Publications by authors named "Samuel O Purvine"

95 Publications

Decrypting bacterial polyphenol metabolism in an anoxic wetland soil.

Nat Commun 2021 04 29;12(1):2466. Epub 2021 Apr 29.

Colorado State University, Fort Collins, CO, USA.

Microorganisms play vital roles in modulating organic matter decomposition and nutrient cycling in soil ecosystems. The enzyme latch paradigm posits microbial degradation of polyphenols is hindered in anoxic peat leading to polyphenol accumulation, and consequently diminished microbial activity. This model assumes that polyphenols are microbially unavailable under anoxia, a supposition that has not been thoroughly investigated in any soil type. Here, we use anoxic soil reactors amended with and without a chemically defined polyphenol to test this hypothesis, employing metabolomics and genome-resolved metaproteomics to interrogate soil microbial polyphenol metabolism. Challenging the idea that polyphenols are not bioavailable under anoxia, we provide metabolite evidence that polyphenols are depolymerized, resulting in monomer accumulation, followed by the generation of small phenolic degradation products. Further, we show that soil microbiome function is maintained, and possibly enhanced, with polyphenol addition. In summary, this study provides chemical and enzymatic evidence that some soil microbiota can degrade polyphenols under anoxia and subvert the assumed polyphenol lock on soil microbial metabolism.
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http://dx.doi.org/10.1038/s41467-021-22765-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8084988PMC
April 2021

Anaerobic gut fungi are an untapped reservoir of natural products.

Proc Natl Acad Sci U S A 2021 May;118(18)

Department of Chemical Engineering, University of California, Santa Barbara, CA 93106;

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: (), (), (), and (). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases. In addition, and genomes encoded seven putative bacteriocins, a class of antimicrobial peptides typically produced by bacteria. During standard laboratory growth on plant biomass or soluble substrates, 26% of total core biosynthetic genes in all four strains were transcribed. Across all four fungal strains, 30% of total biosynthetic gene products were detected via proteomics when grown on cellobiose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of fungal supernatants detected 72 likely natural products from alone. A compound produced by all four strains of anaerobic fungi was putatively identified as the polyketide-related styrylpyrone baumin. Molecular networking quantified similarities between tandem mass spectrometry (MS/MS) spectra among these fungi, enabling three groups of natural products to be identified that are unique to anaerobic fungi. Overall, these results support the finding that anaerobic gut fungi synthesize natural products, which could be harnessed as a source of antimicrobials, therapeutics, and other bioactive compounds.
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http://dx.doi.org/10.1073/pnas.2019855118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106346PMC
May 2021

Mechanisms of Manganese(II) Oxidation by Filamentous Ascomycete Fungi Vary With Species and Time as a Function of Secretome Composition.

Front Microbiol 2021 10;12:610497. Epub 2021 Feb 10.

Department of Marine Chemistry & Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA, United States.

Manganese (Mn) oxides are among the strongest oxidants and sorbents in the environment, and Mn(II) oxidation to Mn(III/IV) (hydr)oxides includes both abiotic and microbially-mediated processes. While white-rot Basidiomycete fungi oxidize Mn(II) using laccases and manganese peroxidases in association with lignocellulose degradation, the mechanisms by which filamentous Ascomycete fungi oxidize Mn(II) and a physiological role for Mn(II) oxidation in these organisms remain poorly understood. Here we use a combination of chemical and in-gel assays and bulk mass spectrometry to demonstrate secretome-based Mn(II) oxidation in three phylogenetically diverse Ascomycetes that is mechanistically distinct from hyphal-associated Mn(II) oxidation on solid substrates. We show that Mn(II) oxidative capacity of these fungi is dictated by species-specific secreted enzymes and varies with secretome age, and we reveal the presence of both Cu-based and FAD-based Mn(II) oxidation mechanisms in all 3 species, demonstrating mechanistic redundancy. Specifically, we identify candidate Mn(II)-oxidizing enzymes as tyrosinase and glyoxal oxidase in sp. SRC1lsM3a, bilirubin oxidase in sp. and AP3s5-JAC2a, and GMC oxidoreductase in all 3 species, including sp. DS3sAY3a. The diversity of the candidate Mn(II)-oxidizing enzymes identified in this study suggests that the ability of fungal secretomes to oxidize Mn(II) may be more widespread than previously thought.
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http://dx.doi.org/10.3389/fmicb.2021.610497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902709PMC
February 2021

Intracellular pathways for lignin catabolism in white-rot fungi.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401;

Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, and , funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts-a key step toward enabling a sustainable bioeconomy.
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http://dx.doi.org/10.1073/pnas.2017381118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936344PMC
March 2021

Widespread polycistronic gene expression in green algae.

