Diversity and evolution of nitric oxide reduction in bacteria and archaea.

Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration.

Balance and imbalance in biogeochemical cycles reflect the operation of closed, exchange, and open sets.

Biogeochemical reactions modulate the chemical composition of the oceans and atmosphere, providing feedbacks that sustain planetary habitability over geological time. Here, we mathematically evaluate a suite of biogeochemical processes to identify combinations of reactions that stabilize atmospheric carbon dioxide by balancing fluxes of chemical species among the ocean, atmosphere, and geosphere. Unlike prior modeling efforts, this approach does not prescribe functional relationships between the rates of biogeochemical processes and environmental conditions. Our agnostic framework generates three types of stable reaction combinations: closed sets, where sources and sinks mutually cancel for all chemical reservoirs; exchange sets, where constant ocean-atmosphere conditions are maintained through the growth or destruction of crustal reservoirs; and open sets, where balance in alkalinity and carbon fluxes is accommodated by changes in other chemical components of seawater or the atmosphere. These three modes of operation have different characteristic timescales and may leave distinct evidence in the rock record. To provide a practical example of this theoretical framework, we applied the model to recast existing hypotheses for Cenozoic climate change based on feedbacks or shared forcing mechanisms. Overall, this work provides a systematic and simplified conceptual framework for understanding the function and evolution of global biogeochemical cycles.

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.

A Bacterial Form I' Rubisco Has a 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) capping 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 L8 complex can assemble 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‱ vs. 22.42 ± 2.37‱, 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.

Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO2 through geologic time should be reevaluated.

The history of Earth's 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. Therefore, we measured both biomass (εp) and enzymatic (εRubisco) 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 εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2.

Response to comment on "Reexamination of 2.5-Ga 'whiff' of oxygen interval points to anoxic ocean before GOE".

Sedimentological, textural, and microscale analyses of the Mount McRae Shale revealed a complex postdepositional history, previously unrecognized in bulk geochemical studies. We found that metal enrichments in the shale do not reside with depositional organic carbon, as previously proposed by Anbar et al., but with late-stage pyrite, compromising claims for a "whiff" of oxygen ~50 million years before the Great Oxygenation Event.

Trajectories for the evolution of bacterial CO2-concentrating mechanisms.

Cyanobacteria rely on CO2-concentrating mechanisms (CCMs) to grow in today's atmosphere (0.04% CO2). These complex physiological adaptations require ≈15 genes to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+ nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air. If any plausible ancestral form-i.e., lacking a single gene-cannot grow, how did the CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was "catalyzed" by historically high CO2 levels that decreased over geologic time. Using an E. coli reconstitution of a bacterial CCM, we constructed strains lacking one or more CCM components and evaluated their growth across CO2 concentrations. We expected these experiments to demonstrate the importance of the carboxysome. Instead, we found that partial CCMs expressing CA or Ci uptake genes grew better than controls in intermediate CO2 levels (≈1%) and observed similar phenotypes in two autotrophic bacteria, Halothiobacillus neapolitanus and Cupriavidus necator. To understand how CA and Ci uptake improve growth, we model autotrophy as colimited by CO2 and HCO3-, as both are required to produce biomass. Our experiments and model delineated a viable trajectory for CCM evolution where decreasing atmospheric CO2 induces an HCO3- deficiency that is alleviated by acquisition of CA or Ci uptake, 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.

Oceanic nutrient rise and the late Miocene inception of Pacific oxygen-deficient zones.

The modern Pacific Ocean hosts the largest oxygen-deficient zones (ODZs), where oxygen concentrations are so low that nitrate is used to respire organic matter. The history of the ODZs may offer key insights into ocean deoxygenation under future global warming. In a 12-My record from the southeastern Pacific, we observe a >10‰ increase in foraminifera-bound nitrogen isotopes (15N/14N) since the late Miocene (8 to 9 Mya), indicating large ODZs expansion. Coinciding with this change, we find a major increase in the nutrient content of the ocean, reconstructed from phosphorus and iron measurements of hydrothermal sediments at the same site. Whereas global warming studies cast seawater oxygen concentrations as mainly dependent on climate and ocean circulation, our findings indicate that modern ODZs are underpinned by historically high concentrations of seawater phosphate.

