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Growth ofChlamydomonas reinhardtiiin zinc (Zn) limited medium leads to disruption of copper (Cu) homeostasis, resulting in up to 40-fold Cu over-accumulation relative to its typical Cu quota. We show that Chlamydomonas controls its Cu quota by balancing Cu import and export, which is disrupted in a Zn deficient cell, thus establishing a mechanistic connection between Cu and Zn homeostasis. Transcriptomics, proteomics and elemental profiling revealed that Zn-limited Chlamydomonas cells up-regulate a subset of genes encoding “first responder” proteins involved in sulfur (S) assimilation and consequently accumulate more intracellular S, which is incorporated into L-cysteine, γ-glutamylcysteine and homocysteine. Most prominently, in the absence of Zn, free L-cysteine is increased ~80-fold, corresponding to ~ 2.8 × 109molecules/cell. Interestingly, classic S-containing metal binding ligands like glutathione and phytochelatins do not increase. X-ray fluorescence microscopy showed foci of S accumulation in Zn-limited cells that co-localize with Cu, phosphorus and calcium, consistent with Cu-thiol complexes in the acidocalcisome, the site of Cu(I) accumulation. Notably, cells that have been previously starved for Cu do not accumulate S or Cys, causally connecting cysteine synthesis with Cu accumulation. We suggest that cysteine is anin vivoCu(I) ligand, perhaps ancestral, that buffers cytosolic Cu.

Using tandem mass spectrometry (MS/MS), we analyzed the proteome of Sinorhizobium medicae WSM419 growing as free-living cells and in symbiosis with Medicago truncatula. In all, 3,215 proteins were identified, over half of the open reading frames predicted from the genomic sequence. The abundance of 1,361 proteins displayed strong lifestyle bias. In total, 1,131 proteins had similar levels in bacteroids and free-living cells, and the low levels of 723 proteins prevented statistically significant assignments. Nitrogenase subunits comprised approximately 12% of quantified bacteroid proteins. Other major bacteroid proteins included symbiosis-specific cytochromes and FixABCX, which transfer electrons to nitrogenase. Bacteroids had normal levels of proteins involved in amino acid biosynthesis, glycolysis or gluconeogenesis, and the pentose phosphate pathway; however, several amino acid degradation pathways were repressed. This suggests that bacteroids maintain a relatively independent anabolic metabolism. Tricarboxylic acid cycle proteins were highly expressed in bacteroids and no other catabolic pathway emerged as an obvious candidate to supply energy and reductant to nitrogen fixation. Bacterial stress response proteins were induced in bacteroids. Many WSM419 proteins that are not encoded in S. meliloti Rm1021 were detected, and understanding the functions of these proteins might clarify why S. medicae WSM419 forms a more effective symbiosis with M. truncatula than S. meliloti Rm1021. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Microbial decomposition drives the transformation of plant-derived substrates into microbial products that form stable soil organic matter (SOM). Recent theories have posited that decomposition depends on an interaction between SOM chemistry with microbial diversity and resulting function (e.g., enzymatic capabilities, growth rates). Here, we explicitly test these theories by coupling quantitative stable isotope probing and metabolomics to track the fate of 13C enriched substrates that vary in chemical composition as they are assimilated by microbes and transformed into new metabolic products in soil. We found that differences in forest nutrient economies (e.g., nutrient cycling, microbial competition) led to arbuscular mycorrhizal (AM) soils harboring greater diversity of fungi and bacteria than ectomycorrhizal (ECM) soils. When incubated with 13C enriched substrates, substrate type drove shifts in which species were active decomposers and the abundance of metabolic products that were reduced or saturated in the highly diverse AM soils. The decomposition pathways were more static in the less diverse, ECM soil. Importantly, the majority of these shifts were driven by taxa only present in the AM soil suggesting a strong link between microbial identity and their ability to decompose and assimilate substrates. Collectively, these results highlight an important interaction between ecosystem-level processes and microbial diversity; whereby the identity and function of active decomposers impacts the composition of decomposition products that can form stable SOM.

