Amanda K. Garcia

University of Wisconsin–Madison

Author Statistics

Papers

Citation

H-Index

i-10 index

Research Trends

Author Order

Document Type

Co-Authors

Betül Kaçar

University of Wisconsin–Madison

Alex Rivier

Lawrence Berkeley National Laboratory

Brooke M. Carruthers

University of Wisconsin–Madison

Bryan Kolaczkowski

University of Florida

Lance C. Seefeldt

Utah State University

Derek F. Harris

Utah State University

Arnaud Taton

University of California, San Diego

Jodi N. Young

University of Washington

Mateusz Kędzior

Duke University

Hanon McShea

Stanford University

Cooperative Institutions

University of Wisconsin–Madison

Georgia Institute of Technology

Planetary Science Institute

Pennsylvania State University

University of Arizona

Texas Tech University Health Sciences Center

Texas Tech University

Arizona State University

Universidade de São Paulo

University of Washington

Author Statistics

Papers

Citation

H-Index

i-10 index

Research Field

Mysteries of metallome evolution: Integrating insights from the Earth and life sciences

Elsevier eBooks (2024)

Kathryn Rico Amanda K. Garcia Mak A. Saito Betül Kaçar Ariel D. Anbar

10.1016/b978-0-323-99762-1.00143-1

Cite

Citations (1)

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

Geobiology (2020)

Amanda K. Garcia Hanon McShea Bryan Kolaczkowski Betül Kaçar

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.

10.1111/gbi.12381

Cite

Citations (91)

Chapter 1: The Astrobiology Primer 3.0

Astrobiology (2024)

Micah J. Schaible Nadia Szeinbaum G. Ozan Bozdag Luoth Chou Natalie Grefenstette

The Astrobiology Primer 3.0 (ABP3.0) is a concise introduction to the field of astrobiology for students and others who are new to the field of astrobiology. It provides an entry into the broader materials in this supplementary issue of Astrobiology and an overview of the investigations and driving hypotheses that make up this interdisciplinary field. The content of this chapter was adapted from the other 10 articles in this supplementary issue and thus represents the contribution of all the authors who worked on these introductory articles. The content of this chapter is not exhaustive and represents the topics that the authors found to be the most important and compelling in a dynamic and changing field.

10.1089/ast.2021.0129

Cite

Citations (1)

Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation

Molecular Biology and Evolution (2024)

Bruno Cuevas-Zuviría Amanda K. Garcia Alex Rivier Holly R. Rucker Brooke M. Carruthers

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.

Molecular evolution

10.1093/molbev/msae067

Cite

Citations (5)

Reconstructed ancestral enzymes suggest long-term cooling of Earth’s photic zone since the Archean

Proceedings of the National Academy of Sciences (2017)

Amanda K. Garcia J. William Schopf Shin‐ichi Yokobori Satoshi Akanuma Akihiko Yamagishi

Paleotemperatures inferred from the isotopic compositions (δ18O and δ30Si) of marine cherts suggest that Earth's oceans cooled from 70 ± 15 °C in the Archean to the present ∼15 °C. This interpretation, however, has been subject to question due to uncertainties regarding oceanic isotopic compositions, diagenetic or metamorphic resetting of the isotopic record, and depositional environments. Analyses of the thermostability of reconstructed ancestral enzymes provide an independent method by which to assess the temperature history inferred from the isotopic evidence. Although previous studies have demonstrated extreme thermostability in reconstructed archaeal and bacterial proteins compatible with a hot early Earth, taxa investigated may have inhabited local thermal environments that differed significantly from average surface conditions. We here present thermostability measurements of reconstructed ancestral enzymatically active nucleoside diphosphate kinases (NDKs) derived from light-requiring prokaryotic and eukaryotic phototrophs having widely separated fossil-based divergence ages. The ancestral environmental temperatures thereby determined for these photic-zone organisms--shown in modern taxa to correlate strongly with NDK thermostability--are inferred to reflect ancient surface-environment paleotemperatures. Our results suggest that Earth's surface temperature decreased over geological time from ∼65-80 °C in the Archean, a finding consistent both with previous isotope-based and protein reconstruction-based interpretations. Interdisciplinary studies such as those reported here integrating genomic, geologic, and paleontologic data hold promise for providing new insight into the coevolution of life and environment over Earth history.