Proc Natl Acad Sci U S A 2021 Feb;118(7)

UCLA DOE Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095;

Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: and Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between , , and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.
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http://dx.doi.org/10.1073/pnas.2017714118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896298PMC
February 2021

The Specific Carbohydrate Diet and Diet Modification as Induction Therapy for Pediatric Crohn's Disease: A Randomized Diet Controlled Trial.

Nutrients 2020 Dec 6;12(12). Epub 2020 Dec 6.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.

Background: Crohn's disease (CD) is a chronic inflammatory intestinal disorder associated with intestinal dysbiosis. Diet modulates the intestinal microbiome and therefore has a therapeutic potential. The aim of this study is to determine the potential efficacy of three versions of the specific carbohydrate diet (SCD) in active Crohn's Disease.

Methods: 18 patients with mild/moderate CD (PCDAI 15-45) aged 7 to 18 years were enrolled. Patients were randomized to either SCD, modified SCD(MSCD) or whole foods (WF) diet. Patients were evaluated at baseline, 2, 4, 8 and 12 weeks. PCDAI, inflammatory labs and multi-omics evaluations were assessed.

Results: Mean age was 14.3 ± 2.9 years. At week 12, all participants (n = 10) who completed the study achieved clinical remission. The C-reactive protein decreased from 1.3 ± 0.7 at enrollment to 0.9 ± 0.5 at 12 weeks in the SCD group. In the MSCD group, the CRP decreased from 1.6 ± 1.1 at enrollment to 0.7 ± 0.1 at 12 weeks. In the WF group, the CRP decreased from 3.9 ± 4.3 at enrollment to 1.6 ± 1.3 at 12 weeks. In addition, the microbiome composition shifted in all patients across the study period. While the nature of the changes was largely patient specific, the predicted metabolic mode of the organisms increasing and decreasing in activity was consistent across patients.

Conclusions: This study emphasizes the impact of diet in CD. Each diet had a positive effect on symptoms and inflammatory burden; the more exclusionary diets were associated with a better resolution of inflammation.
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http://dx.doi.org/10.3390/nu12123749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762109PMC
December 2020

Functional Genomics, Transcriptomics, and Proteomics Reveal Distinct Combat Strategies Between Lineages of Wood-Degrading Fungi With Redundant Wood Decay Mechanisms.

Front Microbiol 2020 28;11:1646. Epub 2020 Jul 28.

Department of Plant and Microbial Biology, University of Minnesota, Saint Paul, MN, United States.

Wood-degrading fungi vary in their strategies for deconstructing wood, and their competitive successes shape the rate and fate of carbon released from wood, Earth's largest pool of aboveground terrestrial carbon. In this study, one-on-one interspecific interactions between two model brown rot (carbohydrate-selective) fungi, and () , were studied on wood wafers where a clearly resolved interaction zone (IZ) could be generated, reproducibly. Comparative RNAseq and proteomics between the IZ and non-interacting hyphae of each species identified combative strategies for each fungus. Glycoside hydrolases were a relatively smaller portion of the interaction secretome compared to non-interacting hyphae. The interaction zone showed higher pectinase specific activity than all other sampling locations, and higher laminarinase specific activity (branched β-glucan proxy) was seen in the IZ secretome relative to equivalent hyphae in single-species cultures. Our efforts also identified two distinct competitive strategies in these two fungi with a shared nutritional mode (brown rot) but polyphyletic ancestral lineages. (Gloeophyllum clade) upregulated more secondary metabolite (SM) synthesis genes in response to a competitor than did . (Antrodia clade) upregulated a larger variety of uncharacterized oxidoreductases in interacting hyphae, suggesting that these may play a role in mediating competitor response in this fungus. Both species produced several hypothetical proteins exclusively in the interaction zone, leaving questions as to the function of these proteins. This work supports the existence of multiple interaction strategies among brown rot fungi and highlights the functional diversity among wood decay fungi.
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http://dx.doi.org/10.3389/fmicb.2020.01646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399148PMC
July 2020

Outer membrane vesicles catabolize lignin-derived aromatic compounds in KT2440.

Proc Natl Acad Sci U S A 2020 04 3;117(17):9302-9310. Epub 2020 Apr 3.

National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401;

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic-catabolic bacteria: KT2440, RHA1, and sp. ATCC 39116. cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.
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http://dx.doi.org/10.1073/pnas.1921073117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196908PMC
April 2020

Structure Dependent Determination of Organophosphate Targets in Mammalian Tissues Using Activity-Based Protein Profiling.

