Environment-specific virocell metabolic reprogramming.

Viruses impact microbial systems through killing hosts, horizontal gene transfer, and altering cellular metabolism, consequently impacting nutrient cycles. A virus-infected cell, a "virocell", is distinct from its uninfected sister cell as the virus commandeers cellular machinery to produce viruses rather than replicate cells. Problematically, virocell responses to the nutrient-limited conditions that abound in nature are poorly understood. Here we used a systems biology approach to investigate virocell metabolic reprogramming under nutrient limitation. Using transcriptomics, proteomics, lipidomics, and endo- and exo-metabolomics, we assessed how low phosphate (low-P) conditions impacted virocells of a marine Pseudoalteromonas host when independently infected by two unrelated phages (HP1 and HS2). With the combined stresses of infection and nutrient limitation, a set of nested responses were observed. First, low-P imposed common cellular responses on all cells (virocells and uninfected cells), including activating the canonical P-stress response, and decreasing transcription, translation, and extracellular organic matter consumption. Second, low-P imposed infection-specific responses (for both virocells), including enhancing nitrogen assimilation and fatty acid degradation, and decreasing extracellular lipid relative abundance. Third, low-P suggested virocell-specific strategies. Specifically, HS2-virocells regulated gene expression by increasing transcription and ribosomal protein production, whereas HP1-virocells accumulated host proteins, decreased extracellular peptide relative abundance, and invested in broader energy and resource acquisition. These results suggest that although environmental conditions shape metabolism in common ways regardless of infection, virocell-specific strategies exist to support viral replication during nutrient limitation, and a framework now exists for identifying metabolic strategies of nutrient-limited virocells in nature.

The mobilization and transport of newly fixed carbon are driven by plant water use in an experimental rainforest under drought.

Non-structural carbohydrates (NSCs) are building blocks for biomass and fuel metabolic processes. However, it remains unclear how tropical forests mobilize, export, and transport NSCs to cope with extreme droughts. We combined drought manipulation and ecosystem 13CO2 pulse-labeling in an enclosed rainforest at Biosphere 2, assessed changes in NSCs, and traced newly assimilated carbohydrates in plant species with diverse hydraulic traits and canopy positions. We show that drought caused a depletion of leaf starch reserves and slowed export and transport of newly assimilated carbohydrates below ground. Drought effects were more pronounced in conservative canopy trees with limited supply of new photosynthates and relatively constant water status than in those with continual photosynthetic supply and deteriorated water status. We provide experimental evidence that local utilization, export, and transport of newly assimilated carbon are closely coupled with plant water use in canopy trees. We highlight that these processes are critical for understanding and predicting tree resistance and ecosystem fluxes in tropical forest under drought.

Dispersal, habitat filtering, and eco-evolutionary dynamics as drivers of local and global wetland viral biogeography.

Wetlands store 20-30% of the world's soil carbon, and identifying the microbial controls on these carbon reserves is essential to predicting feedbacks to climate change. Although viral infections likely play important roles in wetland ecosystem dynamics, we lack a basic understanding of wetland viral ecology. Here 63 viral size-fraction metagenomes (viromes) and paired total metagenomes were generated from three time points in 2021 at seven fresh- and saltwater wetlands in the California Bodega Marine Reserve. We recovered 12,826 viral population genomic sequences (vOTUs), only 4.4% of which were detected at the same field site two years prior, indicating a small degree of population stability or recurrence. Viral communities differed most significantly among the seven wetland sites and were also structured by habitat (plant community composition and salinity). Read mapping to a new version of our reference database, PIGEONv2.0 (515,763 vOTUs), revealed 196 vOTUs present over large geographic distances, often reflecting shared habitat characteristics. Wetland vOTU microdiversity was significantly lower locally than globally and lower within than between time points, indicating greater divergence with increasing spatiotemporal distance. Viruses tended to have broad predicted host ranges via CRISPR spacer linkages to metagenome-assembled genomes, and increased SNP frequencies in CRISPR-targeted major tail protein genes suggest potential viral eco-evolutionary dynamics in response to both immune targeting and changes in host cell receptors involved in viral attachment. Together, these results highlight the importance of dispersal, environmental selection, and eco-evolutionary dynamics as drivers of local and global wetland viral biogeography.