Sulfur cycling at natural hydrocarbon and sulfur seeps in Santa Paula Creek, CA.

Biogeochemical cycling of sulfur is relatively understudied in terrestrial environments compared to marine environments. However, the comparative ease of access, observation, and sampling of terrestrial settings can expand our understanding of organisms and processes important in the modern sulfur cycle. Furthermore, these sites may allow for the discovery of useful process analogs for ancient sulfur-metabolizing microbial communities at times in Earth's past when atmospheric O2 concentrations were lower and sulfide was more prevalent in Earth surface environments. We identified a new site at Santa Paula Creek (SPC) in Ventura County, CA-a remarkable freshwater, gravel-bedded mountain stream charged with a range of oxidized and reduced sulfur species and heavy hydrocarbons from the emergence of subsurface fluids within the underlying sulfur- and organic-rich Miocene-age Monterey Formation. SPC hosts a suite of morphologically distinct microbial biofacies that form in association with the naturally occurring hydrocarbon seeps and sulfur springs. We characterized the geology, stream geochemistry, and microbial facies and diversity of the Santa Paula Creek ecosystem. Using geochemical analyses and 16S rRNA gene sequencing, we found that SPC supports a dynamic sulfur cycle that is largely driven by sulfide-oxidizing microbial taxa, with contributions from smaller populations of sulfate-reducing and sulfur-disproportionating taxa. This preliminary characterization of SPC revealed an intriguing site in which to study geological and geochemical controls on microbial community composition and to expand our understanding of sulfur cycling in terrestrial environments.

Early impacts of climate change on a coastal marine microbial mat ecosystem.

Among the earliest consequences of climate change are extreme weather and rising sea levels-two challenges to which coastal environments are particularly vulnerable. Often found in coastal settings are microbial mats-complex, stratified microbial ecosystems that drive massive nutrient fluxes through biogeochemical cycles and have been important constituents of Earth's biosphere for eons. Little Ambergris Cay, in the Turks and Caicos Islands, supports extensive mats that vary sharply with relative water level. We characterized the microbial communities across this variation to understand better the emerging threat of sea level rise. In September 2017, the eyewall of category 5 Hurricane Irma transited the island. We monitored the impact and recovery from this devastating storm event. New mat growth proceeded rapidly, with patterns suggesting that storm perturbation may facilitate the adaptation of these ecosystems to changing sea level. Sulfur cycling, however, displayed hysteresis, stalling for >10 months after the hurricane and likely altering carbon storage potential.

Reexamination of 2.5-Ga "whiff" of oxygen interval points to anoxic ocean before GOE.

Transient appearances of oxygen have been inferred before the Great Oxygenation Event (GOE) [∼2.3 billion years (Ga) ago] based on redox-sensitive elements such as Mo and S­most prominently from the ∼2.5-Ga Mount McRae Shale in Western Australia. We present new spatially resolved data including synchrotron-based x-ray spectroscopy and secondary ion mass spectrometry to characterize the petrogenesis of the Mount McRae Shale. Sediments were primarily composed of organic matter and volcanic ash (a potential source of Mo), with U-Pb ages revealing extremely low sedimentation rates. Catagenesis created bedding-parallel microfractures, which subsequently acted as fluid pathways for metasomatic alteration and recent oxidative weathering. Our collective observations suggest that the bulk chemical datasets pointing toward a "whiff" of oxygen developed during postdepositional events. Nonzero Δ33S in trace-metal­poor, early diagenetic pyrite and the unusually enriched organic carbon at low sedimentation rates instead suggest that environmental oxygen levels were negligible ∼150 million years before the GOE.

Inflammatory breast cancer defined: proposed common diagnostic criteria to guide treatment and research.