Chem Res Toxicol 2020 02 10;33(2):414-425. Epub 2020 Jan 10.

Biological Sciences Division , Pacific Northwest National Laboratory , Richland , Washington 99354 , United States.

Acute and chronic exposures to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity. FOP features a fluorophosphonate moiety, and PODA and CODA utilize a dialkynyl phosphate ester; both reactive group types target serine hydrolase activity. As the oxon represents the highly reactive and toxic functional group of many OPs, the new probes described herein enhance our understanding of tissue-specific reactivity of OPs. Chemoproteomic analysis of mouse tissues treated with the probes revealed divergent protein profiles, demonstrating the influence of probe structure on protein targeting. These targets also vary in sensitivity toward different OPs. The simultaneous use of multiple probes in ABPP experiments may therefore offer more comprehensive coverage of OP targets; FOP consistently labeled more targets in both brain and liver than PODA or CODA, suggesting the dialkyne warhead is more selective for enzymes in major signaling pathways than the more reactive fluorophosphonate warhead. Additionally, the probes can be used to assess reactivation of OP-inhibited enzymes by N-oximes and may serve as diagnostic tools for screening of therapeutic candidates in a panel of protein targets. These applications will help clarify the short- and long-term effects of OP toxicity beyond acetylcholinesterase inhibition, investigate potential points of convergence for broad spectrum therapeutic development, and support future efforts to screen candidate molecules for efficacy in various model systems.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00344DOI Listing
February 2020

In situ transformation of ethoxylate and glycol surfactants by shale-colonizing microorganisms during hydraulic fracturing.

ISME J 2019 11 26;13(11):2690-2700. Epub 2019 Jun 26.

Department of Civil and Environmental Engineering, University of New Hampshire, Durham, NH, 03824, USA.

In the last decade, extensive application of hydraulic fracturing technologies to unconventional low-permeability hydrocarbon-rich formations has significantly increased natural-gas production in the United States and abroad. The injection of surface-sourced fluids to generate fractures in the deep subsurface introduces microbial cells and substrates to low-permeability rock. A subset of injected organic additives has been investigated for their ability to support biological growth in shale microbial community members; however, to date, little is known on how complex xenobiotic organic compounds undergo biotransformations in this deep rock ecosystem. Here, high-resolution chemical, metagenomic, and proteomic analyses reveal that widely-used surfactants are degraded by the shale-associated taxa Halanaerobium, both in situ and under laboratory conditions. These halotolerant bacteria exhibit surfactant substrate specificities, preferring polymeric propoxylated glycols (PPGs) and longer alkyl polyethoxylates (AEOs) over polyethylene glycols (PEGs) and shorter AEOs. Enzymatic transformation occurs through repeated terminal-end polyglycol chain shortening during co-metabolic growth through the methylglyoxal bypass. This work provides the first evidence that shale microorganisms can transform xenobiotic surfactants in fracture fluid formulations, potentially affecting the efficiency of hydrocarbon recovery, and demonstrating an important association between injected substrates and microbial growth in an engineered subsurface ecosystem.
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http://dx.doi.org/10.1038/s41396-019-0466-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794257PMC
November 2019

Deep-Subsurface Pressure Stimulates Metabolic Plasticity in Shale-Colonizing spp.

Appl Environ Microbiol 2019 06 30;85(12). Epub 2019 May 30.

Department of Soil and Crop Sciences, Colorado State University, Fort Collins, Colorado, USA

Bacterial strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales. is believed to be inadvertently introduced into this environment during the drilling and fracturing process and must therefore tolerate large changes in pressure, temperature, and salinity. Here, we used a strain isolated from a natural gas well in the Utica Point Pleasant formation to investigate metabolic and physiological responses to growth under high-pressure subsurface conditions. Laboratory incubations confirmed the ability of strain WG8 to grow under pressures representative of deep shale formations (21 to 48 MPa). Under these conditions, broad metabolic and physiological shifts were identified, including higher abundances of proteins associated with the production of extracellular polymeric substances. Confocal laser scanning microscopy indicated that extracellular polymeric substance (EPS) production was associated with greater cell aggregation when biomass was cultured at high pressure. Changes in central carbon metabolism under the same conditions were inferred from nuclear magnetic resonance (NMR) and gas chromatography measurements, revealing large per-cell increases in production of ethanol, acetate, and propanol and cessation of hydrogen production. These metabolic shifts were associated with carbon flux through 1,2-propanediol in response to slower fluxes of carbon through stage 3 of glycolysis. Together, these results reveal the potential for bioclogging and corrosion (via organic acid fermentation products) associated with persistent growth in deep, hydraulically fractured shale ecosystems, and offer new insights into cellular mechanisms that enable these strains to dominate deep-shale microbiomes. The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demonstrate that a representative species, frequently the dominant microbial taxon in hydraulically fractured shales, responds to pressures characteristic of the deep subsurface by shifting its metabolism to generate more corrosive organic acids and produce more polymeric substances that cause "clumping" of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly impact hydrocarbon recovery, these data also offer new insights for microbial control in these ecosystems.
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http://dx.doi.org/10.1128/AEM.00018-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544827PMC
June 2019

