Faculty & Staff

Start and Promotion Dates

  • Assistant Professor: 2021

Education

PhD: Emory University, Department of Chemistry
Postdoctoral research: NASA Astrobiology Institute and Harvard University

Areas of Study

Origins of life, astrobiology, early life and evolution, systems and synthetic biology, molecular paleobiology

Research Overview

The Kaçar Lab investigates the origins of life, the biology of early Earth and how understanding life’s emergence and early mechanisms may assist finding life beyond Earth. We are home to the NASA Astrobiology Center for Early Life and Evolution. Our integrative approach enables the study of biomolecule-scale macroevolutionary trends that span billions of years of history and is a fundamentally new methodology with which to study the origins and early evolution of life. The overall goal of our work is to assess the possible environmental impacts of ancient enzymes on global-scale signatures that record biological activity. Our work has been recognized by various media outlets, such as the UN Women, UNICEF, Library of Congress, European Union Delegation on Education, NOVA Science, BBC, NPR Science Friday, MIT Technology Review, Vice News, Wired, PBS, CNN and others. 

 

For more please visit the following links:

 

Lab Personnel

Picture of Adam
Zachary Adam
Associate Scientist
zadam2@wisc.edu
Picture of Amritkar
Kaustubh Amritkar
Grad Student
amritkar@wisc.edu
Picture of Cuevas Zuviria
Bruno Cuevas Zuviria
Honorary Researcher
cuevaszuviri@wisc.edu
Picture of Fer
Evrim Fer
Grad Student
fer@wisc.edu
Picture of Garcia
Amanda Garcia
Scientist
akgarcia3@wisc.edu
Picture of Katsoulidis
Maria Katsoulidis
Project Coordinator
katsoulidis@wisc.edu
Picture of Klos
Aya Klos
Honorary Associate
asklos@wisc.edu
Picture of McGrath
Kaitlyn McGrath
Honorary Associate
kmcgrath5@wisc.edu
Picture of Rucker
Holly Rucker
Grad Student
hrucker@wisc.edu
Picture of Sobol
Morgan Sobol
Postdoc
msobol@wisc.edu
Picture of Wozniak
Chris Wozniak
Scientist
cewozniak@wisc.edu
Picture of Yao
Tony Yao
Grad Student
tyao42@wisc.edu

Research Papers

  • Harris DF, Rucker HR, Garcia AK, Yang Z-Y, Chang SD, Feinsilber H, Kaçar B, Seefeldt LC (2024) Ancient nitrogenases are ATP dependent. mBio 15((7)):e0127124 PMC9490801 · Pubmed · DOI

    Life depends on a conserved set of chemical energy currencies that are relics of early biochemistry. One of these is ATP, a molecule that, when paired with a divalent metal ion such as Mg, can be hydrolyzed to support numerous cellular and molecular processes. Despite its centrality to extant biochemistry, it is unclear whether ATP supported the function of ancient enzymes. We investigate the evolutionary necessity of ATP by experimentally reconstructing an ancestral variant of the N-reducing enzyme nitrogenase. The Proterozoic ancestor is predicted to be ~540-2,300 million years old, post-dating the Great Oxidation Event. Growth rates under nitrogen-fixing conditions are ~80% of those of wild type in Azotobacter vinelandii . In the extant enzyme, the hydrolysis of two MgATP is coupled to electron transfer to support substrate reduction. The ancestor has a strict requirement for ATP with no other nucleotide triphosphate analogs (GTP, ITP, and UTP) supporting activity. Alternative divalent metal ions (Fe, Co, and Mn) support activity with ATP but with diminished activities compared to Mg, similar to the extant enzyme. Additionally, it is shown that the ancestor has an identical efficiency in ATP hydrolyzed per electron transferred to the extant of two. Our results provide direct laboratory evidence of ATP usage by an ancient enzyme.IMPORTANCELife depends on energy-carrying molecules to power many sustaining processes. There is evidence that these molecules may predate the rise of life on Earth, but how and when these dependencies formed is unknown. The resurrection of ancient enzymes provides a unique tool to probe the enzyme's function and usage of energy-carrying molecules, shedding light on their biochemical origins. Through experimental reconstruction, this research investigates the ancestral dependence of a nitrogen-fixing enzyme on the energy carrier ATP, a requirement for function in the modern enzyme. We show that the resurrected ancestor does not have generalist nucleotide specificity. Rather, the ancestor has a strict requirement for ATP, like the modern enzyme, with similar function and efficiency. The findings elucidate the early-evolved necessity of energy-yielding molecules, delineating their role in ancient biochemical processes. Ultimately, these insights contribute to unraveling the intricate tapestry of evolutionary biology and the origins of life-sustaining dependencies.

