The Chlamydomonas Genome Project, version 6: Reference assemblies for mating-type plus and minus strains reveal extensive structural mutation in the laboratory.

Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating-type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive manual curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of the helicase RECQ3, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference organism.

Pumping iron: A multi-omics analysis of two extremophilic algae reveals iron economy management.

Marine algae are responsible for half of the world's primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multiomics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a unique family of siderophore-iron-uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, which is closely related but identifiably distinct from the chlorophyll binding protein, LHCA3. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms.

Genome-centric view of carbon processing in thawing permafrost.

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.

Intracellular pathways for lignin catabolism in white-rot fungi.

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 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora, 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.

Anaerobic gut fungi are an untapped reservoir of natural products.

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: Anaeromyces robustus (Arobustus), Caecomyces churrovis (Cchurrovis), Neocallimastix californiae (Ncaliforniae), and Piromyces finnis (Pfinnis). 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, N. californiae and C. churrovis 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 A. robustus 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.

Widespread polycistronic gene expression in green algae.

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: Chlamydomonas reinhardtii and Chromochloris zofingiensis 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 C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.

Soil metabolome response to whole-ecosystem warming at the Spruce and Peatland Responses under Changing Environments experiment.

In this study, a suite of complementary environmental geochemical analyses, including NMR and gas chromatography-mass spectrometry (GC-MS) analyses of central metabolites, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of secondary metabolites, and lipidomics, was used to investigate the influence of organic matter (OM) quality on the heterotrophic microbial mechanisms controlling peatland CO2, CH4, and CO2:CH4 porewater production ratios in response to climate warming. Our investigations leverage the Spruce and Peatland Responses under Changing Environments (SPRUCE) experiment, where air and peat warming were combined in a whole-ecosystem warming treatment. We hypothesized that warming would enhance the production of plant-derived metabolites, resulting in increased labile OM inputs to the surface peat, thereby enhancing microbial activity and greenhouse gas production. Because shallow peat is most susceptible to enhanced warming, increases in labile OM inputs to the surface, in particular, are likely to result in significant changes to CO2 and CH4 dynamics and methanogenic pathways. In support of this hypothesis, significant correlations were observed between metabolites and temperature consistent with increased availability of labile substrates, which may stimulate more rapid turnover of microbial proteins. An increase in the abundance of methanogenic genes in response to the increase in the abundance of labile substrates was accompanied by a shift toward acetoclastic and methylotrophic methanogenesis. Our results suggest that as peatland vegetation trends toward increasing vascular plant cover with warming, we can expect a concomitant shift toward increasingly methanogenic conditions and amplified climate-peatland feedbacks.

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

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: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida 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.

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

The unicellular green alga Chlamydomonas reinhardtii 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 Chlamydomonas 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 PSBS, LHCSR1, and LHCSR3 show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while LHCSR3 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.

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

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.

Coupled laboratory and field investigations resolve microbial interactions that underpin persistence in hydraulically fractured shales.

Hydraulic fracturing is one of the industrial processes behind the surging natural gas output in the United States. This technology inadvertently creates an engineered microbial ecosystem thousands of meters below Earth's surface. Here, we used laboratory reactors to perform manipulations of persisting shale microbial communities that are currently not feasible in field scenarios. Metaproteomic and metabolite findings from the laboratory were then corroborated using regression-based modeling performed on metagenomic and metabolite data from more than 40 produced fluids from five hydraulically fractured shale wells. Collectively, our findings show that Halanaerobium, Geotoga, and Methanohalophilus strain abundances predict a significant fraction of nitrogen and carbon metabolites in the field. Our laboratory findings also exposed cryptic predatory, cooperative, and competitive interactions that impact microorganisms across fractured shales. Scaling these results from the laboratory to the field identified mechanisms underpinning biogeochemical reactions, yielding knowledge that can be harnessed to potentially increase energy yields and inform management practices in hydraulically fractured shales.

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

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.

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.

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.

Genomes and secretomes of Ascomycota fungi reveal diverse functions in plant biomass decomposition and pathogenesis.

