Biology subjects

Fischer, W. W.

Publications and source records attributed to Fischer, W. W..

10 recordsLinked to original sources

bioRxiv2024

The proteome is a terminal electron acceptor

Microbial metabolism is impressively flexible, enabling growth even when available nutrients differ greatly from biomass in redox state. E. coli, for example, rearranges its physiology to grow on reduced and oxidized carbon sources through several forms of fermentation and respiration. To understand the limits on and evolutionary consequences of metabolic flexibility, we developed a mathematical model coupling redox chemistry with principles of cellular resource allocation. Our integrated model clarifies key phenomena, including demonstrating that autotrophs grow slower than heterotrophs because of constraints imposed by intracellular production of reduced carbon. Our model further indicates that growth is improved by adapting the redox state of biomass to nutrients, revealing an unexpected mode of evolution where proteins accumulate mutations benefiting organismal redox balance. One sentence summaryMicrobial proteins adapt their composition on physiological and evolutionary timescales to ensure organismal redox balance.

microbiology↗

bioRxiv2024

Annotation-free prediction of microbial dioxygen utilization

Aerobes require dioxygen (O2) to grow; anaerobes do not. But nearly all microbes -- aerobes, anaerobes, and facultative organisms alike -- express enzymes whose substrates include O2, if only for detoxification. This presents a challenge when trying to assess which organisms are aerobic from genomic data alone. This challenge can be overcome by noting that O2 utilization has wide-ranging effects on microbes: aerobes typically have larger genomes, encode more O2-utilizing enzymes, and tend to use different amino acids in their proteins. Here we show that these effects permit high-quality prediction of O2 utilization from genome sequences, with several models displaying >70% balanced accuracy on a ternary classification task wherein blind guessing is only 33.3% accurate. Since genome annotation is compute-intensive and relies on many assumptions, we asked if annotation-free methods also perform well. We discovered that simple and efficient models based entirely on genome sequence content -- e.g. triplets of amino acids -- perform about as well as intensive annotation-based algorithms, enabling the rapid processing of global-scale sequence data to predict aerobic physiology. To demonstrate the utility of efficient physiological predictions we estimated the prevalence of aerobes and anaerobes along a well-studied O2 gradient in the Black Sea, finding strong quantitative correspondence between local chemistry (O2:sulfide concentration ratio) and the composition of microbial communities. We therefore suggest that statistical methods like ours can be used to estimate, or "sense," pivotal features of the environment from DNA sequencing data. ImportanceWe now have access to sequence data from a wide variety of natural environments. These data document a bewildering diversity of microbes, many known only from their genomes. Physiology -- an organisms capacity to engage metabolically with its environment -- may provide a more useful lens than taxonomy for understanding microbial communities. As an example of this broader principle, we developed algorithms that accurately predict microbial dioxygen utilization directly from genome sequences without first annotating genes, e.g. by considering only the amino acids in protein sequences. Annotation-free algorithms enabled rapid characterization of natural samples, demonstrating a quantitative correspondence between sequences and local O2 levels. These results suggest that DNA sequencing can be repurposed as a multi-pronged chemical sensor, estimating concentrations of O2 and other key facets of complex natural settings.

microbiology↗

bioRxiv2023

Metabolic evolution of pyranopterin-dependent biochemistry

Molybdenum (Mo)-dependent biochemistry is essential for many key metabolic pathways. However, theory and geological evidence suggests that its solubility during long intervals with low dioxygen would have limited its availability on early Earth. We developed models of metabolic evolution and found that reactions employing tungsten (W)-dependent biochemistry likely preceded Mo-dependent reactions, where Mo-usage increased dramatically after the production of dioxygen. Consistent with this finding, we analyzed genomes from over 65,000 phylogenetically diverse microbes and metagenomes from an environmental dataset, and we observed that dioxygen-utilizing prokaryotes living in aerobic niches are enriched with Mo-dependent enzymes as compared to anaerobic microbes. As an independent evaluation of this hypothesis, we combined protein language models, machine learning, and phylogenomic analysis to build a classifier for W- or Mo-pterin dependence in the DMSO reductase superfamily, and we found that W-pterin-dependent enzymes cluster near the root of the tree and that a subset of late-evolving aldehyde oxidoreductases (AORs) from aerobes are predicted to rely on Mo instead of W. Overall, our combination of metabolic modeling, phenotypic analysis, machine learning, and phylogenomic analysis suggest that Mo-pterin-dependent biochemistry likely derived from W-pterin-dependent biochemistry, and that Mo-usage increased drastically after the rise of oxygen.