  • Timmis K, Hallsworth JE, McGenity TJ, Armstrong R, Colom MF, Karahan ZC, Chavarría M, Bernal P, Boyd ES, Ramos JL, Kaltenpoth M, Pruzzo C, Clarke G, López-Garcia P, Yakimov MM, Perlmutter J, Greening C, Eloe-Fadrosh E, Verstraete W, Nunes OC, Kotsyurbenko O, Nikel PI, Scavone P, Häggblom MM, Lavigne R, Le Roux F, Timmis JK, Parro V, Michán C, García JL, Casadevall A, Payne SM, Frey J, Koren O, Prosser JI, Lahti L, Lal R, Anand S, Sood U, Offre P, Bryce CC, Mswaka AY, Jores J, Kaçar B, Blank LM, Maaßen N, Pope PB, Banciu HL, Armitage J, Lee SY, Wang F, Makhalanyane TP, Gilbert JA, Wood TK, Vasiljevic B, Soberón M, Udaondo Z, Rojo F, Tamang JP, Giraud T, Ropars J, Ezeji T, Müller V, Danbara H, Averhoff B, Sessitsch A, Partida-Martínez LP, Huang W, Molin S, Junier P, Amils R, Wu XL, Ron E, Erten H, de Martinis ECP, Rapoport A, Öpik M, Pokatong WDR, Stairs C, Amoozegar MA, Serna JG (2024) A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society. Microbial biotechnology 17((5)):e14456 PMC9595278 · Pubmed · DOI

    Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient.

  • Cuevas-Zuviría B, Garcia AK, Rivier AJ, Rucker HR, Carruthers BM, Kaçar B (2024) Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation. Molecular biology and evolution 41((4)): PMC176766 · Pubmed · DOI

    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.

  • Russell SJ, Garcia AK, Kaçar B (2024) A CRISPR interference system for engineering biological nitrogen fixation. mSystems 9((3)):e0015524 PMC4837600 · Pubmed · DOI

    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.

  • McGrath KM, Russell SJ, Fer E, Garmendia E, Hosgel A, Baltrus DA, Kaçar B (2024) Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background. Journal of bacteriology 206((2)):e0032923 PMC8419925 · Pubmed · DOI

    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.

  • Marshall B, Amritkar K, Wolfe M, Kaçar B, Landick R (2023) Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway. Frontiers in microbiology 14:1286626 PMC3487848 · Pubmed · DOI

    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.

  • Cuevas-Zuviría B, Fer E, Adam ZR, Kaçar B (2023) The modular biochemical reaction network structure of cellular translation. NPJ systems biology and applications 9((1)):52 PMC6504708 · Pubmed · DOI

    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.

  • McGrath KM, Russell SJ, Fer E, Garmendia E, Hosgel A, Baltrus DA, Kaçar B (2023) A beneficial synonymous substitution in EF-Tu is contingent on genetic background. bioRxiv : the preprint server for biology : PMC8419925 · Pubmed · DOI

    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.

  • Peng Z, Adam ZR, Fahrenbach AC, Kaçar B (2023) Assessment of Stoichiometric Autocatalysis across Element Groups. Journal of the American Chemical Society 145((41)):22483-22493 PMC7216921 · Pubmed · DOI

    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 Comp roportionation-based A utocatalytic C ycle (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.

  • Rivier AJ, Myers KS, Garcia AK, Sobol MS, Kaçar B (2023) Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii . Microbiology spectrum 11((5)):e0281523 PMC3339379 · Pubmed · DOI

    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.

  • Cuevas-Zuviría B, Adam ZR, Goldman AD, Kaçar B (2023) Informatic Capabilities of Translation and Its Implications for the Origins of Life. Journal of molecular evolution 91((5)):567-569 · Pubmed · DOI

    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.