BACKGROUND: The dominant fungi in arid grasslands and shrublands are members of the Ascomycota phylum. Ascomycota fungi are important drivers in carbon and nitrogen cycling in arid ecosystems. These fungi play roles in soil stability, plant biomass decomposition, and endophytic interactions with plants. They may also form symbiotic associations with biocrust components or be latent saprotrophs or pathogens that live on plant tissues. However, their functional potential in arid soils, where organic matter, nutrients and water are very low or only periodically available, is poorly characterized. RESULTS: Five Ascomycota fungi were isolated from different soil crust microhabitats and rhizosphere soils around the native bunchgrass Pleuraphis jamesii in an arid grassland near Moab, UT, USA. Putative genera were Coniochaeta, isolated from lichen biocrust, Embellisia from cyanobacteria biocrust, Chaetomium from below lichen biocrust, Phoma from a moss microhabitat, and Aspergillus from the soil. The fungi were grown in replicate cultures on different carbon sources (chitin, native bunchgrass or pine wood) relevant to plant biomass and soil carbon sources. Secretomes produced by the fungi on each substrate were characterized. Results demonstrate that these fungi likely interact with primary producers (biocrust or plants) by secreting a wide range of proteins that facilitate symbiotic associations. Each of the fungal isolates secreted enzymes that degrade plant biomass, small secreted effector proteins, and proteins involved in either beneficial plant interactions or virulence. Aspergillus and Phoma expressed more plant biomass degrading enzymes when grown in grass- and pine-containing cultures than in chitin. Coniochaeta and Embellisia expressed similar numbers of these enzymes under all conditions, while Chaetomium secreted more of these enzymes in grass-containing cultures. CONCLUSIONS: This study of Ascomycota genomes and secretomes provides important insights about the lifestyles and the roles that Ascomycota fungi likely play in arid grassland, ecosystems. However, the exact nature of those interactions, whether any or all of the isolates are true endophytes, latent saprotrophs or opportunistic phytopathogens, will be the topic of future studies.

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

Bacterial Halanaerobium strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales. Halanaerobium 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 Halanaerobium 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 Halanaerobium congolense 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 Halanaerobium 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 Halanaerobium growth in deep, hydraulically fractured shale ecosystems, and offer new insights into cellular mechanisms that enable these strains to dominate deep-shale microbiomes.IMPORTANCE 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 Halanaerobium 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.

Mineral Surfaces as Agents of Environmental Proteolysis: Mechanisms and Controls.

We investigated the extent to which contact with mineral surfaces affected the molecular integrity of a model protein, with an emphasis on identifying the mechanisms (hydrolysis, oxidation) and conditions leading to protein alteration. To this end, we studied the ability of four mineral surface archetypes (negatively charged, positively charged, neutral, redox-active) to abiotically fragment a well-characterized protein (GB1) as a function of pH and contact time. GB1 was exposed to the soil minerals montmorillonite, goethite, kaolinite, and birnessite at pH 5 and pH 7 for 1, 8, 24, and 168 h and the supernatant was screened for peptide fragments using Tandem Mass Spectrometry. To distinguish between products of oxidative and hydrolytic cleavage, we combined results from the SEQUEST algorithm, which identifies protein fragments that were cleaved hydrolytically, with the output of a deconvolution algorithm (DECON-Routine) designed to identify oxidation fragments. All four minerals were able to induce protein cleavage. Manganese oxide was effective at both hydrolytic and oxidative cleavage. The fact that phyllosilicates-which are not redox active-induced oxidative cleavage indicates that surfaces acted as catalysts and not as reactants. Our results extend previous observations of proteolytic capabilities in soil minerals to the groups of phyllosilicates and Fe-oxides. We identified structural regions of the protein with particularly high susceptibility to cleavage (loops and ß strands) as well as regions that were entirely unaffected (α helix).

Individual Variability of Protein Expression in Human Tissues.

Human tissues are known to exhibit interindividual variability, but a deeper understanding of the different factors affecting protein expression is necessary to further apply this knowledge. Our goal was to explore the proteomic variability between individuals as well as between healthy and diseased samples, and to test the efficacy of machine learning classifiers. In order to investigate whether disparate proteomics data sets may be combined, we performed a retrospective analysis of proteomics data from 9 different human tissues. These data sets represent several different sample prep methods, mass spectrometry instruments, and tissue health. Using these data, we examined interindividual and intertissue variability in peptide expression, and analyzed the methods required to build accurate tissue classifiers. We also evaluated the limits of tissue classification by downsampling the peptide data to simulate situations where less data is available, such as clinical biopsies, laser capture microdissection or potentially single-cell proteomics. Our findings reveal the strong potential for utilizing proteomics data to build robust tissue classifiers, which has many prospective clinical applications for evaluating the applicability of model clinical systems.

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

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.

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

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 (Postia placenta and Gloeophyllum trabeum) and two white rot (WR) fungi (Stereum hirsutum and Trametes versicolor). 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, S. hirsutum 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 T. versicolor 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.IMPORTANCE 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.

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

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.