Soil nitrogen (N) transformations constrain terrestrial net primary productivity and are driven by the activity of soil microorganisms. Free-living N fixation (FLNF) is an important soil N transformation and key N input to terrestrial systems, but the forms of N contributed to soil by FLNF are poorly understood. To address this knowledge gap, a focus on microorganisms and microbial scale processes is needed that links N-fixing bacteria and their contributed N sources to FLNF process rates. However, studying the activity of soil microorganisms

Soil microorganisms have adapted to compete and exploit different metabolic niches in their physically and chemically diverse environment via evolution and acquisition of distinct physiological and biochemical traits. As the interface for most carbon and nutrient exchange between plants and microorganisms, the rhizosphere has received substantial attention. By comparison, what is commonly termed bulk-soil (soil free of living roots) represents a far greater volume and surface area throughout the season, and substantially higher taxonomic and phylogenetic diversity; the traits and activity of its inhabitants may also have a significant impact on overall soil function. We used a combination of comparative genomics and exoproteomics to identify metabolic traits of bacteria adapted to life in bulk soil and compared these with traits of bacteria living in the rhizosphere of wild oat, Avena barbata. In bulk soil bacteria, we observed: (i) greater investment in extracellular polymer-degrading enzyme production; (ii) greater potential for secretion (presence of signal peptides) of polymer-degrading enzymes; (iii) production of accessory proteins (carbohydrate binding modules) fused with glycoside hydrolases that enhance substrate affinity, stabilize, and increase reaction rates of polymer degrading enzymes; and (iv) organization of polymer degradation machinery within gene clusters that facilitate co-transcription of enzymes, transcription factors and transporters for polymer depolymerization products. Together, these findings suggest that unlike rhizosphere-adapted bacteria—which specialize on small molecules released primarily as root exudates—bulk soil-adapted bacteria have evolved to exploit plant polymers. This biochemically costly strategy may be mitigated by protein-level adaptations that enhance the efficiency of extracellular enzyme-mediated substrate acquisition.IMPORTANCEPlant-soil-microbe interactions are dynamic and complex, with significant implications for ecosystem functioning. Microbial traits, such as nutrient acquisition and growth yield, combined with soil and climate parameters, impact major biogeochemical processes and can define the future fate of soil carbon. Diverse soil microorganisms occupy different physical habitats within soil and exploit distinct niches by expressing different metabolic traits. Identifying and quantifying traits that underlie their fitness and function is key for understanding and predicting how soil carbon transformation and stabilization will change in the future or can be managed through intervention.

Despite the promise of the gut microbiome to forecast human health, few studies expose the microbial functions underpinning such predictions. To comprehensively inventory gut microorganisms and their gene content that control trimethylamine induced cardiovascular disease, we mined over 200,000 gut-derived genomes from cultivated and uncultivated microbial lineages. Creating MAGICdb (Methylated Amine Gene Inventory of Catabolism database), we designated an atherosclerotic profile for 6,341 microbial genomes that encoded metabolisms associated with heart disease. We used MAGICdb to evaluate diverse human fecal metatranscriptome and metaproteome datasets, demonstrating how this resource eases the recovery of methylated amine gene content previously obscured in microbiome datasets. From the feces of healthy and diseased subjects, we show MAGICdb gene markers predicted cardiovascular disease as effectively as traditional blood diagnostics. This functional microbiome catalog is a public, exploitable resource, enabling a new era of microbiota-based therapeutics.