evolutionary biology↗

bioRxiv2023

An evolutionary timescale for Bacteria calibrated using the Great Oxidation Event

Most of lifes diversity and history is microbial but it has left a meagre fossil record, greatly hindering understanding of evolution in deep time. However, the co-evolution of life and the Earth system has left signatures of bacterial metabolism in the geochemical record, most conspicuously the Great Oxidation Event (GOE) [~]2.33 billion years ago (Ga, (Poulton et al. 2021)), in which oxygenic photosynthesis and tectonism (Eguchi, Seales, and Dasgupta 2019) transformed Earths biosphere from dominantly anaerobic to aerobic. Here, we combine machine learning and phylogenetic reconciliation to infer ancestral transitions to aerobic lifestyles during bacterial evolution. Linking these transitions to the GOE provides new constraints to infer the timetree of Bacteria. We find that extant bacterial phyla are truly ancient, having radiated in the Archaean and the Proterozoic: the oldest include Bacillota (Firmicutes), which radiated 3.1-3.7 Ga, Cyanobacteria (3.3-3.5 Ga) and Patescibacteria (3-3.5 Ga). We show that most bacterial phyla were ancestrally anaerobic and that most transitions to an aerobic lifestyle post-dated the GOE. Our analyses trace oxygen production and consumption back to Cyanobacteria. From that starting point, horizontal transfer seeded aerobic lifestyles across bacterial diversity over hundreds of millions of years. Our analyses demonstrate that the diversification of aerobes proceeded in two waves corresponding to the GOE and to a second sustained rise in atmospheric O2 at the dawn of the Palezoic (Krause et al. 2022).

evolutionary biology↗

bioRxiv2023

Bacterial Form I' rubisco has smaller carbon isotope fractionation than its Form I counterpart

Form I rubiscos evolved in Cyanobacteria [≥]2.5 billion years ago and are enzymatically unique due to the presence of small subunits (RbcS) that cap both ends of an octameric large subunit (RbcL) rubisco assembly to form a hexadecameric (L8S8) holoenzyme. Although RbcS was previously thought to be integral to Form I rubisco stability, the recent discovery of a closely related sister clade of octameric rubiscos (Form I; L8) demonstrates that the enzyme complex assembles without small subunits (Banda et al. 2020). Rubisco also displays a kinetic isotope effect (KIE) where the 3PG product is depleted in 13C relative to 12C. In Cyanobacteria only two Form I KIE measurements exist, making interpretation of bacterial carbon isotope data difficult. To aid comparison, we measured in vitro the KIEs of Form I (Candidatus Promineofilum breve) and Form I (Synechococcus elongatus PCC 6301) rubiscos and found the KIE to be smaller in the L8 rubisco (16.25 {+/-} 1.36{per thousand} vs. 22.42 {+/-} 2.37{per thousand} respectively). Therefore, while small subunits may not be necessary for protein stability, they may affect the KIE. Our findings may provide insight into the function of RbcS and allow more refined interpretation of environmental carbon isotope data.