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures.

J Vis Exp 2019 01 7(143). Epub 2019 Jan 7.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).
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http://dx.doi.org/10.3791/59035DOI Listing
January 2019

Multiomics resolution of molecular events during a day in the life of Chlamydomonas.

Proc Natl Acad Sci U S A 2019 02 18;116(6):2374-2383. Epub 2019 Jan 18.

Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095;

The unicellular green alga displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes , , and show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.
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http://dx.doi.org/10.1073/pnas.1815238116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369806PMC
February 2019

Circadian Proteomic Analysis Uncovers Mechanisms of Post-Transcriptional Regulation in Metabolic Pathways.

Cell Syst 2018 12 12;7(6):613-626.e5. Epub 2018 Dec 12.

Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA. Electronic address:

Transcriptional and translational feedback loops in fungi and animals drive circadian rhythms in transcript levels that provide output from the clock, but post-transcriptional mechanisms also contribute. To determine the extent and underlying source of this regulation, we applied newly developed analytical tools to a long-duration, deeply sampled, circadian proteomics time course comprising half of the proteome. We found a quarter of expressed proteins are clock regulated, but >40% of these do not arise from clock-regulated transcripts, and our analysis predicts that these protein rhythms arise from oscillations in translational rates. Our data highlighted the impact of the clock on metabolic regulation, with central carbon metabolism reflecting both transcriptional and post-transcriptional control and opposing metabolic pathways showing peak activities at different times of day. The transcription factor CSP-1 plays a role in this metabolic regulation, contributing to the rhythmicity and phase of clock-regulated proteins.
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http://dx.doi.org/10.1016/j.cels.2018.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433121PMC
December 2018

Dichomitus squalens partially tailors its molecular responses to the composition of solid wood.

Environ Microbiol 2018 11 18;20(11):4141-4156. Epub 2018 Oct 18.

Fungal Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Utrecht, The Netherlands.

White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.
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http://dx.doi.org/10.1111/1462-2920.14416DOI Listing
November 2018

Genome-centric view of carbon processing in thawing permafrost.

Nature 2018 08 16;560(7716):49-54. Epub 2018 Jul 16.

Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia.

As global temperatures rise, large amounts of carbon sequestered in permafrost are becoming available for microbial degradation. Accurate prediction of carbon gas emissions from thawing permafrost is limited by our understanding of these microbial communities. Here we use metagenomic sequencing of 214 samples from a permafrost thaw gradient to recover 1,529 metagenome-assembled genomes, including many from phyla with poor genomic representation. These genomes reflect the diversity of this complex ecosystem, with genus-level representatives for more than sixty per cent of the community. Meta-omic analysis revealed key populations involved in the degradation of organic matter, including bacteria whose genomes encode a previously undescribed fungal pathway for xylose degradation. Microbial and geochemical data highlight lineages that correlate with the production of greenhouse gases and indicate novel syntrophic relationships. Our findings link changing biogeochemistry to specific microbial lineages involved in carbon processing, and provide key information for predicting the effects of climate change on permafrost systems.
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http://dx.doi.org/10.1038/s41586-018-0338-1DOI Listing
August 2018

Specialized proteomic responses and an ancient photoprotection mechanism sustain marine green algal growth during phosphate limitation.

Nat Microbiol 2018 07 25;3(7):781-790. Epub 2018 Jun 25.

Monterey Bay Aquarium Research Institute, Moss Landing, CA, USA.

