern counterparts. Here we reconstructed the shared ancestral trajectory and subsequent evolution of essential translation factor GTPases, elongation factor EF-Tu (aEF-1A/eEF-1A), and initiation factor IF2 (aIF5B/eIF5B). Based upon their similar functions and structural homologies, it has been proposed that EF-Tu and IF2 emerged from an ancient common ancestor. We generated the phylogenetic tree of IF2 and EF-Tu proteins and reconstructed ancestral sequences corresponding to the deepest nodes in their shared evolutionary history, including the last common IF2 and EF-Tu ancestor. By identifying the residue and domain substitutions, as well as structural changes along the phylogenetic history, we developed an evolutionary scenario for the origins, divergence and functional refinement of EF-Tu and IF2 proteins. Our analyses suggest that the common ancestor of IF2 and EF-Tu was an IF2-like GTPase protein. Given the central importance of the translation machinery to all cellular life, its earliest evolutionary constraints and trajectories are key to characterizing the universal constraints and capabilities of cellular evolution.

An Integrated Method to Reconstruct Ancient Proteins.

Proteins have played a fundamental role throughout life's history on Earth. Despite their biological importance, ancient origin, early function, and evolution of proteins are seldom able to be directly studied because few of these attributes are preserved across geologic timescales. Ancestral sequence reconstruction (ASR) provides a method to infer ancestral amino acid sequences and determine the evolutionary predecessors of modern-day proteins using phylogenetic tools. Laboratory application of ASR allows ancient sequences to be deduced from genetic information available in extant organisms and then experimentally resurrected to elucidate ancestral characteristics. In this article, we provide a generalized, stepwise protocol that considers the major elements of a well-designed ASR study and details potential sources of reconstruction bias that can reduce the relevance of historical inferences. We underscore key stages in our approach so that it may be broadly utilized to reconstruct the evolutionary histories of proteins.

Earliest Photic Zone Niches Probed by Ancestral Microbial Rhodopsins.

For billions of years, life has continuously adapted to dynamic physical conditions near the Earth's surface. Fossils and other preserved biosignatures in the paleontological record are the most direct evidence for reconstructing the broad historical contours of this adaptive interplay. However, biosignatures dating to Earth's earliest history are exceedingly rare. Here, we combine phylogenetic inference of primordial rhodopsin proteins with modeled spectral features of the Precambrian Earth environment to reconstruct the paleobiological history of this essential family of photoactive transmembrane proteins. Our results suggest that ancestral microbial rhodopsins likely acted as light-driven proton pumps and were spectrally tuned toward the absorption of green light, which would have enabled their hosts to occupy depths in a water column or biofilm where UV wavelengths were attenuated. Subsequent diversification of rhodopsin functions and peak absorption frequencies was enabled by the expansion of surface ecological niches induced by the accumulation of atmospheric oxygen. Inferred ancestors retain distinct associations between extant functions and peak absorption frequencies. Our findings suggest that novel information encoded by biomolecules can be used as "paleosensors" for conditions of ancient, inhabited niches of host organisms not represented elsewhere in the paleontological record. The coupling of functional diversification and spectral tuning of this taxonomically diverse protein family underscores the utility of rhodopsins as universal testbeds for inferring remotely detectable biosignatures on inhabited planetary bodies.

Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time.

The earliest geochemical indicators of microbes-and the enzymes that powered them-extend back ∼3.8 Ga on Earth. Paleobiologists often attempt to understand these indicators by assuming that the behaviors of extant microbes and enzymes are uniform with those of their predecessors. This consistency in behavior seems at odds with our understanding of the inherent variability of living systems. Here, we examine whether a uniformitarian assumption for an enzyme thought to generate carbon isotope indicators of biological activity, RuBisCO, can be corroborated by independently studying the history of changes recorded within RuBisCO's genetic sequences. We resurrected a Precambrian-age RuBisCO by engineering its ancient DNA inside a cyanobacterium genome and measured the engineered organism's fitness and carbon-isotope-discrimination profile. Results indicate that Precambrian uniformitarian assumptions may be warranted but with important caveats. Experimental studies illuminating early innovations are crucial to explore the molecular foundations of life's earliest traces.

Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins.

The evolution of biological nitrogen fixation, uniquely catalyzed by nitrogenase enzymes, has been one of the most consequential biogeochemical innovations over life's history. Though understanding the early evolution of nitrogen fixation has been a longstanding goal from molecular, biogeochemical, and planetary perspectives, its origins remain enigmatic. In this study, we reconstructed the evolutionary histories of nitrogenases, as well as homologous maturase proteins that participate in the assembly of the nitrogenase active-site cofactor but are not able to fix nitrogen. We combined phylogenetic and ancestral sequence inference with an analysis of predicted functionally divergent sites between nitrogenases and maturases to infer the nitrogen-fixing capabilities of their shared ancestors. Our results provide phylogenetic constraints to the emergence of nitrogen fixation and are consistent with a model wherein nitrogenases emerged from maturase-like predecessors. Though the precise functional role of such a predecessor protein remains speculative, our results highlight evolutionary contingency as a significant factor shaping the evolution of a biogeochemically essential enzyme.