PURPOSE: Inflammatory breast cancer is a deadly and aggressive type of breast cancer. A key challenge relates to the need for a more detailed, formal, objective definition of IBC, the lack of which compromises clinical care, hampers the conduct of clinical trials, and hinders the search for IBC-specific biomarkers and treatments because of the heterogeneity of patients considered to have IBC. METHODS: Susan G. Komen, the Inflammatory Breast Cancer Research Foundation, and the Milburn Foundation convened patient advocates, clinicians, and researchers to review the state of IBC and to propose initiatives to advance the field. After literature review of the defining clinical, pathologic, and imaging characteristics of IBC, the experts developed a novel quantitative scoring system for diagnosis. RESULTS: The experts identified through consensus several "defining characteristics" of IBC, including factors related to timing of onset and specific symptoms. These reflect common pathophysiologic changes, sometimes detectable on biopsy in the form of dermal lymphovascular tumor emboli and often reflected in imaging findings. Based on the importance and extent of these characteristics, the experts developed a scoring scale that yields a continuous score from 0 to 48 and proposed cut-points for categorization that can be tested in subsequent validation studies. CONCLUSION: To move beyond subjective 'clinical diagnosis' of IBC, we propose a quantitative scoring system to define IBC, based on clinical, pathologic, and imaging features. This system is intended to predict outcome and biology, guide treatment decisions and inclusion in clinical trials, and increase diagnostic accuracy to aid basic research; future validation studies are necessary to evaluate its performance.

Using C-DFT to develop an e-ReaxFF force field for acetophenone radical anion.

Increased electricity usage over the past several decades has accelerated the need for efficient high-voltage power transmission with reliable insulating materials. Cross-linked polyethylene (XLPE) prepared via dicumyl peroxide (DCP) cross-linking has emerged as the insulator of choice for modern power cables. Although DCP cross-linking generates the desired XLPE product in high yield, other by-products are also produced. One such by-product, acetophenone, is particularly intriguing due to its aromaticity and positive electron affinity. In this work, constrained density functional theory (C-DFT) was utilized to develop an e-ReaxFF force field suitable for describing the acetophenone radical anion. Initial parameters were taken from the 2021 Akbarian e-ReaxFF force field, which was developed to describe XLPE chemistry. Then, C-DFT geometry optimizations were performed wherein an excess electron was constrained to each atom of acetophenone. The resulting C-DFT energy values for the various electronic positions were added to the e-ReaxFF training set. Next, an analogous set of structures was energy-minimized using e-ReaxFF, and equilibrium mixture compositions for the two methods were compared at multiple temperatures. Iterative fitting against C-DFT energy data was performed until satisfactory agreement was achieved. To test force field performance, molecular dynamics simulations were performed in e-ReaxFF and the resulting electronic distributions were qualitatively compared to unconstrained-DFT spin density data. By expanding our e-ReaxFF force field for XLPE, namely, adding the capability to describe acetophenone and its interactions with an excess electron, we move one step closer to a comprehensive molecular understanding of XLPE chemistry in a high-voltage power cable.

Prebiotic photoredox synthesis from carbon dioxide and sulfite.

Carbon dioxide (CO2) is the major carbonaceous component of many planetary atmospheres, which includes the Earth throughout its history. Carbon fixation chemistry-which reduces CO2 to organics, utilizing hydrogen as the stoichiometric reductant-usually requires high pressures and temperatures, and the yields of products of potential use to nascent biology are low. Here we demonstrate an efficient ultraviolet photoredox chemistry between CO2 and sulfite that generates organics and sulfate. The chemistry is initiated by electron photodetachment from sulfite to give sulfite radicals and hydrated electrons, which reduce CO2 to its radical anion. A network of reactions that generates citrate, malate, succinate and tartrate by irradiation of glycolate in the presence of sulfite was also revealed. The simplicity of this carboxysulfitic chemistry and the widespread occurrence and abundance of its feedstocks suggest that it could have readily taken place on the surfaces of rocky planets. The availability of the carboxylate products on early Earth could have driven the development of central carbon metabolism before the advent of biological CO2 fixation.

Formation of Magnesium Carbonates on Earth and Implications for Mars.