Marine algae perform approximately half of global carbon fixation, but their growth is often limited by the availability of phosphate or other nutrients. As oceans warm, the area of phosphate-limited surface waters is predicted to increase, resulting in ocean desertification. Understanding the responses of key eukaryotic phytoplankton to nutrient limitation is therefore critical. We used advanced photo-bioreactors to investigate how the widespread marine green alga Micromonas commoda grows under transitions from replete nutrients to chronic phosphate limitation and subsequent relief, analysing photosystem changes and broad cellular responses using proteomics, transcriptomics and biophysical measurements. We find that physiological and protein expression responses previously attributed to stress are critical to supporting stable exponential growth when phosphate is limiting. Unexpectedly, the abundance of most proteins involved in light harvesting does not change, but an ancient light-harvesting-related protein, LHCSR, is induced and dissipates damaging excess absorbed light as heat throughout phosphate limitation. Concurrently, a suite of uncharacterized proteins with narrow phylogenetic distributions increase multifold. Notably, of the proteins that exhibit significant changes, 70% are not differentially expressed at the mRNA transcript level, highlighting the importance of post-transcriptional processes in microbial eukaryotes. Nevertheless, transcript-protein pairs with concordant changes were identified that will enable more robust interpretation of eukaryotic phytoplankton responses in the field from metatranscriptomic studies. Our results show that P-limited Micromonas responds quickly to a fresh pulse of phosphate by rapidly increasing replication, and that the protein network associated with this ability is composed of both conserved and phylogenetically recent proteome systems that promote dynamic phosphate homeostasis. That an ancient mechanism for mitigating light stress is central to sustaining growth during extended phosphate limitation highlights the possibility of interactive effects arising from combined stressors under ocean change, which could reduce the efficacy of algal strategies for optimizing marine photosynthesis.
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http://dx.doi.org/10.1038/s41564-018-0178-7DOI Listing
July 2018

Multi 'omics comparison reveals metabolome biochemistry, not microbiome composition or gene expression, corresponds to elevated biogeochemical function in the hyporheic zone.

Sci Total Environ 2018 Nov 17;642:742-753. Epub 2018 Jun 17.

Pacific Northwest National Laboratory, Richland, WA, USA.

Biogeochemical hotspots are pervasive at terrestrial-aquatic interfaces, particularly within groundwater-surface water mixing zones (hyporheic zones), and they are critical to understanding spatiotemporal variation in biogeochemical cycling. Here, we use multi 'omic comparisons of hotspots to low-activity sediments to gain mechanistic insight into hyporheic zone organic matter processing. We hypothesized that microbiome structure and function, as described by metagenomics and metaproteomics, would distinguish hotspots from low-activity sediments by shifting metabolism towards carbohydrate-utilizing pathways and elucidate discrete mechanisms governing organic matter processing in each location. We also expected these differences to be reflected in the metabolome, whereby hotspot carbon (C) pools and metabolite transformations therein would be enriched in sugar-associated compounds. In contrast to expectations, we found pronounced phenotypic plasticity in the hyporheic zone microbiome that was denoted by similar microbiome structure, functional potential, and expression across sediments with dissimilar metabolic rates. Instead, diverse nitrogenous metabolites and biochemical transformations characterized hotspots. Metabolomes also corresponded more strongly to aerobic metabolism than bulk C or N content only (explaining 67% vs. 42% and 37% of variation respectively), and bulk C and N did not improve statistical models based on metabolome composition alone. These results point to organic nitrogen as a significant regulatory factor influencing hyporheic zone organic matter processing. Based on our findings, we propose incorporating knowledge of metabolic pathways associated with different chemical fractions of C pools into ecosystem models will enhance prediction accuracy.
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http://dx.doi.org/10.1016/j.scitotenv.2018.05.256DOI Listing
November 2018

Coupling Secretomics with Enzyme Activities To Compare the Temporal Processes of Wood Metabolism among White and Brown Rot Fungi.

Appl Environ Microbiol 2018 08 1;84(16). Epub 2018 Aug 1.

University of Minnesota, Department of Bioproducts and Biosystems Engineering, St. Paul, Minnesota, USA

