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DOE OSTI2013

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes

Background: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. Methods: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. Results: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. Conclusions: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components

Background: Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. Results: To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples. Conclusions: Camelid antibody VHH domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development. Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data. We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Single-cell proteomics of Arabidopsis leaf mesophyll reveals dynamic protein responses to water-deficit stress

Background The application of single-cell omics tools to biological systems can provide unique insights into diverse cellular populations and their heterogeneous responses to internal and external perturbations. Thus far, most single-cell studies in plant systems have been limited to RNA-sequencing approaches, which only provide indirect readouts of cellular functions. Results Here, we present a single-cell proteomics workflow for plant cells that integrates tape-sandwich protoplasting, piezoelectric cell sorting, nanoPOTS sample preparation, and ion mobility-based MS data acquisition method for label-free single-cell proteomics analysis of Arabidopsis leaf mesophyll cells. From a single leaf protoplast, over 3,000 proteins were quantified with high precision. The workflow is demonstrated to identify stress associated changes in protein abundance by analyzing 117 protoplasts from well-watered and water-deficit stressed plants. Additionally, we describe a new approach for constructing covarying protein networks at the single-cell level and demonstrate how single-cell protein covariation analysis can reveal previously unrecognized protein functions while also capturing stress-induced changes in protein–protein dynamics. Conclusions The label-free scProteomic approach presented here represents a significant advance through the demonstration of a facile protoplast isolation method combined with deep and precise proteomic coverage of Arabidopsis leaf mesophyll cell types. We believe this study will serve as an informative reference to future plant scProteomic investigations.

Arabidopsis↗

DOE OSTI2014

Systematically programmed adaptive evolution reveals potential role of carbon and nitrogen pathways during lipid accumulation in Chlamydomonas reinhardtii

Background: The concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen-(N) depleted and -replete conditions. Results: The successive sorting and regeneration of the top 25,000 high-lipid content cells of CC124 and sta6-1, combined with nitrogen starvation, led to the generation of a new population with an improved lipid content when compared to the original populations (approximately 175% and 50% lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes, and the enzymes involved in carbon fixation and uptake, including chlorophyll-ab-binding proteins and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically overexpressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution. Conclusions: Adaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning, flux, and fixation and carbon-nitrogen metabolism during lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation.

09 BIOMASS FUELS↗

DOE OSTI2019

A finalized determinant for complete lignocellulose enzymatic saccharification potential to maximize bioethanol production in bioenergy Miscanthus

Miscanthus is a leading bioenergy crop with enormous lignocellulose production potential for biofuels and chemicals. However, lignocellulose recalcitrance leads to biomass process difficulty for an efficient bioethanol production. Hence, it becomes essential to identify the integrative impact of lignocellulose recalcitrant factors on cellulose accessibility for biomass enzymatic hydrolysis. In this study, we analyzed four typical pairs of Miscanthus accessions that showed distinct cell wall compositions and sorted out three major factors that affected biomass saccharification for maximum bioethanol production. Among the three optimal (i.e., liquid hot water, H 2 SO 4 and NaOH) pretreatments performed, mild alkali pretreatment (4% NaOH at 50 °C) led to almost complete biomass saccharification when 1% Tween-80 was co-supplied into enzymatic hydrolysis in the desirable Miscanthus accessions. Consequently, the highest bioethanol yields were obtained at 19% (% dry matter) from yeast fermentation, with much higher sugar–ethanol conversion rates by 94–98%, compared to the other Miscanthus species subjected to stronger pretreatments as reported in previous studies. By comparison, three optimized pretreatments distinctively extracted wall polymers and specifically altered polymer features and inter-linkage styles, but the alkali pretreatment caused much increased biomass porosity than that of the other pretreatments. Based on integrative analyses, excellent equations were generated to precisely estimate hexoses and ethanol yields under various pretreatments and a hypothetical model was proposed to outline an integrative impact on biomass saccharification and bioethanol production subjective to a predominate factor (CR stain) of biomass porosity and four additional minor factors (DY stain, cellulose DP, hemicellulose X/A, lignin G-monomer). Using four pairs of Miscanthus samples with distinct cell wall composition and varied biomass saccharification, this study has determined three main factors of lignocellulose recalcitrance that could be significantly reduced for much-increased biomass porosity upon optimal pretreatments. It has also established a novel standard that should be applicable to judge any types of biomass process technology for high biofuel production in distinct lignocellulose substrates. Hence, this study provides a potential strategy for precise genetic modification of lignocellulose in all bioenergy crops.