Many organisms process carbon and other nutrients to generate energy through aerobic respiration where organic carbon compounds are broken down and oxygen is consumed, producing carbon dioxide and water. Respiration is indicative of active metabolism, and respiration rates are proportional to the amount of living biomass in an ecosystem. Although there are many methods for measuring respiration rates in the laboratory, current systems, such as infrared gas analyzers, are limited in their ability to independently resolve isotopomer fluxes across a range of relevant gases including both CO2 and O2 in real-time. Therefore, monitoring of biological respiration in real time under controlled laboratory conditions would enable better understanding of cellular physiology. To address this challenge, we developed a real time mass spectrometry (RTMS) manifold that simultaneously measures production and consumption of multiple gases and their isotopologues in seconds with the speed and sensitivity necessary to characterize rapidly changing respiration events as they occur. This universal manifold can be fitted to a variety of instruments and affords the same analytical precision and accuracy of the instrument while allowing for the real time measurements. Here, we paired the manifold to a single quad MS with an electron impact (EI) source operated in scan mode to detect extracted target gases by their respective masses (e.g., 12CO2 at mass 44, 13CO2 at 45). We demonstrated applicability of the RTMS instrument to different biological ecosystems (bacterial cultures, plants, and soil), and in all cases, we were able to detect simultaneous and rapid measurements of multiple gases in real time, providing novel insights into complex respiratory metabolism and the influence of biological and environmental factors.

Communities of soil microorganisms (soil microbiomes) play a major role in biogeochemical cycles and support of plant growth. Here we focus primarily on the roles that the soil microbiome plays in cycling soil organic carbon and the impact of climate change on the soil carbon cycle. We first discuss current challenges in understanding the roles carried out by highly diverse and heterogeneous soil microbiomes and review existing knowledge gaps in understanding how climate change will impact soil carbon cycling by the soil microbiome. Because soil microbiome stability is a key metric to understand as the climate changes, we discuss different aspects of stability, including resistance, resilience, and functional redundancy.We then review recent research pertaining to the impact of major climate perturbations on the soil microbiome and the functions that they carry out. Finally, we review new experimental methodologies and modeling approaches under development that should facilitate our understanding of the complex nature of the soil microbiome to better predict its future responses to climate change.

Soil microorganisms drive ecosystem function, but challenges of scale between microbe and ecosystem hinder our ability to accurately quantify and predictively model the soil microbe-ecosystem function relationship. Quantifying this relationship necessitates studies that systematically characterize multi-omics of soil microorganisms and their activity across sampling scales from spatially resolved to bulk measures, and structural complexity, from liquid pure culture to in situ. To address this need, we cultured two diazotrophic bacteria in liquid and solid media, with and without nitrogen (N) to quantify differences in extracellular metabolites associated with nitrogen fixation under increasing environmental structural complexity. We also quantified extracellular metabolites across sampling scales including bulk sampling via GC-MS analysis and spatially resolved analysis via MALDI mass spectrometry imaging. We found extracellular production of inorganic and organic N during free-living nitrogen fixation activity, highlighting a key mechanism of terrestrial N contributions from this process. Additionally, our results emphasize the need to consider the structural complexity of the environment and spatial scale when quantifying microbial activity. We found differences in metabolite profiles between culture conditions, supporting previous work indicating environmental structure influences microbial function, and across scales, underscoring the need to quantify microbial scale conditions to accurately interpret microbial function.ImportanceStudying soil microorganisms, both who is present and what they are doing, is a challenge because of vast differences in scale between microorganism and ecosystem and because of inherent complexities of the soil system (e.g., opacity, chemical complexity). This makes measuring and predicting important ecosystem processes driven by soil microorganisms, like free-living nitrogen fixation, difficult. Free-living nitrogen fixing bacteria play a key role in terrestrial nitrogen contributions and may represent a significant, yet overlooked, nitrogen source in agricultural systems like bioenergy crops. However, we still know very little about how free-living nitrogen fixation contributes nitrogen to terrestrial systems. Our work provides key insight by hierarchically increasing structural complexity (liquid vs. solid culture) and scale (spatially resolved vs. bulk) to address the impact of environmental structure and sampling scale on detection of free-living nitrogen fixation and to identify the forms of nitrogen contributed to terrestrial systems by free-living nitrogen bacteria.

