Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii.

Interactions between plants and soil microbial communities that benefit plant growth and enhance nutrient acquisition are driven by the selective release of metabolites from plant roots, or root exudation. To investigate these plant-microbe interactions, we developed a photoaffinity probe based on sorgoleone (sorgoleone diazirine alkyne for photoaffinity labeling, SoDA-PAL), a hydrophobic secondary metabolite and allelochemical produced in Sorghum bicolor root exudates. We applied SoDA-PAL to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth-promoting microbe isolated from sorghum rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins, including putative transporters, transcriptional regulators, and a subset of proteins with predicted enzymatic functions. We performed computational protein modeling and docking with sorgoleone to prioritize candidates for experimental validation and then confirmed binding of sorgoleone to four of these proteins in vitro: the α/ß fold hydrolase SrgB (OH685_09420), a fumarylacetoacetase (OH685_02300), a lysophospholipase (OH685_14215), and an unannotated hypothetical protein (OH685_18625). Our application of this specialized sorgoleone-based probe coupled with structural bioinformatics streamlines the identification of microbial proteins involved in metabolite recognition, metabolism, and toxicity, widening our understanding of the range of cellular pathways that can be affected by a plant secondary metabolite.IMPORTANCEHere, we demonstrate that a photoaffinity-based chemical probe modeled after sorgoleone, an important secondary metabolite released by sorghum roots, can be used to identify microbial proteins that directly interact with sorgoleone. We applied this probe to the sorghum-associated bacterium Acinetobacter pittii and showed that probe labeling is dose-dependent and sensitive to competition with purified sorgoleone. Coupling the probe with proteomics and computational analysis facilitated the identification of putative sorgoleone binders, including a protein implicated in a conserved pathway essential for sorgoleone catabolism. We anticipate that discoveries seeded by this workflow will expand our understanding of the molecular mechanisms by which specific metabolites in root exudates shape the sorghum rhizosphere microbiome.

Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia-host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX2 system of proximity-dependent biotinylation. APEX2 is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX2 fused to known inclusion membrane proteins, allowing biotinylation and purification of inclusion-associated proteins. Using quantitative mass spectrometry against APEX2 labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX2 is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.

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.

A Global Survey of ATPase Activity in Plasmodium falciparum Asexual Blood Stages and Gametocytes.

Effective malaria control and elimination in hyperendemic areas of the world will require treatment of the Plasmodium falciparum (Pf) blood stage that causes disease as well as the gametocyte stage that is required for transmission from humans to the mosquito vector. Most currently used therapies do not kill gametocytes, a highly specialized, non-replicating sexual parasite stage. Further confounding next generation drug development against Pf is the unknown metabolic state of the gametocyte and the lack of known biochemical activity for most parasite gene products in general. Here, we take a systematic activity-based proteomics approach to survey the activity of the large and druggable ATPase family in replicating blood stage asexual parasites and transmissible, non-replicating sexual gametocytes. ATPase activity broadly changes during the transition from asexual schizonts to sexual gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. We further experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.

Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism.

Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12 Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.

Uniformly 15N-Labeled Recombinant Ricin A-Chain as an Internal Retention Time Standard for Increased Confidence in Forensic Identification of Ricin by Untargeted Nanoflow Liquid Chromatography-Tandem Mass Spectrometry.

Ricin, a toxic protein from the castor plant, is of forensic and biosecurity interest because of its high toxicity and common occurrence in crimes and attempted crimes. Qualitative methods to detect ricin are therefore needed. Untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics methods are well suited because of their high specificity. Specificity in LC-MS/MS comes from both the LC and MS components. However, modern untargeted proteomics methods often use nanoflow LC, which has less reproducible retention times than standard-flow LC, making it challenging to use retention time as a point of identification in a forensic assay. We address this challenge by using retention times relative to a standard, namely, the uniformly 15N-labeled ricin A-chain produced recombinantly in a bacterial expression system. This material, added as an internal standard prior to trypsin digestion, produces a stable-isotope-labeled standard for every ricin tryptic peptide in the sample. We show that the MS signals for 15N and natural isotopic abundance ricin peptides are distinct, with mass shifts that correspond to the numbers of nitrogen atoms in each peptide or fragment. We also show that, as expected, labeled and unlabeled peptides coelute, with relative retention time differences of less than 0.2%.

Profiling microbial lignocellulose degradation and utilization by emergent omics technologies.