Wood-degrading fungi use a sequence of oxidative and hydrolytic mechanisms to loosen lignocellulose and then release and metabolize embedded sugars. These temporal sequences have recently been mapped at high resolution using directional growth on wood wafers, revealing previously obscured dynamics as fungi progressively colonize wood. Here, we applied secretomics in the same wafer design to track temporal trends on aspen decayed by fungi with distinct nutritional modes: two brown rot (BR) fungi ( and ) and two white rot (WR) fungi ( and ). We matched secretomic data from three zones of decay (early, middle, and late) with enzyme activities in these zones, and we included measures of total protein and ergosterol as measures of fungal biomass. In line with previous transcriptomics data, the fungi tested showed an initial investment in pectinases and a delayed investment in glycoside hydrolases (GHs). Brown rot fungi also staggered the abundance of some oxidoreductases ahead of GHs to produce a familiar two-step mechanism. White rot fungi, however, showed late-stage investment in pectinases as well, unlike brown rot fungi. Ligninolytic enzyme activities and abundances were also different between the two white rot fungi. Specifically, ligninolytic activity was delayed, which was explained almost entirely by the activity and abundance of five atypical manganese peroxidases, unlike more varied peroxidases and laccases in These secretomic analyses support brown rot patterns generated via transcriptomics, they reveal distinct patterns among and within rot types, and they link spectral counts with activities to help functionalize these multistrain secretomic data. Wood decay, driven primarily by wood-degrading basidiomycetes, is an essential component of global carbon cycles, and decay mechanisms are essential for understanding forest ecosystem function. These fungi efficiently consolidate pretreatment and saccharification of wood under mild conditions, making them promising templates for low-cost lignocellulose conversion. Species are categorized as ligninolytic white rots and polysaccharide-selective brown rots, with considerable undescribed variability in decay mechanism that may manifest in the sequential variation in protein secretion over the progression of decay. Here we resolved spatially a temporal progression of decay on intact wood wafers and compared secretome dynamics in two white and two brown rot fungi. We identified several universal mechanistic components among decay types, including early pectinolytic "pretreatment" and later-stage glycoside hydrolase-mediated saccharification. Interspecific comparisons also identified considerable mechanistic diversity within rot types, indicating that there are multiple avenues to facilitate white and brown rots.
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http://dx.doi.org/10.1128/AEM.00159-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070766PMC
August 2018

MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape.

Proc Natl Acad Sci U S A 2018 01 16;115(5):E1012-E1021. Epub 2018 Jan 16.

Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems-based approach, we examined differential regulation of IFN-γ-dependent genes following infection with robust respiratory viruses including influenza viruses [A/influenza/Vietnam/1203/2004 (H5N1-VN1203) and A/influenza/California/04/2009 (H1N1-CA04)] and coronaviruses [severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV)]. Categorizing by function, we observed down-regulation of gene expression associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down-regulation of antigen-presentation gene expression, which was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation, rather than histone modification, plays a crucial role in MERS-CoV-mediated antagonism of antigen-presentation gene expression; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Together, the results indicate a common mechanism utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.
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http://dx.doi.org/10.1073/pnas.1706928115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798318PMC
January 2018

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates.

Plant J 2018 02 7;93(3):545-565. Epub 2018 Jan 7.

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA.

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.
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http://dx.doi.org/10.1111/tpj.13788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775909PMC
February 2018

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases.

J Am Chem Soc 2017 11 1;139(45):16032-16035. Epub 2017 Nov 1.

Chemical Biology and Exposure Sciences, Biological Sciences Division, Pacific Northwest National Laboratory , 902 Battelle Boulevard, Richland, Washington 99352, United States.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.
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http://dx.doi.org/10.1021/jacs.7b07378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279235PMC
November 2017

Feathermoss and epiphytic Nostoc cooperate differently: expanding the spectrum of plant-cyanobacteria symbiosis.

ISME J 2017 12 11;11(12):2821-2833. Epub 2017 Aug 11.

Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, Sweden.

Dinitrogen (N)-fixation by cyanobacteria in symbiosis with feathermosses is the primary pathway of biological nitrogen (N) input into boreal forests. Despite its significance, little is known about the cyanobacterial gene repertoire and regulatory rewiring needed for the establishment and maintenance of the symbiosis. To determine gene acquisitions and regulatory changes allowing cyanobacteria to form and maintain this symbiosis, we compared genomically closely related symbiotic-competent and -incompetent Nostoc strains using a proteogenomics approach and an experimental set up allowing for controlled chemical and physical contact between partners. Thirty-two gene families were found only in the genomes of symbiotic strains, including some never before associated with cyanobacterial symbiosis. We identified conserved orthologs that were differentially expressed in symbiotic strains, including protein families involved in chemotaxis and motility, NO regulation, sulfate/phosphate transport, and glycosyl-modifying and oxidative stress-mediating exoenzymes. The physical moss-cyanobacteria epiphytic symbiosis is distinct from other cyanobacteria-plant symbioses, with Nostoc retaining motility, and lacking modulation of N-fixation, photosynthesis, GS-GOGAT cycle and heterocyst formation. The results expand our knowledge base of plant-cyanobacterial symbioses, provide a model of information and material exchange in this ecologically significant symbiosis, and suggest new currencies, namely nitric oxide and aliphatic sulfonates, may be involved in establishing and maintaining the cyanobacteria-feathermoss symbiosis.
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http://dx.doi.org/10.1038/ismej.2017.134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702739PMC
December 2017

Sulfide Generation by Dominant Microorganisms in Hydraulically Fractured Shales.

mSphere 2017 Jul-Aug;2(4). Epub 2017 Jul 5.