  • Evans GN, Coogan LA, Kaçar B, Seyfried WE (2023) Molybdenum in basalt-hosted seafloor hydrothermal systems: Experimental, theoretical, and field sampling approaches Geochimica et Cosmochimica Acta 353:28-44 · DOI

    Seafloor hydrothermal vents represent potential sources of Mo and other biologically relevant transition metals to the global ocean, complementing continental runoff. Here, we use a combination of experimental, theoretical, and field-sampling approaches to investigate the behavior of Mo in basalt-hosted seafloor hydrothermal systems to provide insight into the processes controlling Mo concentrations in hydrothermal fluids and to derive estimates of vent fluid Mo concentrations and fluxes. Results of this study demonstrate that reaction fluids generated from 350 °C, 500 bar hydrothermal basalt alteration experiments contain 775–801 nmol/kg Mo and are thus comparable to a recently collected time series of natural seafloor vent fluids that contained 200–220 nmol/kg Mo at 302 °C and 29–30 nmol/kg at 281–282 °C (Evans et al., 2023). Synchrotron-based analyses of experimentally altered basalt produced in this study and additional natural samples of altered oceanic crust originally collected from Pito Deep Rift reveal the presence of Mo-rich particles consistent with trace molybdenite. Comparisons of Mo:Cu ratios in natural vent fluids and near-vent sediment trap samples from Main Endeavour Field indicate that vent fluid Mo is readily incorporated into buoyant plume particles and advected out of the near-vent field, analogous to previous mass balance studies of Cu in this region. Thermodynamic calculations of molybdenite solubility in the context of mineral-buffered hydrothermal fluids and comparisons with natural and experimental hydrothermal fluids suggest that high-temperature vent fluids contain 30–1500 nmol/kg Mo. While a minor component of the modern Mo budget, hydrothermal Mo fluxes are estimated to have constituted 0.3–200× the contemporaneous continental weathering fluxes prior to the ∼2.4 Ga ago “Great Oxidation Event” and widespread oxidative continental weathering. Overall, identification of hydrothermal vents as a source of Mo-rich plume particles with potential for dispersal into the wider marine environment has significant implications for hypotheses regarding the co-evolution of Life and Earth’s environments, specifically the form and availability of Mo in anoxic Archean-Eon oceans, where Mo-dependent enzymatic pathways are thought to have emerged and subsequently evolved.

  • Rucker HR, Kaçar B (2023) Enigmatic evolution of microbial nitrogen fixation: insights from Earth's past. Trends in microbiology 32((6)):554-564 · Pubmed · DOI

    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.

  • Garcia AK, Harris DF, Rivier AJ, Carruthers BM, Pinochet-Barros A, Seefeldt LC, Kaçar B (2023) Nitrogenase resurrection and the evolution of a singular enzymatic mechanism. eLife 12: PMC3103320 · Pubmed · DOI

    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.

  • Amritkar KM, Cuevas-Zuviría B, Kaçar B (2023) Biophysical models for studying patterns of RuBisCO small subunit evolution. Biophysical journal 122((3S1)):45a · Pubmed · DOI

    No abstract available.

  • Garcia AK, Kędzior M, Taton A, Li M, Young JN, Kaçar B (2023) Effects of RuBisCO and CO concentration on cyanobacterial growth and carbon isotope fractionation. Geobiology 21((3)):390-403 · Pubmed · DOI

    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 CO 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 CO concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO fixation versus CO supply, and thus whole-cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO 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 C/ C isotopic discrimination (ε ) at all tested CO concentrations, yielding ε of ≈ 23‰ for both wild-type and mutant strains at elevated CO . 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.

  • Schwartz SL, Garcia AK, Kaçar B, Fournier GP (2022) Early Nitrogenase Ancestors Encompassed Novel Active Site Diversity. Molecular biology and evolution 39((11)): PMC9549741 · Pubmed · DOI

    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.

  • Fer E, McGrath KM, Guy L, Hockenberry AJ, Kaçar B (2022) Early divergence of translation initiation and elongation factors. Protein science : a publication of the Protein Society 31((9)):e4393 PMC1570152 · Pubmed · DOI

    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.

  • Garcia AK, Fer E, Sephus C, Kacar B (2022) An Integrated Method to Reconstruct Ancient Proteins. Methods in molecular biology (Clifton, N.J.) 2569:267-281 · Pubmed · DOI

    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.

  • Goldman AD, Kaçar B (2022) Very early evolution from the perspective of microbial ecology. Environmental microbiology 25((1)):5-10 · Pubmed · DOI

    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.