The use of plant materials to generate renewable biofuels and other high-value chemicals is the sustainable and preferable option, but will require considerable improvements to increase the rate and efficiency of lignocellulose depolymerization. This review highlights novel and emerging technologies that are being developed and deployed to characterize the process of lignocellulose degradation. The review will also illustrate how microbial communities deconstruct and metabolize lignocellulose by identifying the necessary genes and enzyme activities along with the reaction products. These technologies include multi-omic measurements, cell sorting and isolation, nuclear magnetic resonance spectroscopy (NMR), activity-based protein profiling, and direct measurement of enzyme activity. The recalcitrant nature of lignocellulose necessitates the need to characterize the methods microbes employ to deconstruct lignocellulose to inform new strategies on how to greatly improve biofuel conversion processes. New technologies are yielding important insights into microbial functions and strategies employed to degrade lignocellulose, providing a mechanistic blueprint in order to advance biofuel production.

Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch.

The majority of Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by drug tolerance and little or no bacterial replication. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability and gross morphological defects in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle-active disease, latency, and reactivation.

Dinitrogenase-Driven Photobiological Hydrogen Production Combats Oxidative Stress in Cyanothece sp. Strain ATCC 51142.

Photobiologically synthesized hydrogen (H2) gas is carbon neutral to produce and clean to combust, making it an ideal biofuel. Cyanothece sp. strain ATCC 51142 is a cyanobacterium capable of performing simultaneous oxygenic photosynthesis and H2 production, a highly perplexing phenomenon because H2 evolving enzymes are O2 sensitive. We employed a system-level in vivo chemoproteomic profiling approach to explore the cellular dynamics of protein thiol redox and how thiol redox mediates the function of the dinitrogenase NifHDK, an enzyme complex capable of aerobic hydrogenase activity. We found that NifHDK responds to intracellular redox conditions and may act as an emergency electron valve to prevent harmful reactive oxygen species formation in concert with other cell strategies for maintaining redox homeostasis. These results provide new insight into cellular redox dynamics useful for advancing photolytic bioenergy technology and reveal a new understanding for the biological function of NifHDK. IMPORTANCE: Here, we demonstrate that high levels of hydrogen synthesis can be induced as a protection mechanism against oxidative stress via the dinitrogenase enzyme complex in Cyanothece sp. strain ATCC 51142. This is a previously unknown feature of cyanobacterial dinitrogenase, and we anticipate that it may represent a strategy to exploit cyanobacteria for efficient and scalable hydrogen production. We utilized a chemoproteomic approach to capture the in situ dynamics of reductant partitioning within the cell, revealing proteins and reactive thiols that may be involved in redox sensing and signaling. Additionally, this method is widely applicable across biological systems to achieve a greater understanding of how cells navigate their environment and how redox chemistry can be utilized to alter metabolism and achieve homeostasis.

Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development.

Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography-mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis.

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of the pathways/networks that contribute to pathophysiological outcomes. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced inducible tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol reactive probes to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent Gorlin syndrome patients, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and ALDH1A1 protein deficiency in GDFs was confirmed by Western blot. A number of additional protein thiol differences in GDFs were identified, including radiation responsive annexin family members and lamin A/C. Collectively, candidates identified in our study have plausible implications for radiation health effects and cancer susceptibility.

Organelle-specific activity-based protein profiling in living cells.

A multimodal activity-based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin-reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle-targeting of activity-based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.

Longitudinal analysis of host protein serum signatures of treatment and recovery in pulmonary tuberculosis.

BACKGROUND: A better understanding of treatment progression and recovery in pulmonary tuberculosis (TB) infectious disease is crucial. This study analyzed longitudinal serum samples from pulmonary TB patients undergoing interventional treatment to identify surrogate markers for TB-related outcomes. METHODS: Serum that was collected at baseline and 8, 17, 26, and 52 weeks from 30 TB patients experiencing durable cure were evaluated and compared using a sensitive LC-MS/MS proteomic platform for the detection and quantification of differential host protein signatures relative to timepoint. The global proteome signature was analyzed for statistical differences across the time course and between disease severity and treatment groups. RESULTS: A total of 676 proteins showed differential expression in the serum over these timepoints relative to baseline. Comparisons to understand serum protein dynamics at 8 weeks, treatment endpoints at 17 and 26 weeks, and post-treatment at 52 weeks were performed. The largest protein abundance changes were observed at 8 weeks as the initial effects of antibiotic treatment strongly impacted inflammatory and immune modulated responses. However, the largest number of proteome changes was observed at the end of treatment time points 17 and 26 weeks respectively. Post-treatment 52-week results showed an abatement of differential proteome signatures from end of treatment, though interestingly those proteins uniquely significant at post-treatment were almost exclusively downregulated. Patients were additionally stratified based upon disease severity and compared across all timepoints, identifying 461 discriminating proteome signatures. These proteome signatures collapsed into discrete expression profiles with distinct pathways across immune activation and signaling, hemostasis, and metabolism annotations. Insulin-like growth factor (IGF) and Integrin signaling maintained a severity signature through 52 weeks, implying an intrinsic disease severity signature well into the post-treatment timeframe. CONCLUSION: Previous proteome studies have primarily focused on the 8-week timepoint in relation to culture conversion status. While this study confirms previous observations, it also highlights some differences. The inclusion of additional end of treatment and post-treatment time points offers a more comprehensive assessment of treatment progression within the serum proteome. Examining the expression dynamics at these later time periods will help in the investigation of relapse patients and has provided indicative markers of response and recovery.