09 BIOMASS FUELS↗

DOE OSTI2018

The orphan nuclear receptor EAR-2 (NR2F6) inhibits hematopoietic cell differentiation and induces myeloid dysplasia in vivo

Background: In patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased predisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)–suggesting that factors regulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as a gene involved in control of growth ability. Methods: Bone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP. Ex vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments cells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin RNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo. Results: Here, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant chimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating experiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization of immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were cytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed that EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic differentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid and myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential in EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone deacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain. Conclusions: This data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the NR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2016

SNaPP: Simplified Nanoproteomics Platform for Reproducible Global Proteomic Analysis of Nanogram Protein Quantities

Global proteomic analyses are now widely applied across biological research to study changes in an organism(s) proteome correlated with a perturbation, phenotype and/or time series of interest.[1-3] Further, it has been broadly established that efficient and reproducible sample preparation workflows are crucial to successful quantitative proteome comparisons, especially when applying label free methods.[4-8] However, clinical samples are often severely limited in quantity and can preclude the application of more robust bulk sample processing workflows due to e.g. contamination, carry-over, or sample losses.[9] This has limited the effective application of global proteomics for many sample types of great interest, e.g. LCM dissected tissues, FACS sorted cells, circulating tumor cells (CTC), and early embryos. In a typical proteomics experiment, bulk homogenization is applied to generate sufficient protein for processing (> 10 µg protein), and can blend the proteomes of many different cell types and disparate tissue regions. The resulting “average” proteome, can effectively render unobservable proteome changes of interest, and preclude important applications. Global proteomic analyses of complex protein samples in nanogram quantities require a fastidious approach to achieve in-depth protein coverage and quantitative reproducibility.

automation↗

DOE OSTI2020

Scenario Grouping and Decomposition Algorithms for Chance-Constrained Programs

A lower bound for a finite-scenario-based chance-constrained program is the quantile value corresponding to the sorted optimal objective values of scenario subproblems. This quantile bound can be improved by grouping subsets of scenarios at the expense of solving larger subproblems. The quality of the bound depends on how the scenarios are grouped. In this paper, we formulate a mixed-integer bilevel program that optimally groups scenarios to tighten the quantile bounds. For general chance-constrained programs, we propose a branch-and-cut algorithm to optimize the bilevel program, and for chance-constrained linear programs, a mixed-integer linear-programming reformulation is derived. Here, we also propose several heuristics for grouping similar or dissimilar scenarios. Our computational results demonstrate that optimal grouping bounds are much tighter than heuristic bounds, resulting in smaller root-node gaps and better performance of scenario decomposition for solving chance-constrained 0-1 programs. Also, the optimal grouping bounds can be greatly strengthened using larger group size.

97 MATHEMATICS AND COMPUTING↗

DOE OSTI2024

Summit Darshan Archival Dataset

Summit Darshan Archival Dataset contains 2021 Summit Darshan log data for 25 applications and is grouped into science domains. The dataset is processed, and all the propriety fields are anonymized. The resultant data is converted into a tabular structure and saved in parquet file format. In this notebook, we demonstrate how to access the data. Data Organization: The data is organized into two directories: Darshan total (`darshan_total`): List all the high levels generated by the `darshan-parser --total` command on `.darshan` files. There is one parquet file for each application. Note: `uid` and `exe` field are masked Darshan detail (`darshan_detail`): This data contains detailed job level log information extracted by command `darshan-parser` on the raw `.darshan` files. The data is sorted by directory hierarchy in the order of `year/month/day (2021/12/07)`. For instance, to get the data for a `job_id` 3819766 of application `App11`, which was executed on `2021-12-07`can be accessed as follows. Note:`uid` and `filename` fields are masked