  • Sephus CD, Fer E, Garcia AK, Adam ZR, Schwieterman EW, Kacar B (2022) Earliest Photic Zone Niches Probed by Ancestral Microbial Rhodopsins. Molecular biology and evolution 39((5)): PMC9117797 · Pubmed · DOI

    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.

  • Kędzior M, Garcia AK, Li M, Taton A, Adam ZR, Young JN, Kaçar B (2022) Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time. Cell reports 39((4)):110726 · Pubmed · DOI

    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.

  • Garcia AK, Kolaczkowski B, Kaçar B (2022) Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins. Genome biology and evolution 14((3)): PMC8890362 · Pubmed · DOI

    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.

  • Carruthers BM, Garcia AK, Rivier A, Kacar B (2022) Correction: Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii. Current protocols 2((1)):e363 PMC9113642 · Pubmed · DOI

    No abstract available.

  • Kędzior M, Kacar B (2021) Quantification of RuBisCO Expression and Photosynthetic Oxygen Evolution in Cyanobacteria. Bio-protocol 11((20)):e4199 PMC8554809 · Pubmed · DOI

    Phototrophic microorganisms are frequently engineered to regulate the expression and the activity of targeted enzymes of interest for specific biotechnological and agricultural applications. This protocol describes a method to evaluate the expression of RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) in the model cyanobacterium Synechococcus elongatus PCC 7942, at both the transcript and protein levels by quantitative PCR and Western blot, respectively. We further describe an experimental method to determine photosynthetic activity using an oxygen electrode that measures the rate of molecular oxygen production by cyanobacterial cultures. Our protocol can be utilized to assess the effects of RuBisCO engineering at the metabolic and physiological levels.

  • De Tarafder A, Parajuli NP, Majumdar S, Kaçar B, Sanyal S (2021) Kinetic Analysis Suggests Evolution of Ribosome Specificity in Modern Elongation Factor-Tus from "Generalist" Ancestors. Molecular biology and evolution 38((8)):3436-3444 PMC8321524 · Pubmed · DOI

    It has been hypothesized that early enzymes are more promiscuous than their extant orthologs. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing presteady state fast kinetics using quench flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3- to 3.3-Gy-old ancestral EF-Tus toward two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. Although the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. In addition, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remains virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, "generalist" ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.

  • Garcia AK, Cavanaugh CM, Kacar B (2021) The curious consistency of carbon biosignatures over billions of years of Earth-life coevolution. The ISME journal 15((8)):2183-2194 PMC8319343 · Pubmed · DOI

    The oldest and most wide-ranging signal of biological activity (biosignature) on our planet is the carbon isotope composition of organic materials preserved in rocks. These biosignatures preserve the long-term evolution of the microorganism-hosted metabolic machinery responsible for producing deviations in the isotopic compositions of inorganic and organic carbon. Despite billions of years of ecosystem turnover, evolutionary innovation, organismic complexification, and geological events, the organic carbon that is a residuum of the global marine biosphere in the rock record tells an essentially static story. The ~25‰ mean deviation between inorganic and organic C/C values has remained remarkably unchanged over >3.5 billion years. The bulk of this record is conventionally attributed to early-evolved, RuBisCO-mediated CO fixation that, in extant oxygenic phototrophs, produces comparable isotopic effects and dominates modern primary production. However, billions of years of environmental transition, for example, in the progressive oxygenation of the Earth's atmosphere, would be expected to have accompanied shifts in the predominant RuBisCO forms as well as enzyme-level adaptive responses in RuBisCO CO-specificity. These factors would also be expected to result in preserved isotopic signatures deviating from those produced by extant RuBisCO in oxygenic phototrophs. Why does the bulk carbon isotope record not reflect these expected environmental transitions and evolutionary innovations? Here, we discuss this apparent discrepancy and highlight the need for greater quantitative understanding of carbon isotope fractionation behavior in extant metabolic pathways. We propose novel, laboratory-based approaches to reconstructing ancestral states of carbon metabolisms and associated enzymes that can constrain isotopic biosignature production in ancient biological systems. Together, these strategies are crucial for integrating the complementary toolsets of biological and geological sciences and for interpretation of the oldest record of life on Earth.