Department of Microbiology, The Ohio State University, Columbus, Ohio, USA.

Hydraulic fracturing of black shale formations has greatly increased United States oil and natural gas recovery. However, the accumulation of biomass in subsurface reservoirs and pipelines is detrimental because of possible well souring, microbially induced corrosion, and pore clogging. Temporal sampling of produced fluids from a well in the Utica Shale revealed the dominance of strains within the microbial community and the potential for these microorganisms to catalyze thiosulfate-dependent sulfidogenesis. From these field data, we investigated biogenic sulfide production catalyzed by a strain isolated from the produced fluids using proteogenomics and laboratory growth experiments. Analysis of isolate genomes and reconstructed genomes from metagenomic data sets revealed the conserved presence of rhodanese-like proteins and anaerobic sulfite reductase complexes capable of converting thiosulfate to sulfide. Shotgun proteomics measurements using a isolate verified that these proteins were more abundant when thiosulfate was present in the growth medium, and culture-based assays identified thiosulfate-dependent sulfide production by the same isolate. Increased production of sulfide and organic acids during the stationary growth phase suggests that fermentative uses thiosulfate to remove excess reductant. These findings emphasize the potential detrimental effects that could arise from thiosulfate-reducing microorganisms in hydraulically fractured shales, which are undetected by current industry-wide corrosion diagnostics. Although thousands of wells in deep shale formations across the United States have been hydraulically fractured for oil and gas recovery, the impact of microbial metabolism within these environments is poorly understood. Our research demonstrates that dominant microbial populations in these subsurface ecosystems contain the conserved capacity for the reduction of thiosulfate to sulfide and that this process is likely occurring in the environment. Sulfide generation (also known as "souring") is considered deleterious in the oil and gas industry because of both toxicity issues and impacts on corrosion of the subsurface infrastructure. Critically, the capacity for sulfide generation via reduction of sulfate was not detected in our data sets. Given that current industry wellhead tests for sulfidogenesis target canonical sulfate-reducing microorganisms, these data suggest that new approaches to the detection of sulfide-producing microorganisms may be necessary.
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http://dx.doi.org/10.1128/mSphereDirect.00257-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497025PMC
July 2017

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi.

Fungal Genet Biol 2017 09 1;106:61-75. Epub 2017 Jul 1.

Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, MA 02543, USA. Electronic address:

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.
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http://dx.doi.org/10.1016/j.fgb.2017.06.004DOI Listing
September 2017

Leucine Biosynthesis Is Involved in Regulating High Lipid Accumulation in .

mBio 2017 06 20;8(3). Epub 2017 Jun 20.

Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden

The yeast is a potent accumulator of lipids, and lipogenesis in this organism can be influenced by a variety of factors, such as genetics and environmental conditions. Using a multifactorial study, we elucidated the effects of both genetic and environmental factors on regulation of lipogenesis in and identified how two opposite regulatory states both result in lipid accumulation. This study involved comparison of a strain overexpressing diacylglycerol acyltransferase () with a control strain grown under either nitrogen or carbon limitation conditions. A strong correlation was observed between the responses on the transcript and protein levels. Combination of overexpression with nitrogen limitation resulted in a high level of lipid accumulation accompanied by downregulation of several amino acid biosynthetic pathways, including that of leucine in particular, and these changes were further correlated with a decrease in metabolic fluxes. This downregulation was supported by the measured decrease in the level of 2-isopropylmalate, an intermediate of leucine biosynthesis. Combining the multi-omics data with putative transcription factor binding motifs uncovered a contradictory role for TORC1 in controlling lipid accumulation, likely mediated through 2-isopropylmalate and a Leu3-like transcription factor. The ubiquitous metabolism of lipids involves refined regulation, and an enriched understanding of this regulation would have wide implications. Various factors can influence lipid metabolism, including the environment and genetics. We demonstrated, using a multi-omics and multifactorial experimental setup, that multiple factors affect lipid accumulation in the yeast Using integrative analysis, we identified novel interactions between nutrient restriction and genetic factors involving regulators that are highly conserved among eukaryotes. Given that lipid metabolism is involved in many diseases but is also vital to the development of microbial cell factories that can provide us with sustainable fuels and oleochemicals, we envision that our report introduces foundational work to further unravel the regulation of lipid accumulation in eukaryal cells.
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http://dx.doi.org/10.1128/mBio.00857-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478895PMC
June 2017

A parts list for fungal cellulosomes revealed by comparative genomics.