97 MATHEMATICS AND COMPUTING↗

DOE OSTI2015

Classification methodology for tritiated waste requiring interim storage

Fusion machines like the ITER experimental research facility will use tritium as fuel. Therefore, most of the solid radioactive waste will result not only from activation by 14 MeV neutrons, but also from contamination by tritium. As a consequence, optimizing the treatment process for waste containing tritium (tritiated waste) is a major challenge. This paper summarizes the studies conducted in France within the framework of the French national plan for the management of radioactive materials and waste. The paper recommends a reference program for managing this waste based on its sorting, treatment and packaging by the producer. It also recommends setting up a 50-year temporary storage facility to allow for tritium decay and designing future disposal facilities using tritiated radwaste characteristics as input data. This paper first describes this waste program and then details an optimized classification methodology which takes into account tritium decay over a 50-year storage period. The paper also describes a specific application for purely tritiated waste and discusses the set-up expected to be implemented for ITER decommissioning waste (current assumption). Comparison between this optimized approach and other viable detritiation techniques will be drawn. (authors)

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

DOE OSTI2018

X-ray tomography of extended objects: a comparison of data acquisition approaches

The penetration power of x-rays allows one to image large objects while their short wavelength allows for high spatial resolution. As a result, with synchrotron sources one has the potential to obtain tomographic images of centimeter-sized specimens with sub-micrometer pixel sizes. However, limitations on beam and detector size make it difficult to acquire such data of this sort in a single take, necessitating strategies for combining data from multiple regions. One strategy is to acquire a tiled set of local tomograms by rotating the specimen around each of the local tomogram center positions. Another strategy, sinogram oriented acquisition, involves the collection of projections at multiple offset positions from the rotation axis followed by data merging and reconstruction. We have carried out a simulation study to compare these two approaches in terms of radiation dose applied to the specimen, and reconstructed image quality. Local tomography acquisition involves an easier data alignment problem, and immediate viewing of subregions before the entire dataset has been acquired. Sinogram oriented acquisition involves a more difficult data assembly and alignment procedure, and it is more sensitive to accumulative registration error. Furthermore, sinogram oriented acquisition is more dose-efficient, it involves fewer translation motions of the object, and it avoids certain artifacts of local tomography.

47 OTHER INSTRUMENTATION↗

DOE OSTI2019

Initial observations of the femtosecond timing jitter at the European XFEL

Intense, ultrashort, and high-repetition-rate X-ray pulses, combined with a femtosecond optical laser, allow pump-probe experiments with fast data acquisition and femtosecond time resolution. However, the relative timing of the X-ray pulses and the optical laser pulses can be controlled only to a level of the intrinsic error of the instrument which, without characterization, limits the time resolution of experiments. This limitation inevitably calls for a precise determination of the relative arrival time, which can be used after measurement for sorting and tagging the experimental data to a much finer resolution than it can be controlled to. The observed root-mean-square timing jitter between the X-ray and the optical laser at the SPB/SFX instrument at European XFEL was 308 fs. This first measurement of timing jitter at the European XFEL provides an important step in realizing ultrafast experiments at this novel X-ray source. Lastly, a method for determining the change in the complex refractive index of samples is also presented.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

DOE OSTI2020

Optical matter machines: angular momentum conversion by collective modes in optically bound nanoparticle arrays

The creation of optically powered self-assembling nano-to-meso-scale machines that do work is a long-standing goal in photonics. We demonstrate an optical matter (OM) machine that converts the spin angular momentum (SAM) of light into orbital angular momentum (OAM) to do mechanical work. The specific OM machine we study is based on a sixfold symmetric hexagonally ordered nanoparticle array that operates as an OM “gear” that is assembled and made to rotate in a circularly polarized Gaussian beam. The rotational symmetry of the OM gear leads to a selection rule for the allowed scattering modes based on their angular momentum. Electrodynamics calculations show that the collective scattering modes with the largest angular momentum scatter strongly in the transverse direction. Simulations and experiments show that the angular momentum that accompanies the scattered light causes a “negative torque” response on the OM gear and drives a “probe” particle placed outside the OM gear around the gear in an asymmetric force field analogously to Brownian ratchets. The gear–probe OM machine concept can be expanded to applications in nanofluidics and particle sorting.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