Background:Rivers serve as a nexus for nutrient transfer between terrestrial and marine ecosystems and as such, have a significant impact on global carbon and nitrogen cycles. In river ecosystems, the sediments found within the hyporheic zone are microbial hotspots that can account for a significant portion of ecosystem respiration and have profound impacts on system biogeochemistry. Despite this, studies using genome-resolved analyses linking microbial and viral communities to nitrogen and carbon biogeochemistry are limited.Results:Here, we characterized the microbial and viral communities of Columbia River hyporheic zone sediments to reveal the metabolisms that actively cycle carbon and nitrogen. Using genome-resolved metagenomics, we created the Hyporheic Uncultured Microbial and Viral (HUM-V) database, containing a dereplicated database of 55 microbial Metagenome-Assembled Genomes (MAGs), representing 12 distinct phyla. We also sampled 111 viral Metagenome Assembled Genomes (vMAGs) from 26 distinct and novel genera. The HUM-V recruited metaproteomes from these same samples, providing the first inventory of microbial gene expression in hyporheic zone sediments. Combining this data with metabolite data, we generated a conceptual model where heterotrophic and autotrophic metabolisms co-occur to drive an integrated carbon and nitrogen cycle, revealing microbial sources and sinks for carbon dioxide and ammonium in these sediments. We uncovered the metabolic handoffs underpinning these processes including mutualistic nitrification by Thermoproteota (formerly Thaumarchaeota) and Nitrospirota, as well as identified possible cooperative and cheating behavior impacting nitrogen mineralization. Finally, by linking vMAGs to microbial genome hosts, we reveal possible viral controls on microbial nitrification and organic carbon degradation.Conclusions:Our multi-omics analyses provide new mechanistic insight into coupled carbon-nitrogen cycling in the hyporheic zone. This is a key step in developing predictive hydrobiogeochemical models that account for microbial cross-feeding and viral influences over potential and expressed microbial metabolisms. Furthermore, the publicly available HUM-V genome resource can be queried and expanded by researchers working in other ecosystems to assess the transferability of our results to other parts of the globe.

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.

Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet.

Lipids are essential to all living organisms, as an energy source, as an important cellular structural component, and as a communication tool. In this study, we used global lipidomic methods to evaluate the lipids in leaf-cutter ant fungal gardens. Leaf-cutter ants and their coevolved fungal cultivar, Leucoagaricus gongylophorus, are a model mutualistic system. The fungus enzymatically digests fresh plant material that the ants cut and deliver, converting energy and nutrients from plants, and providing them to the ants through specialized hyphal swellings called gongylidia. Using combined liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry we evaluated differences between the molecular speciation of lipids in the leaf-cutter ant fungal garden ecosystem. This lipidomic study characterized leaves that are fed to the gardens, gongylidia that are produced by the fungus to feed the ants, and spatially resolved regions of the fungal garden through stages of leaf degradation. Lipids containing alpha-linolenic acid (18:3) were enriched in leaves and the top of the gardens, but not dominant in the middle or bottom regions. Gongylidia were dominated by lipids containing linoleic acid (18:2). To evaluate the communicative potential of the lipids in fungal gardens we conducted a behavioral experiment that showed Atta leaf-cutter ants responded differently to 18:3 and 18:2 fatty acids, with aggression towards 18:3 and attraction for 18:2. This work demonstrates the role of lipids in both the transfer of energy and as an inter-kingdom communication tool in leaf-cutter ant fungal gardens.ImportanceIn this work we examined the role of lipids in the mutualism between leaf-cutter ants and fungus. These ants cut fresh leaf material, which they provide to their fungal cultivar, that converts energy and nutrients from the plants and provides it to the ants in specialized hyphal swellings called gongylidia. This work constitutes the first example of a global lipidomics study of a symbiotic system and provides insights as to how the fungus modifies plant lipids into a usable source for the ants. Through a behavioral experiment, this work also demonstrates how lipids can be used as an inter-kingdom communication tool, in this case an attractant, rather than as a repellant, which is more often seen.

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., It is thought that polyphenols inhibit organic matter decomposition in soils devoid of oxygen. Here the authors use metabolomics and genome-resolved metaproteomics to provide experimental evidence of polyphenol biodegradation and maintained soil microbial community metabolism despite anoxia.