Magnesium carbonates have been identified within the landing site of the Perseverance rover mission. This study reviews terrestrial analog environments and textural, mineral assemblage, isotopic, and elemental analyses that have been applied to establish formation conditions of magnesium carbonates. Magnesium carbonates form in five distinct settings: ultramafic rock-hosted veins, the matrix of carbonated peridotite, nodules in soil, alkaline lake, and playa deposits, and as diagenetic replacements within lime-and dolostones. Dominant textures include fine-grained or microcrystalline veins, nodules, and crusts. Microbial influences on formation are recorded in thrombolites, stromatolites, crinkly, and pustular laminites, spheroids, and filamentous microstructures. Mineral assemblages, fluid inclusions, and carbon, oxygen, magnesium, and clumped isotopes of carbon and oxygen have been used to determine the sources of carbon, magnesium, and fluid for magnesium carbonates as well as their temperatures of formation. Isotopic signatures in ultramafic rock-hosted magnesium carbonates reveal that they form by either low-temperature meteoric water infiltration and alteration, hydrothermal alteration, or metamorphic processes. Isotopic compositions of lacustrine magnesium carbonate record precipitation from lake water, evaporation processes, and ambient formation temperatures. Assessment of these features with similar analytical techniques applied to returned Martian samples can establish whether carbonates on ancient Mars were formed at high or low temperature conditions in the surface or subsurface through abiotic or biotic processes. The timing of carbonate formation processes could be constrained by 147Sm-143Nd isochron, U-Pb concordia, 207Pb-206Pb isochron radiometric dating as well as 3He, 21Ne, 22Ne, or 36Ar surface exposure dating of returned Martian magnesium carbonate samples.

Impact of River Channel Lateral Migration on Microbial Communities across a Discontinuous Permafrost Floodplain.

Permafrost soils store approximately twice the amount of carbon currently present in Earth's atmosphere and are acutely impacted by climate change due to the polar amplification of increasing global temperature. Many organic-rich permafrost sediments are located on large river floodplains, where river channel migration periodically erodes and redeposits the upper tens of meters of sediment. Channel migration exerts a first-order control on the geographic distribution of permafrost and floodplain stratigraphy and thus may affect microbial habitats. To examine how river channel migration in discontinuous permafrost environments affects microbial community composition, we used amplicon sequencing of the 16S rRNA gene on sediment samples from floodplain cores and exposed riverbanks along the Koyukuk River, a large tributary of the Yukon River in west-central Alaska. Microbial communities are sensitive to permafrost thaw: communities found in deep samples thawed by the river closely resembled near-surface active-layer communities in nonmetric multidimensional scaling analyses but did not resemble floodplain permafrost communities at the same depth. Microbial communities also displayed lower diversity and evenness in permafrost than in both the active layer and permafrost-free point bars recently deposited by river channel migration. Taxonomic assignments based on 16S and quantitative PCR for the methyl coenzyme M reductase functional gene demonstrated that methanogens and methanotrophs are abundant in older permafrost-bearing deposits but not in younger, nonpermafrost point bar deposits. The results suggested that river migration, which regulates the distribution of permafrost, also modulates the distribution of microbes potentially capable of producing and consuming methane on the Koyukuk River floodplain. IMPORTANCE Arctic lowlands contain large quantities of soil organic carbon that is currently sequestered in permafrost. With rising temperatures, permafrost thaw may allow this carbon to be consumed by microbial communities and released to the atmosphere as carbon dioxide or methane. We used gene sequencing to determine the microbial communities present in the floodplain of a river running through discontinuous permafrost. We found that the river's lateral movement across its floodplain influences the occurrence of certain microbial communities-in particular, methane-cycling microbes were present on the older, permafrost-bearing eroding riverbank but absent on the newly deposited river bars. Riverbank sediment had microbial communities more similar to those of the floodplain active-layer samples than permafrost samples from the same depth. Therefore, spatial patterns of river migration influence the distribution of microbial taxa relevant to the warming Arctic climate.

Evaluation of overall survival and barriers to surgery for patients with breast cancer treated without surgery: a National Cancer Database analysis.