  • Carruthers BM, Garcia AK, Rivier A, Kacar B (2021) Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii. Current protocols 1((3)):e57 · Pubmed · DOI

    Azotobacter vinelandii (A. vinelandii) is a commonly used model organism for the study of aerobic respiration, the bacterial production of several industrially relevant compounds, and, perhaps most significantly, the genetics and biochemistry of biological nitrogen fixation. Laboratory growth assessments of A. vinelandii are useful for evaluating the impact of environmental and genetic modifications on physiological properties, including diazotrophy. However, researchers typically rely on manual growth methods that are oftentimes laborious and inefficient. We present a protocol for the automated growth assessment of A. vinelandii on a microplate reader, particularly well-suited for studies of diazotrophic growth. We discuss common pitfalls and strategies for protocol optimization, and demonstrate the protocol's application toward growth evaluation of strains carrying modifications to nitrogen-fixation genes. © 2021 The Authors. Basic Protocol 1: Preparation of A. vinelandii plate cultures from frozen stock Basic Protocol 2: Preparation of A. vinelandii liquid precultures Basic Protocol 3: Automated growth rate experiment of A. vinelandii on a microplate reader.

  • Goldman AD, Kacar B (2021) Cofactors are Remnants of Life's Origin and Early Evolution. Journal of molecular evolution 89((3)):127-133 PMC7982383 · Pubmed · DOI

    The RNA World is one of the most widely accepted hypotheses explaining the origin of the genetic system used by all organisms today. It proposes that the tripartite system of DNA, RNA, and proteins was preceded by one consisting solely of RNA, which both stored genetic information and performed the molecular functions encoded by that genetic information. Current research into a potential RNA World revolves around the catalytic properties of RNA-based enzymes, or ribozymes. Well before the discovery of ribozymes, Harold White proposed that evidence for a precursor RNA world could be found within modern proteins in the form of coenzymes, the majority of which contain nucleobases or nucleoside moieties, such as Coenzyme A and S-adenosyl methionine, or are themselves nucleotides, such as ATP and NADH (a dinucleotide). These coenzymes, White suggested, had been the catalytic active sites of ancient ribozymes, which transitioned to their current forms after the surrounding ribozyme scaffolds had been replaced by protein apoenzymes during the evolution of translation. Since its proposal four decades ago, this groundbreaking hypothesis has garnered support from several different research disciplines and motivated similar hypotheses about other classes of cofactors, most notably iron-sulfur cluster cofactors as remnants of the geochemical setting of the origin of life. Evidence from prebiotic geochemistry, ribozyme biochemistry, and evolutionary biology, increasingly supports these hypotheses. Certain coenzymes and cofactors may bridge modern biology with the past and can thus provide insights into the elusive and poorly-recorded period of the origin and early evolution of life.

  • Adam ZR, Fahrenbach AC, Jacobson SM, Kacar B, Zubarev DY (2021) Radiolysis generates a complex organosynthetic chemical network. Scientific reports 11((1)):1743 PMC7813863 · Pubmed · DOI

    The architectural features of cellular life and its ecologies at larger scales are built upon foundational networks of reactions between molecules that avoid a collapse to equilibrium. The search for life's origins is, in some respects, a search for biotic network attributes in abiotic chemical systems. Radiation chemistry has long been employed to model prebiotic reaction networks, and here we report network-level analyses carried out on a compiled database of radiolysis reactions, acquired by the scientific community over decades of research. The resulting network shows robust connections between abundant geochemical reservoirs and the production of carboxylic acids, amino acids, and ribonucleotide precursors-the chemistry of which is predominantly dependent on radicals. Moreover, the network exhibits the following measurable attributes associated with biological systems: (1) the species connectivity histogram exhibits a heterogeneous (heavy-tailed) distribution, (2) overlapping families of closed-loop cycles, and (3) a hierarchical arrangement of chemical species with a bottom-heavy energy-size spectrum. The latter attribute is implicated with stability and entropy production in complex systems, notably in ecology where it is known as a trophic pyramid. Radiolysis is implicated as a driver of abiotic chemical organization and could provide insights about the complex and perhaps radical-dependent mechanisms associated with life's origins.