evolutionary biology↗

bioRxiv2022

Evolution of Carbon Isotope Fractionation in Cyanobacteria

The history of Earths carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. We tested the intersection of environment and evolution by measuring both biomass ({varepsilon}p) and enzymatic ({varepsilon}Rubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to >>1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger {varepsilon}p values than WT, despite having a much smaller {varepsilon}Rubisco (17.23 {+/-} 0.61{per thousand} vs. 25.18 {+/-} 0.31{per thousand} respectively). Measuring both enzymatic and biomass fractionation revealed a surprising result--ANC {varepsilon}p exceeded ANC {varepsilon}Rubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. However, these models were corrected by accounting for cyanobacterial physiology, notably the CO2 concentrating mechanism (CCM). Our model suggested that additional fractionating processes like powered inorganic carbon uptake systems contribute to {varepsilon}p, and this effect is exacerbated in ANC. Understanding the evolution of rubisco and the CCM is therefore critical for interpreting the carbon isotope record. Large fluctuations in that record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2. Significance StatementEarth scientists rely on chemical fossils like the carbon isotope record to derive ancient atmospheric CO2 concentrations, but interpretation of this record is calibrated using modern organisms. We tested this assumption by measuring the carbon isotope fractionation of a reconstructed ancestral rubisco enzyme (>1 billion years old) in vivo and in vitro. Our results contradicted prevailing models of carbon flow in Cyanobacteria, but our data could be rationalized if light-driven uptake of CO2 is taken into account. Our study showed that the carbon isotope record tracks both the evolution of photosynthesis physiology as well as changes in atmospheric CO2, highlighting the value of considering both evolution and physiology for comparative biological approaches to understanding Earths history.

evolutionary biology↗

bioRxiv2022

Trajectories for the evolution of bacterial CO2-concentrating mechanisms

Cyanobacteria rely on CO2 concentrating mechanisms (CCMs) that depend on {approx}15 genes to produce two protein complexes: an inorganic carbon (Ci) transporter and a 100+ nm carboxysome compartment that encapsulates rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting CCM components prohibit growth in todays atmosphere (0.04% CO2), indicating that CCMs evolved to cope with declining environmental CO2. Indeed, geochemical data and models indicate that atmospheric CO2 has been generally decreasing from high concentrations over the last {approx}3.5 billion years. We used a synthetic reconstitution of a bacterial CCM in E. coli to study the co-evolution of CCMs with atmospheric CO2. We constructed strains expressing putative ancestors of modern CCMs -- strains lacking one or more CCM components -- and evaluated their growth in a variety of CO2 concentrations. Partial forms expressing CA or Ci uptake genes grew better than controls in intermediate CO2 levels ({approx}1%); we observed similar phenotypes in genetic studies of two autotrophic bacteria, H. neapolitanus and C. necator. To explain how partial CCMs improve growth, we advance a model of co-limitation of autotrophic growth by CO2 and HCO3-, as both are required to produce biomass. Our model and results delineated a viable trajectory for bacterial CCM evolution where decreasing atmospheric CO2 induces an HCO3- deficiency that is alleviated by acquisition of CAs or Ci uptake genes, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity. SignificanceThe greenhouse gas content of the ancient atmosphere is estimated using models and measurements of geochemical proxies. Some inferred high ancient CO2 levels using models of biological CO2 fixation to interpret the C isotopes found in preserved organic matter. Others argued that elevated CO2 could reconcile a faint young Sun with evidence for liquid water on Earth. We took a complementary "synthetic biological" approach to understanding the composition of the ancient atmosphere by studying present-day bacteria engineered to resemble ancient autotrophs. By showing that it is simpler to rationalize the emergence of modern bacterial autotrophs if CO2 was once high, these investigations provided independent evidence for the view that CO2 concentrations were significantly elevated in the atmosphere of early Earth.

microbiology↗

bioRxiv2021

Diversity and evolution of nitric oxide reduction

Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- [->] NO2- [->] NO [->] N2O [->] N2)1. Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone-depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we use phylogenomics to identify a number of previously uncharacterized HCO families and propose that many of them (eNOR, sNOR, gNOR, and nNOR) perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from Rhodothermus marinus, verifying that it performs nitric oxide reduction both in vitro and in vivo. These newly identified NORs exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.