Surgery remains the foundation of curative therapy for non-metastatic breast cancer, but many patients do not undergo surgery. Evidence is limited regarding this population. We sought to assess factors associated with lack of surgery and overall survival (OS) in patients not receiving breast cancer surgery. Retrospective cohort study of patients in the US National Cancer Database treated in 2004-2016. The dataset comprised 2,696,734 patients; excluding patients with unknown surgical status or stage IV, cT0, cTx, or pIS, metastatic or recurrent disease resulted in 1,192,294 patients for analysis. Chi-square and Wilcoxon rank-sum tests were used to assess differences between groups. OS was analyzed using the Kaplan-Meier method with a Cox proportional hazards model performed to assess associated factors. In total 50,626 (4.3%) did not undergo surgery. Black race, age >50 years, lower income, uninsured or public insurance, and lower education were more prevalent in the non-surgical cohort; this group was also more likely to have more comorbidities, higher disease stage, and more aggressive disease biology. Only 3,689 non-surgical patients (7.3%) received radiation therapy (RT). Median OS time for the non-surgical patients was 58 months (3-year and 5-year OS rates 63% and 49%). Median OS times were longer for patients who received chemotherapy (80 vs 50 (no-chemo) months) and RT (85 vs 56 (no-RT) months). On multivariate analysis, age, race, income, insurance status, comorbidity score, disease stage, tumor subtype, treatment facility type and location, and receipt of RT were associated with OS. On subgroup analysis, receipt of chemotherapy improved OS for patients with triple negative (HR 0.66, 95% CI 0.59-0.75, P < 0.001) and HER2+ (HR 0.74, 95% CI 0.65-0.84, P < 0.001) subgroups while RT improved OS for ER+ (HR 0.72, 95% CI 0.64-0.82, P < 0.001) and favorable-disease (ER+, early-stage, age >60) (HR 0.61, 95% CI 0.45-0.83, P = 0.002) subgroups. Approximately 4% of women with breast cancer do not undergo surgery, particularly those with more aggressive disease and lower socioeconomic status. Despite its benefits, RT was underutilized. This study provides a benchmark of survival outcomes for patients who do not undergo surgery and highlights a potential role for use of RT.

An ecophysiological explanation for manganese enrichment in rock varnish.

Desert varnish is a dark rock coating that forms in arid environments worldwide. It is highly and selectively enriched in manganese, the mechanism for which has been a long-standing geological mystery. We collected varnish samples from diverse sites across the western United States, examined them in petrographic thin section using microscale chemical imaging techniques, and investigated the associated microbial communities using 16S amplicon and shotgun metagenomic DNA sequencing. Our analyses described a material governed by sunlight, water, and manganese redox cycling that hosts an unusually aerobic microbial ecosystem characterized by a remarkable abundance of photosynthetic Cyanobacteria in the genus Chroococcidiopsis as the major autotrophic constituent. We then showed that diverse Cyanobacteria, including the relevant Chroococcidiopsis taxon, accumulate extraordinary amounts of intracellular manganese-over two orders of magnitude higher manganese content than other cells. The speciation of this manganese determined by advanced paramagnetic resonance techniques suggested that the Cyanobacteria use it as a catalytic antioxidant-a valuable adaptation for coping with the substantial oxidative stress present in this environment. Taken together, these results indicated that the manganese enrichment in varnish is related to its specific uptake and use by likely founding members of varnish microbial communities.

Non-lithifying microbial ecosystem dissolves peritidal lime sand.

Microbialites accrete where environmental conditions and microbial metabolisms promote lithification, commonly through carbonate cementation. On Little Ambergris Cay, Turks and Caicos Islands, microbial mats occur widely in peritidal environments above ooid sand but do not become lithified or preserved. Sediment cores and porewater geochemistry indicated that aerobic respiration and sulfide oxidation inhibit lithification and dissolve calcium carbonate sand despite widespread aragonite precipitation from platform surface waters. Here, we report that in tidally pumped environments, microbial metabolisms can negate the effects of taphonomically-favorable seawater chemistry on carbonate mineral saturation and microbialite development.