Hydration levels influence carbon and nutrient cycles in soils. The rapid carbon release and nutrient utilization upon hydration in desiccated soils has been observed for decades, but little is known about the controls and timing of the underlying molecular events. This research is aimed at developing and using novel isotope based techniques to map the interdependence of carbon and nutrient cycling in soils using 13C labeled substrates as a tracer of nutrient fluxes in desiccated soil microbiomes after rewetting in combination with our novel Real Time Mass Spectrometry (RTMS) system 1, isotope ratio mass spectrometry (IRMS) and omics measurements.Our experiments involve mapping the initial microbial response to wetting, by using a combination of an atmospheric monitoring RTMS instrument that measures the levels of CO2, O2, N2, H2O and their isotopologues, simultaneously in real time and IRMS which will be used to determine the biological fate of the carbon from each substrate. In each experiment desiccated soil that has been kept at drought conditions for 2 weeks was placed in the incubation chamber. Deionized water (control), 13C glucose and 13C alanine dissolved in water was added to the soil through a syringe to mimic a rewetting event. An identical initial hydration response measured by tracing the production of 12CO2 every 5 sec for the first 10 minutes after the addition of water was seen for all the samples potentially indicating that the carbon respired in this initial burst had formed an association with the cell (either intracellular or EPS bound) during dry down. The metabolism and respiration of glucose and alanine, measured by the production of 13CO2 occurred at a much slower time frame (60 to 90 minutes) where the rate of 13CO2 production of the glucose was about 10x that of alanine.IRMS measurements were used to determine the preferential metabolic pathway of each substrate. The soil was removed from the chamber, treated with a mixture of methanol/chloroform/water, beadbeat, sonicated and centrifuged in a modified Folch extraction. The resulting supernatant was allowed to separate into 3 fractions of polar metabolites (methanol layer), proteins (middle layer) and lipids (chloroform layer). Each fraction was analyzed by IRMS to quantify the extent of 13C label incorporation into the soil phase to help guide interpretation of the 13CO2 production rates (measured via RTMS), the preferential metabolic pathway of each substrate will be determined. While it was seen that glucose was taken up into all of the biomass partitions as well as respired into CO2, alanine was metabolized to a lesser extent and was predominantly used in protein synthesis.These studies represent approaches that showcase advanced stable isotope analyses to determine molecular provenance in ecosystems by developing an understanding of the molecular mechanisms involved in carbon and nutrient cycles in terrestrial ecosystems.

Extensive spatial variability combined with analytical challenges associated with soil sampling complicate efforts to elucidate plant-microbe interactions within the rhizosphere, changes in these relationships over time, and the impacts of shifting microenvironmental conditions on microbial community membership and activity. Proteomics analysis of root or soil samples can provide insights to the taxonomy and functional capability of microbial populations and be used to augment various genomic and imaging techniques. Historically, however, proteomics relies on bulk level sampling by removing rhizosphere over relatively large lengths of root surface and is, therefore, neither spatially specific nor non-destructive. We are employing spatially resolved, non-destructive harvesting of mobile proteins onto a membrane to enable both two-dimensional protein mapping and proteomic analysis within rhizosphere while preserving the sample for either timeseries measurements or complementary, destructive techniques.We are using rhizoboxes planted with switchgrass (variety Cave-in-rock) and constructed with natural soil (Kellogg Biological Station, Hickory Corners, Michigan, USA) to develop the approach. We are coupling membrane extraction with specialized sample digestion, purification, and analysis to enable proteomic interpretation. Through its non-destructive nature, this approach permits timeseries analyses for tracking specific taxa and, in some cases, functions associated with rhizosphere processes before and after a system perturbation or over plant growth phases during a growing season. The method’s high sensitivity enables spatial analysis at the up to two-millimeter diameter scale along the rhizobox sampling plane and samples can be manually selected based on proximity to specific root structure, metabolic hotspots, or other parameters of choice. We are using this analysis to track statistically significant shifts in plant and microbe contributions to rhizosphere proteome associated with roots at different growth stages. We are also linking this approach to a 13C tracer to identify specific taxonomic groups having the closest metabolic association with a host plant to identify shifts in plant-microbe interactions associated with nutrient availability. For instance, we are using a split root rhizobox approach to monitor the plasticity of plant-microbe C exchange associated with P availability. Combined, the spatial and 13C tracer components of this proteomic technique can help illuminate understanding of the complex inter-kingdom interactions within the rhizosphere and the implications these interactions have on driving C cycling and plant performance.