biochemistry↗

bioRxiv2019

Phototrophic Methane Oxidation in a Member of the Chloroflexi Phylum

Biological methane cycling plays an important role in Earths climate and the global carbon cycle, with biological methane oxidation (methanotrophy) modulating methane release from numerous environments including soils, sediments, and water columns. Methanotrophy is typically coupled to aerobic respiration or anaerobically via the reduction of sulfate, nitrate, or metal oxides, and while the possibility of coupling methane oxidation to phototrophy (photomethanotrophy) has been proposed, no organism has ever been described that is capable of this metabolism. Here we described a new bacterial genome from a member of the Chloroflexi phylum--termed here Candidatus Chlorolinea photomethanotrophicum--with cooccurring methanotrophy and phototrophy pathways, suggesting a novel link between these two metabolisms. Recovered as a metagenome-assembled genome from microbial mats in an iron-rich hot spring in Japan, Ca. C. photomethanotrophicum forms a new lineage within the Chloroflexi phylum and expands the known metabolic diversity of this already diverse clade. Ca. C. photomethanotrophicum appears to be metabolically versatile, capable of phototrophy (via a Type 2 reaction center), aerobic respiration, nitrite reduction, oxidation of methane and carbon monoxide, and potentially carbon fixation via a novel pathway composed of hybridized components of the serine cycle and the 3-hydroxypropionate bicycle. The biochemical network of this organism is constructed from components from multiple organisms and pathways, further demonstrating the modular nature of metabolic machinery and the ecological and evolutionary importance of horizontal gene transfer in the establishment of novel pathways. SignificanceMethane is a major greenhouse gas, and the production and consumption of methane is largely driven by the metabolism of microorganisms. Although it has been hypothesized for decades that some bacteria may be capable of growth by eating methane and conserving energy from sunlight (photomethanotrophy), this metabolism has never been discovered in nature. Here, we describe the first genetic evidence for a bacterium capable of photomethanotrophy, adding a new pathway to the known diversity of how microbes can make a living. This discovery also adds a new link to the global carbon cycle, and may provide new opportunities for designing biotechnological tools for processing methane.

microbiology↗

bioRxiv2018

Thermophilic Lithotrophy and Phototrophy in an Intertidal, Iron-rich, Geothermal Spring

Hydrothermal systems, including terrestrial hot springs, contain diverse geochemical conditions that vary over short spatial scales due to progressive interaction between the reducing hydrothermal fluids, the oxygenated atmosphere, and in some cases seawater. At Jinata Onsen, on Shikinejima Island, Japan, an intertidal, anoxic, iron-rich hot spring mixes with the oxygenated atmosphere and seawater over short spatial scales, creating a diversity of chemical potentials and redox pairs over a distance ~10 m. We characterized the geochemical conditions along the outflow of Jinata Onsen as well as the microbial communities present in biofilms, mats, and mineral crusts along its traverse via 16S rDNA amplicon and genome-resolved shotgun metagenomic sequencing. The microbial community changed significantly downstream as temperatures and dissolved iron concentrations decreased and dissolved oxygen increased. Near the spring source, biomass is limited relative to downstream, and primary productivity may be fueled by oxidation of ferrous iron and molecular hydrogen by members of the Zetaproteobacteria and Aquificae. Downstream, the microbial community is dominated by oxygenic Cyanobacteria. Cyanobacteria are abundant and active even at ferrous iron concentrations of ~150 M, which challenges the idea that iron toxicity limited cyanobacterial expansion in Precambrian oceans. Several novel lineages of Bacteria are also present at Jinata Onsen, including previously uncharacterized members of the Chloroflexi and Caldithrichaeota phyla, positioning Jinata Onsen as a valuable site for future characterization of these clades. ImportanceHigh temperatures and reducing conditions allow hot springs to support microbial communities that are very different from those found elsewhere on the surface of the Earth today; in some ways, these environments and the communities they support can be similar to environments that existed on the early Earth and that may exist on other planets. Here, we describe a novel hot spring system where hot, iron-rich but oxygen-poor water flows into the ocean, supporting a range of unique microbial communities. Metagenomic sequencing recovered many novel microbial lineages, including deep-branching and uniquely thermotolerant members of known groups. Comparison of the biological communities in the upstream part of the hot spring, potentially supported by biological iron and hydrogen oxidizing metabolisms, to downstream microbial mats, supported by oxygenic photosynthesis, provides insight into the potential productivity of life during Proterozoic time and on other planets where oxygenic photosynthesis is not possible.

microbiology↗