10.1073/pnas.1702729114

Cite

Citations (64)

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

Geobiology (2023)

Amanda K. Garcia Mateusz Kędzior Arnaud Taton Meng Li Jodi N. Young

Abstract 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 2 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 2 concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO 2 fixation versus CO 2 supply, and thus whole‐cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO 2 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 (ε p ) at all tested CO 2 concentrations, yielding ε p of ≈ 23‰ for both wild‐type and mutant strains at elevated CO 2 . 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.

Carbon fixation

10.1111/gbi.12543

Cite

Citations (6)

Evolutionary History of Bioessential Elements Can Guide the Search for Life in the Universe

ChemBioChem (2020)

Betül Kaçar Amanda K. Garcia Ariel D. Anbar

Abstract 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.

Abiogenesis

Extraterrestrial Life

DECIPHER

Artificial life

10.1002/cbic.202000500

Cite

Citations (24)

Reconstruction of nitrogenase predecessors suggests origin from maturase-like proteins

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Amanda K. Garcia Bryan Kolaczkowski Betül Kaçar

ABSTRACT 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. SIGNIFICANCE STATEMENT The origin of nitrogenase-catalyzed nitrogen fixation was a transformative event in life’s history, garnering long-term study from molecular, biogeochemical, and planetary perspectives. Reconstruction of ancestral nitrogenases suggests that the protein sequence space capable of yielding a nitrogen-fixing enzyme in the past was likely more constrained than previously thought. Specifically, here we show that nitrogenases likely evolved from ancestors that resemble maturases, homologs that today participate in nitrogenase cofactor assembly, contrary to the commonly accepted view that maturases evolved from a nitrogenase ancestor. We further submit that the molecular architecture that may have been required for nitrogenase origins was unlikely to have been shaped by the same environmental drivers often implicated in the evolution of nitrogen fixation. If this decoupling is found to be a recurring pattern in metabolic origins, then the presented results would undercut the common, systems-focused rationale of using ancient environmental conditions to explain the timing of critical and singular biogeochemical innovations in life’s past.

10.1101/2021.07.07.451390

Cite

Citations (1)

A CRISPR interference system for engineering biological nitrogen fixation

mSystems (2024)

Steven J. Russell Amanda K. Garcia Betül Kaçar

ABSTRACT 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. IMPORTANCE All 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.

Nitrogen Cycle

10.1128/msystems.00155-24

Cite

Citations (1)

Reconstructed ancient nitrogenases suggest Mo-specific ancestry

Amanda K. Garcia Hanon McShea Bryan Kolaczkowski Betül Kaçar

The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life s key biogeochemical innovations. The three isozymes of nitrogenase differ in their metal dependence, each binding either a FeMo-, FeV-, or FeFe-cofactor for the reduction of nitrogen. 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 combine phylogenetics and ancestral sequence reconstruction, a method by which inferred, historical protein sequence information can be linked to functional molecular properties, to reconstruct the metal dependence of ancient nitrogenases. Inferred ancestral nitrogenase sequences at the deepest nodes of the phylogeny suggest that ancient nitrogenases were Mo-dependent. We find that active-site sequence identity can reliably distinguish extant Mo-nitrogenases from V- and Fe-nitrogenases, as opposed to modeled active-site structural features that cannot be used to reliably classify nitrogenases of unknown metal dependence. Taxa represented by early-branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that are necessary for assembly of the FeMo-cofactor, suggesting that early Mo-dependent nitrogenases may have utilized an alternate pathway for Mo-usage predating the FeMo-cofactor. Our results underscore the profound impacts that protein-level innovations likely had on shaping global biogeochemical cycles throughout Precambrian, in contrast to organism-level innovations which characterize Phanerozoic eon.

Biogeochemical Cycle

Cite

Citations (0)