Significance Historically, it has been understood that for gene expression in eukaryotes, each messenger RNA encodes a single protein. With the recent development of technologies to sequence full-length transcripts en masse, we have discovered hundreds of examples in two species of green algae where two, three, or more proteins are translated from a single transcript. These “polycistronic” transcripts are found in diverse species throughout the green algal lineage, which highlights their biological importance. We have leveraged these findings to coexpress pairs of genes on polycistronic transcripts in vitro, which should facilitate efforts to engineer algae for research and industrial applications., 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.

Anthropogenic inputs of nitrogen into aquatic ecosystems, and especially those of agricultural origin, involve a mix of chemical species. Although it is well-known in general that nitrogen eutrophication markedly influences the metabolism of aquatic phototrophic communities, relatively little is known regarding whether the specific chemical form of nitrogen inputs matter. Our data suggest that the nitrogen form alters the rate of nitrogen uptake significantly, whereas corresponding alterations in carbon uptake were minor. However, differences imposed by uptake of divergent nitrogen forms may result in alterations among phototroph-heterotroph interactions that rewire community metabolism. Furthermore, our data hint that availability of other nutrients (i.e., iron) might mediate the linkage between carbon and nitrogen cycling in these communities. Taken together, our data suggest that different nitrogen forms should be examined for divergent impacts on phototrophic communities in fluvial systems and that these anthropogenic nitrogen inputs may significantly differ in their ultimate biogeochemical impacts., Increasing anthropogenic inputs of fixed nitrogen are leading to greater eutrophication of aquatic environments, but it is unclear how this impacts the flux and fate of carbon in lacustrine and riverine systems. Here, we present evidence that the form of nitrogen governs the partitioning of carbon among members in a genome-sequenced, model phototrophic biofilm of 20 members. Consumption of NO3− as the sole nitrogen source unexpectedly resulted in more rapid transfer of carbon to heterotrophs than when NH4+ was also provided, suggesting alterations in the form of carbon exchanged. The form of nitrogen dramatically impacted net community nitrogen, but not carbon, uptake rates. Furthermore, this alteration in nitrogen form caused very large but focused alterations to community structure, strongly impacting the abundance of only two species within the biofilm and modestly impacting a third member species. Our data suggest that nitrogen metabolism may coordinate coupled carbon-nitrogen biogeochemical cycling in benthic biofilms and, potentially, in phototroph-heterotroph consortia more broadly. It further indicates that the form of nitrogen inputs may significantly impact the contribution of these communities to carbon partitioning across the terrestrial-aquatic interface. IMPORTANCE Anthropogenic inputs of nitrogen into aquatic ecosystems, and especially those of agricultural origin, involve a mix of chemical species. Although it is well-known in general that nitrogen eutrophication markedly influences the metabolism of aquatic phototrophic communities, relatively little is known regarding whether the specific chemical form of nitrogen inputs matter. Our data suggest that the nitrogen form alters the rate of nitrogen uptake significantly, whereas corresponding alterations in carbon uptake were minor. However, differences imposed by uptake of divergent nitrogen forms may result in alterations among phototroph-heterotroph interactions that rewire community metabolism. Furthermore, our data hint that availability of other nutrients (i.e., iron) might mediate the linkage between carbon and nitrogen cycling in these communities. Taken together, our data suggest that different nitrogen forms should be examined for divergent impacts on phototrophic communities in fluvial systems and that these anthropogenic nitrogen inputs may significantly differ in their ultimate biogeochemical impacts.