  • Kacar B, Garcia AK, Anbar AD (2020) Evolutionary History of Bioessential Elements Can Guide the Search for Life in the Universe. Chembiochem : a European journal of chemical biology 22((1)):114-119 · Pubmed · DOI

    Our understanding of life in the universe comes from one sample, life on Earth. Current and next-generation space missions will target exoplanets as well as planets and moons in our own solar system with the primary goal of detecting, interpreting and characterizing indications of possible biological activity. Thus, understanding life's fundamental characteristics is increasingly critical for detecting and interpreting potential biological signatures elsewhere in the universe. Astrobiologists have outlined the essential roles of carbon and water for life, but we have yet to decipher the rules governing the evolution of how living organisms use bioessential elements. Does the suite of life's essential chemical elements on Earth constitute only one possible evolutionary outcome? Are some elements so essential for biological functions that evolution will select for them despite low availability? How would this play out on other worlds that have different relative element abundances? When we look for life in the universe, or the conditions that could give rise to life, we must learn how to recognize it in extremely different chemical and environmental conditions from those on Earth. We argue that by exposing self-organizing biotic chemistries to different combinations of abiotic materials, and by mapping the evolutionary history of metalloenzyme biochemistry onto geological availabilities of metals, alternative element choices that are very different from life's present-day molecular structure might result. A greater understanding of the paleomolecular evolutionary history of life on Earth will create a predictive capacity for detecting and assessing life's existence on worlds where alternate evolutionary paths might have been taken.

  • Venkataram S, Monasky R, Sikaroodi SH, Kryazhimskiy S, Kacar B (2020) Evolutionary stalling and a limit on the power of natural selection to improve a cellular module. Proceedings of the National Academy of Sciences of the United States of America 117((31)):18582-18590 PMC7414050 · Pubmed · DOI

    Cells consist of molecular modules which perform vital biological functions. Cellular modules are key units of adaptive evolution because organismal fitness depends on their performance. Theory shows that in rapidly evolving populations, such as those of many microbes, adaptation is driven primarily by common beneficial mutations with large effects, while other mutations behave as if they are effectively neutral. As a consequence, if a module can be improved only by rare and/or weak beneficial mutations, its adaptive evolution would stall. However, such evolutionary stalling has not been empirically demonstrated, and it is unclear to what extent stalling may limit the power of natural selection to improve modules. Here we empirically characterize how natural selection improves the translation machinery (TM), an essential cellular module. We experimentally evolved populations of Escherichia coli with genetically perturbed TMs for 1,000 generations. Populations with severe TM defects initially adapted via mutations in the TM, but TM adaptation stalled within about 300 generations. We estimate that the genetic load in our populations incurred by residual TM defects ranges from 0.5 to 19%. Finally, we found evidence that both epistasis and the depletion of the pool of beneficial mutations contributed to evolutionary stalling. Our results suggest that cellular modules may not be fully optimized by natural selection despite the availability of adaptive mutations.

  • Garcia AK, McShea H, Kolaczkowski B, Kaçar B (2020) Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum-cofactor utilization. Geobiology 18((3)):394-411 PMC7216921 · Pubmed · DOI

    The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo-, FeV-, or FeFe-cofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active-site sequence features can reliably distinguish extant Mo-nitrogenases from V- and Fe-nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo-nitrogenases. Taxa representing early-branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo-cofactor in studied organisms, suggesting that early Mo-nitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein-level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism-level innovations that characterize the Phanerozoic Eon.

  • Liberles DA, Chang B, Geiler-Samerotte K, Goldman A, Hey J, Kaçar B, Meyer M, Murphy W, Posada D, Storfer A (2020) Emerging Frontiers in the Study of Molecular Evolution. Journal of molecular evolution 88((3)):211-226 PMC7386396 · Pubmed · DOI

    A collection of the editors of Journal of Molecular Evolution have gotten together to pose a set of key challenges and future directions for the field of molecular evolution. Topics include challenges and new directions in prebiotic chemistry and the RNA world, reconstruction of early cellular genomes and proteins, macromolecular and functional evolution, evolutionary cell biology, genome evolution, molecular evolutionary ecology, viral phylodynamics, theoretical population genomics, somatic cell molecular evolution, and directed evolution. While our effort is not meant to be exhaustive, it reflects research questions and problems in the field of molecular evolution that are exciting to our editors.