DOE OSTI2020

3D diffractive imaging of nanoparticle ensembles using an x-ray laser

Single particle imaging at x-ray free electron lasers (XFELs) has the potential to determine the structure and dynamics of single biomolecules at room temperature. Two major hurdles have prevented this potential from being reached, namely, the collection of sufficient high-quality diffraction patterns and robust computational purification to overcome structural heterogeneity. We report the breaking of both of these barriers using gold nanoparticle test samples, recording around 10 million diffraction patterns at the European XFEL and structurally and orientationally sorting the patterns to obtain better than 3-nm-resolution 3D reconstructions for each of four samples. With these new developments, integrating advancements in x-ray sources, fast-framing detectors, efficient sample delivery, and data analysis algorithms, we illuminate the path towards sub-nanometer biomolecular imaging. The methods developed here can also be extended to characterize ensembles that are inherently diverse to obtain their full structural landscape.

47 OTHER INSTRUMENTATION↗

DOE OSTI2026

Astigmatism-free 3D optical tweezer control for rapid atom rearrangement

Reconfigurable neutral-atom arrays are a promising platform for quantum computing, quantum simulation, and quantum metrology, but atom transport using frequency-chirped acousto-optic deflectors (AODs) is limited by chirp-induced acoustic lensing and trajectory distortion. We address these limitations using a three-dimensional acousto-optic deflector lens (3D-AODL), a design predicted to reduce long-range transport times by more than a factor of two. We further introduce fading-Shepard waveforms that circumvent finite AOD bandwidth, enabling sustained axial displacement. We demonstrate unrestricted three-dimensional optical-tweezer motion over a 200 μm × 200 μm × 136 μm volume with velocities exceeding 4.2 m/s. Arbitrary three-dimensional control of optical-tweezer trajectories enables rapid atom rearrangement and dynamical engineering of optical potentials in tweezer arrays and optical lattices. This capability advances quantum control and atom manipulation in neutral-atom quantum processors by enabling faster rearrangement, higher clock rates, and scalable sorting in complex geometries.

Lu, Yue-Hui [University of California, Berkeley, C↗

DOE OSTI2018

Candida albicans biofilm–induced vesicles confer drug resistance through matrix biogenesis

Cells from all kingdoms of life produce extracellular vesicles (EVs). Their cargo is protected from the environment by the surrounding lipid bilayer. EVs from many organisms have been shown to function in cell–cell communication, relaying signals that impact metazoan development, microbial quorum sensing, and pathogenic host–microbe interactions. Here, we have investigated the production and functional activities of EVs in a surface-associated microbial community or biofilm of the fungal pathogen Candida albicans. Crowded communities like biofilms are a context in which EVs are likely to function. Biofilms are noteworthy because they are encased in an extracellular polymeric matrix and because biofilm cells exhibit extreme tolerance to antimicrobial compounds. We found that biofilm EVs are distinct from those produced by free-living planktonic cells and display strong parallels in composition to biofilm matrix material. The functions of biofilm EVs were delineated with a panel of mutants defective in orthologs of endosomal sorting complexes required for transport (ESCRT) subunits, which are required for normal EV production in diverse eukaryotes. Most ESCRT-defective mutations caused reduced biofilm EV production, reduced matrix polysaccharide levels, and greatly increased sensitivity to the antifungal drug fluconazole. Matrix accumulation and drug hypersensitivity of ESCRT mutants were reversed by addition of wild-type (WT) biofilm EVs. Vesicle complementation showed that biofilm EV function derives from specific cargo proteins. Our studies indicate that C. albicans biofilm EVs have a pivotal role in matrix production and biofilm drug resistance. Biofilm matrix synthesis is a community enterprise; prior studies of mixed cell biofilms have demonstrated extracellular complementation. Therefore, EVs function not only in cell–cell communication but also in the sharing of microbial community resources.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota , none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

59 BASIC BIOLOGICAL SCIENCES↗