Clear elucidation of plant-microbe interactions within the rhizosphere and how these relationships change over time can be confounded by the large microbial biodiversity, shifting microenvironmental conditions, and extensive spatial constraints within these complex systems. Proteomics analysis of root or soil samples, when linked with metagenomic interpretation, can provide key insights to both the taxonomy and functional capability of microbial populations within a sample. Yet, existing proteomic approaches may not always be able to provide the needed temporal and spatial resolution to capture fine-scale and short-term interactions between plants and microorganisms. To remedy this limitation, we are developing a suite of methodological adaptations intended to leverage proteomic analysis to help identify key interactions between rhizosphere microorganisms and their host plant.First, we are employing 13C tracers coupled with automated data analysis to identify specific organisms consuming both simulated and natural root exudates. We are specifically exploring microcosms constructed from natural soil (Kellogg Biological Station, Hickory Corners, Michigan, USA) and planted with switchgrass as a platform for developing the techniques. Multiple previous studies have linked key interactions between both free-living and epiphytic microbial members with improved performance of a switchgrass host under nutrient-depleted, natural field conditions. Providing evaluation of the amount and taxonomic recipient of switchgrass-supplied carbon under varying conditions may help link key taxonomic groups with improved plant performance and biomass production.Second, we are leveraging a membrane extraction technique coupled with specialized sample digestion, purification, and analysis to enable non-destructive, spatially-resolved protein extraction from the root-soil interface within our constructed microcosms. Through its non-destructive nature, this approach permits timeseries analysis for tracking specific taxa and, in some cases, functions associated with rhizosphere processes both before and after a system perturbation as well as variations over plant growth phases during a growing season. The high sensitivity of this system enables spatial analysis at the one to two mm scale where samples can be manually selected based on proximity to specific root structure, metabolic hotspots in the system, or other parameter of choice. Spatial analysis can be leveraged to track taxonomic distribution within the rhizospheres associated with roots at different growth stages or levels of maturity.

Soil microbes are highly sensitive to changes in their environment, and rapidly measuring their responses is necessary to fully understand the biological processes. Drought is one of the most common environmental stresses that soil microbiomes experience, and it is important to understand the mechanisms by which the soil microbiome respond to soil dehydration. We used 13C as a tracer of nutrient fluxes in desiccated soil microbiomes after rewetting to simultaneously measure aerobic respiration and track the metabolic state of the community. Here, we describe a Real Time Mass Spectrometry (RTMS) approach for rapid gas monitoring combined with omics approaches to track 13C flow through a soil system.The mechanism(s) behind the burst of rapid mineralization of soil organic matter and increased rate of CO2 release upon rewetting dry soil (termed the ‘Birch Effect’) are yet to be fully defined. One known mechanism used by microbes to protect against dehydration is the production of intracellular compounds known as osmolytes. We evaluated metabolic mechanisms produced upon rewetting a marginal soil testing the hypothesis that the rapid release of CO2 arises from the microbial processing of putative intracellular osmolytes that build up during desiccation. RTMS allows for the simultaneous, rapid and fine scale (every 2 sec) evaluation and deconvolution of the production and consumption of a number of gasses including 12CO2,13CO2, O2, N2 and H2O. We compared the hydration response (production of CO2 in real time) between the addition of water and 13C labeled glucose dissolved in water. The initial burst of 12CO2 followed by a leveling off was identical in both treatments with an additional larger increase in 13CO2 about 20 minutes later in the 13C labeled glucose experiment. Examination of the two minutes after the water addition revealed a rapid rate of 12CO2 (38 sec) and H2O (47 sec) production and slow rate of 13CO2 (56 sec) production followed by the consumption of O2 (67 sec) and N2 (73 sec). Evaluation of the soil metabolomes at specified time points within 3 hours after wetting revealed the immediate release of sugars from the cells into the extracellular matrix. These results provide evidence for respiration of putative intracellular osmolytes as one driving mechanism of the Birch Effect.