  • Garcia AK, Kaçar B (2019) How to resurrect ancestral proteins as proxies for ancient biogeochemistry. Free radical biology & medicine 140:260-269 · Pubmed · DOI

    Throughout the history of life, enzymes have served as the primary molecular mediators of biogeochemical cycles by catalyzing the metabolic pathways that interact with geochemical substrates. The byproducts of enzymatic activities have been preserved as chemical and isotopic signatures in the geologic record. However, interpretations of these signatures are limited by the assumption that such enzymes have remained functionally conserved over billions of years of molecular evolution. By reconstructing ancient genetic sequences in conjunction with laboratory enzyme resurrection, preserved biogeochemical signatures can instead be related to experimentally constrained, ancestral enzymatic properties. We may thereby investigate instances within molecular evolutionary trajectories potentially tied to significant biogeochemical transitions evidenced in the geologic record. Here, we survey recent enzyme resurrection studies to provide a reasoned assessment of areas of success and common pitfalls relevant to ancient biogeochemical applications. We conclude by considering the Great Oxidation Event, which provides a constructive example of a significant biogeochemical transition that warrants investigation with ancestral enzyme resurrection. This event also serves to highlight the pitfalls of facile interpretation of paleophenotype models and data, as applied to two examples of enzymes that likely both influenced and were influenced by the rise of atmospheric oxygen - RuBisCO and nitrogenase.

  • Walker SI, Bains W, Cronin L, DasSarma S, Danielache S, Domagal-Goldman S, Kacar B, Kiang NY, Lenardic A, Reinhard CT, Moore W, Schwieterman EW, Shkolnik EL, Smith HB (2018) Exoplanet Biosignatures: Future Directions. Astrobiology 18((6)):779-824 PMC6016573 · Pubmed · DOI

    No abstract available.

  • Kacar B, Guy L, Smith E, Baross J (2017) Resurrecting ancestral genes in bacteria to interpret ancient biosignatures. Philosophical transactions. Series A, Mathematical, physical, and engineering sciences 375((2109)): PMC5686408 · Pubmed · DOI

    No abstract available.

  • Kacar B, Garmendia E, Tuncbag N, Andersson DI, Hughes D (2017) Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs. mBio 8((4)): PMC5574714 · Pubmed · DOI

    No abstract available.

  • Kacar B, Hanson-Smith V, Adam ZR, Boekelheide N (2017) Constraining the timing of the Great Oxidation Event within the Rubisco phylogenetic tree. Geobiology 15((5)):628-640 PMC5575542 · Pubmed · DOI

    No abstract available.

  • Kacar B, Ge X, Sanyal S, Gaucher EA (2017) Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein. Journal of molecular evolution 84((2-3)):69-84 PMC5371648 · Pubmed · DOI

    No abstract available.

  • Domagal-Goldman SD, Wright KE, Adamala K, Arina de la Rubia L, Bond J, Dartnell LR, Goldman AD, Lynch K, Naud ME, Paulino-Lima IG, Singer K, Walther-Antonio M, Abrevaya XC, Anderson R, Arney G, Atri D, Azúa-Bustos A, Bowman JS, Brazelton WJ, Brennecka GA, Carns R, Chopra A, Colangelo-Lillis J, Crockett CJ, DeMarines J, Frank EA, Frantz C, de la Fuente E, Galante D, Glass J, Gleeson D, Glein CR, Goldblatt C, Horak R, Horodyskyj L, Kaçar B, Kereszturi A, Knowles E, Mayeur P, McGlynn S, Miguel Y, Montgomery M, Neish C, Noack L, Rugheimer S, Stüeken EE, Tamez-Hidalgo P, Imari Walker S, Wong T (2016) The Astrobiology Primer v2.0. Astrobiology 16((8)):561-653 PMC5008114 · Pubmed · DOI

    No abstract available.

  • Kaçar B, Gaucher EA (2013) Experimental evolution of protein-protein interaction networks. The Biochemical journal 453((3)):311-9 PMC3727214 · Pubmed · DOI

    The modern synthesis of evolutionary theory and genetics has enabled us to discover underlying molecular mechanisms of organismal evolution. We know that in order to maximize an organism's fitness in a particular environment, individual interactions among components of protein and nucleic acid networks need to be optimized by natural selection, or sometimes through random processes, as the organism responds to changes and/or challenges in the environment. Despite the significant role of molecular networks in determining an organism's adaptation to its environment, we still do not know how such inter- and intra-molecular interactions within networks change over time and contribute to an organism's evolvability while maintaining overall network functions. One way to address this challenge is to identify connections between molecular networks and their host organisms, to manipulate these connections, and then attempt to understand how such perturbations influence molecular dynamics of the network and thus influence evolutionary paths and organismal fitness. In the present review, we discuss how integrating evolutionary history with experimental systems that combine tools drawn from molecular evolution, synthetic biology and biochemistry allow us to identify the underlying mechanisms of organismal evolution, particularly from the perspective of protein interaction networks.