A compendium of multi-omics data illuminating host responses to lethal human virus infections.

Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology.

Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Affinity- and activity-based probes synthesized from structurally diverse hops-derived xanthohumol flavonoids reveal highly varied protein profiling in Escherichia coli.

Xanthohumol, the principle prenylflavonoid found in hops (Humulus lupulus) and a reported anti-inflammatory agent, has great potential for pharmaceutical interventions related to inflammatory disorders in the gut. A suite of probes was prepared from xanthohumol and its structural isomer isoxanthohumol to enable profiling of both protein affinity binding and catalytic enzyme reactivity. The regiochemistry of the reactive group on the probes was altered to reveal how probe structure dictates protein labeling, and which probes best emulate the natural flavonoids. Affinity- and activity-based probes were applied to Escherichia coli, and protein labeling was measured by chemoproteomics. Structurally dependent activity-based probe protein labeling demonstrates how subtle alterations in flavonoid structure and probe reactive groups can result in considerably different protein interactions. This work lays the groundwork to expand upon unexplored cellular activities related to xanthohumol interactions, metabolism, and anti-inflammatory mechanisms.

Suite of activity-based probes for cellulose-degrading enzymes.

Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry and to increase enzyme active site inclusion for liquid chromatography-mass spectrometry (LC-MS) analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role associated with biofuel development.

A Mineral-Doped Micromodel Platform Demonstrates Fungal Bridging of Carbon Hot Spots and Hyphal Transport of Mineral-Derived Nutrients.

Soil fungi facilitate the translocation of inorganic nutrients from soil minerals to other microorganisms and plants. This ability is particularly advantageous in impoverished soils because fungal mycelial networks can bridge otherwise spatially disconnected and inaccessible nutrient hot spots. However, the molecular mechanisms underlying fungal mineral weathering and transport through soil remains poorly understood primarily due to the lack of a platform for spatially resolved analysis of biotic-driven mineral weathering. Here, we addressed this knowledge gap by demonstrating a mineral-doped soil micromodel platform where mineral weathering mechanisms can be studied. We directly visualize acquisition and transport of inorganic nutrients from minerals through fungal hyphae in the micromodel using a multimodal imaging approach. We found that Fusarium sp. strain DS 682, a representative of common saprotrophic soil fungus, exhibited a mechanosensory response (thigmotropism) around obstacles and through pore spaces (~12 µm) in the presence of minerals. The fungus incorporated and translocated potassium (K) from K-rich mineral interfaces, as evidenced by visualization of mineral-derived nutrient transport and unique K chemical moieties following fungus-induced mineral weathering. Specific membrane transport proteins were expressed in the fungus in the presence of minerals, including those involved in oxidative phosphorylation pathways and the transmembrane transport of small-molecular-weight organic acids. This study establishes the significance of a spatial visualization platform for investigating microbial induced mineral weathering at microbially relevant scales. Moreover, we demonstrate the importance of fungal biology and nutrient translocation in maintaining fungal growth under water and carbon limitations in a reduced-complexity soil-like microenvironment. IMPORTANCE Fungal species are foundational members of soil microbiomes, where their contributions in accessing and transporting vital nutrients is key for community resilience. To date, the molecular mechanisms underlying fungal mineral weathering and nutrient translocation in low-nutrient environments remain poorly resolved due to the lack of a platform for spatial analysis of biotic weathering processes. Here, we addressed this knowledge gap by developing a mineral-doped soil micromodel platform. We demonstrate the function of this platform by directly probing fungal growth using spatially resolved optical and chemical imaging methodologies. We found the presence of minerals was required for fungal thigmotropism around obstacles and through soil-like pore spaces, and this was related to fungal transport of potassium (K) and corresponding K speciation from K-rich minerals. These findings provide new evidence and visualization into hyphal transport of mineral-derived nutrients under nutrient and water stresses.

Draft Genome Sequence of Fusarium sp. Strain DS 682, a Novel Fungal Isolate from the Grass Rhizosphere.

The novel fungal strain, Fusarium sp. strain DS 682, was isolated from the rhizosphere of the perennial grass, Bouteloua gracilis, at the Konza Prairie Biological Station in Kansas. This fungal strain is common across North American grasslands and is resilient to environmental fluctuations. The draft genome is estimated to be 97.2% complete.

Terabase Metagenome Sequencing of Grassland Soil Microbiomes.

To enable an in-depth survey of the metabolic potential of complex soil microbiomes, we performed ultra-deep metagenome sequencing, collecting >1 Tb of sequence data from three grassland soils representing different precipitation regimes.