Because of their agricultural value, there is a great body of research dedicated to understanding the microorganisms responsible for rumen carbon degradation. However, we lack a holistic view of the microbial food web responsible for carbon processing in this ecosystem. Here, we sampled rumen-fistulated moose, allowing access to rumen microbial communities actively degrading woody plant biomass in real time. We resolved 1,193 viral contigs and 77 unique, near-complete microbial metagenome-assembled genomes, many of which lacked previous metabolic insights. Plant-derived metabolites were measured with NMR and carbohydrate microarrays to quantify the carbon nutrient landscape. Network analyses directly linked measured metabolites to expressed proteins from these unique metagenome-assembled genomes, revealing a genome-resolved three-tiered carbohydrate-fuelled trophic system. This provided a glimpse into microbial specialization into functional guilds defined by specific metabolites. To validate our proteomic inferences, the catalytic activity of a polysaccharide utilization locus from a highly connected metabolic hub genome was confirmed using heterologous gene expression. Viral detected proteins and linkages to microbial hosts demonstrated that phage are active controllers of rumen ecosystem function. Our findings elucidate the microbial and viral members, as well as their metabolic interdependencies, that support in situ carbon degradation in the rumen ecosystem., A combination of proteomics, metagenome-assembled genomes and heterologous gene expression experiments reveals a trophic system for carbon utilization in the moose rumen microbiome and provides insights into phage dynamics in this ecosystem.

BackgroundThe 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.ResultsFive Ascomycota fungi were isolated from different soil crust microhabitats and rhizosphere soils around the native bunchgrassPleuraphis jamesiiin an arid grassland near Moab, UT, USA. Putative genera wereConiochaeta, isolated from lichen biocrust,Embellisiafrom cyanobacteria biocrust, Chaetomiumfrom below lichen biocrust,Phomafrom a moss microhabitat, andAspergillusfrom 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.AspergillusandPhomaexpressed more plant biomass degrading enzymes when grown in grass- and pine-containing cultures than in chitin.ConiochaetaandEmbellisiaexpressed similar numbers of these enzymes under all conditions, whileChaetomiumsecreted more of these enzymes in grass-containing cultures.ConclusionsThis 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.

To harness the potential of microbiome science across the broad range of relevant disciplines, new approaches to data infrastructure and transdisciplinary collaboration are necessary. The National Microbiome Data Collaborative (NMDC) is a new initiative to support microbiome data exploration and discovery through a collaborative, integrative data science ecosystem. To harness the potential of microbiome science across the broad range of relevant disciplines, new approaches to data infrastructure and transdisciplinary collaboration are necessary. The National Microbiome Data Collaborative is a new initiative to support microbiome data exploration and discovery through a collaborative, integrative data science ecosystem.

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (C(i)), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of C(i) incorporation into different taxa, mat samples were incubated with [(13)C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of (13)C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with (13)C. Most (13)C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, C(i) fixation, and N(2) fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for C(i) fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to C(i) fixation. Other taxa showed much lower [(13)C]bicarbonate incorporation. IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven C(i) fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in C(i) fixation. Because this pathway imparts a lower degree of selection of isotopically heavy C(i) than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of (13)C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the (13)C/(12)C ratios of mat biomass will help geochemists interpret the (13)C/(12)C ratios of organic carbon in the fossil record.

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unkown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase ofNeurospora crassaaffect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 minutes) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

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.

Significance Chlamydomonas reinhardtii is the premier reference organism for understanding unicellular green algae. Chlamydomonas is an important model for photosynthesis as well as fermentation and other anaerobic pathways under dark anoxic conditions. We have produced a diurnal transcriptome, validated by subproteomic analyses, and matched with measurements of pigments, select metabolites, and physiological parameters. We report that the majority of the algal genome is differentially expressed over the course of the day and the timing of specific genes is dictated by their biological function. We also discovered that fermentation rather than respiration is the preferred metabolic fate of starch-derived glycolytic pyruvate. We offer our rich dataset to the algal and plant communities., 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.