Nat Microbiol 2017 May 30;2:17087. Epub 2017 May 30.

Department of Chemical Engineering, University of California, Santa Barbara, California 93106, USA.

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs). However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including conserved scaffolding proteins unique to the Neocallimastigomycota. High-quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single-molecule technology. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure-an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.
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http://dx.doi.org/10.1038/nmicrobiol.2017.87DOI Listing
May 2017

Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry.

Mol Cell Proteomics 2016 Dec 25;15(12):3694-3705. Epub 2016 Sep 25.

From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington

Current proteomic approaches include both broad discovery measurements and quantitative targeted analyses. In many cases, discovery measurements are initially used to identify potentially important proteins (e.g. candidate biomarkers) and then targeted studies are employed to quantify a limited number of selected proteins. Both approaches, however, suffer from limitations. Discovery measurements aim to sample the whole proteome but have lower sensitivity, accuracy, and quantitation precision than targeted approaches, whereas targeted measurements are significantly more sensitive but only sample a limited portion of the proteome. Herein, we describe a new approach that performs both discovery and targeted monitoring (DTM) in a single analysis by combining liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled target peptides are spiked into tryptic digests and both the labeled and unlabeled peptides are detected using LC-IMS-MS instrumentation. Compared with the broad LC-MS discovery measurements, DTM yields greater peptide/protein coverage and detects lower abundance species. DTM also achieved detection limits similar to selected reaction monitoring (SRM) indicating its potential for combined high quality discovery and targeted analyses, which is a significant step toward the convergence of discovery and targeted approaches.
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http://dx.doi.org/10.1074/mcp.M116.061143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141281PMC
December 2016

Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi.

PLoS One 2016 19;11(7):e0157844. Epub 2016 Jul 19.

Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America.

Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157844PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4951024PMC
July 2017

Evidence-based green algal genomics reveals marine diversity and ancestral characteristics of land plants.

BMC Genomics 2016 Mar 31;17:267. Epub 2016 Mar 31.

Monterey Bay Aquarium Research Institute, 7700 Sandholdt Rd, Moss Landing, CA, 95039, USA.

Background: Prasinophytes are widespread marine green algae that are related to plants. Cellular abundance of the prasinophyte Micromonas has reportedly increased in the Arctic due to climate-induced changes. Thus, studies of these unicellular eukaryotes are important for marine ecology and for understanding Viridiplantae evolution and diversification.

Results: We generated evidence-based Micromonas gene models using proteomics and RNA-Seq to improve prasinophyte genomic resources. First, sequences of four chromosomes in the 22 Mb Micromonas pusilla (CCMP1545) genome were finished. Comparison with the finished 21 Mb genome of Micromonas commoda (RCC299; named herein) shows they share ≤8,141 of ~10,000 protein-encoding genes, depending on the analysis method. Unlike RCC299 and other sequenced eukaryotes, CCMP1545 has two abundant repetitive intron types and a high percent (26 %) GC splice donors. Micromonas has more genus-specific protein families (19 %) than other genome sequenced prasinophytes (11 %). Comparative analyses using predicted proteomes from other prasinophytes reveal proteins likely related to scale formation and ancestral photosynthesis. Our studies also indicate that peptidoglycan (PG) biosynthesis enzymes have been lost in multiple independent events in select prasinophytes and plants. However, CCMP1545, polar Micromonas CCMP2099 and prasinophytes from other classes retain the entire PG pathway, like moss and glaucophyte algae. Surprisingly, multiple vascular plants also have the PG pathway, except the Penicillin-Binding Protein, and share a unique bi-domain protein potentially associated with the pathway. Alongside Micromonas experiments using antibiotics that halt bacterial PG biosynthesis, the findings highlight unrecognized phylogenetic complexity in PG-pathway retention and implicate a role in chloroplast structure or division in several extant Viridiplantae lineages.

Conclusions: Extensive differences in gene loss and architecture between related prasinophytes underscore their divergence. PG biosynthesis genes from the cyanobacterial endosymbiont that became the plastid, have been selectively retained in multiple plants and algae, implying a biological function. Our studies provide robust genomic resources for emerging model algae, advancing knowledge of marine phytoplankton and plant evolution.
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http://dx.doi.org/10.1186/s12864-016-2585-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815162PMC
March 2016