Drought re-routes soil microbial carbon metabolism towards emission of volatile metabolites in an artificial tropical rainforest.

Drought impacts on microbial activity can alter soil carbon fate and lead to the loss of stored carbon to the atmosphere as CO2 and volatile organic compounds (VOCs). Here we examined drought impacts on carbon allocation by soil microbes in the Biosphere 2 artificial tropical rainforest by tracking 13C from position-specific 13C-pyruvate into CO2 and VOCs in parallel with multi-omics. During drought, efflux of 13C-enriched acetate, acetone and C4H6O2 (diacetyl) increased. These changes represent increased production and buildup of intermediate metabolites driven by decreased carbon cycling efficiency. Simultaneously,13C-CO2 efflux decreased, driven by a decrease in microbial activity. However, the microbial carbon allocation to energy gain relative to biosynthesis was unchanged, signifying maintained energy demand for biosynthesis of VOCs and other drought-stress-induced pathways. Overall, while carbon loss to the atmosphere via CO2 decreased during drought, carbon loss via efflux of VOCs increased, indicating microbially induced shifts in soil carbon fate.

Uncovering the dominant role of root metabolism in shaping rhizosphere metabolome under drought in tropical rainforest plants.

Plant-soil-microbe interactions are crucial for driving rhizosphere processes that contribute to metabolite turnover and nutrient cycling. With the increasing frequency and severity of water scarcity due to climate warming, understanding how plant-mediated processes, such as root exudation, influence soil organic matter turnover in the rhizosphere is essential. In this study, we used 16S rRNA gene amplicon sequencing, rhizosphere metabolomics, and position-specific 13C-pyruvate labeling to examine the effects of three different plant species (Piper auritum, Hibiscus rosa sinensis, and Clitoria fairchildiana) and their associated microbial communities on soil organic carbon turnover in the rhizosphere. Our findings indicate that in these tropical plants, the rhizosphere metabolome is primarily shaped by the response of roots to drought rather than direct shifts in the rhizosphere bacterial community composition. Specifically, the reduced exudation of plant roots had a notable effect on the metabolome of the rhizosphere of P. auritum, with less reliance on neighboring microbes. Contrary to P. auritum, H. rosa sinensis and C. fairchildiana experienced changes in their exudate composition during drought, causing alterations to the bacterial communities in the rhizosphere. This, in turn, had a collective impact on the rhizosphere's metabolome. Furthermore, the exclusion of phylogenetically distant microbes from the rhizosphere led to shifts in its metabolome. Additionally, C. fairchildiana appeared to be associated with only a subset of symbiotic bacteria under drought conditions. These results indicate that plant species-specific microbial interactions systematically change with the root metabolome. As roots respond to drought, their associated microbial communities adapt, potentially reinforcing the drought tolerance strategies of plant roots. These findings have significant implications for maintaining plant health and preference during drought stress and improving plant performance under climate change.

MetaboDirect: an analytical pipeline for the processing of FT-ICR MS-based metabolomic data.

BACKGROUND: Microbiomes are now recognized as the main drivers of ecosystem function ranging from the oceans and soils to humans and bioreactors. However, a grand challenge in microbiome science is to characterize and quantify the chemical currencies of organic matter (i.e., metabolites) that microbes respond to and alter. Critical to this has been the development of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which has drastically increased molecular characterization of complex organic matter samples, but challenges users with hundreds of millions of data points where readily available, user-friendly, and customizable software tools are lacking. RESULTS: Here, we build on years of analytical experience with diverse sample types to develop MetaboDirect, an open-source, command-line-based pipeline for the analysis (e.g., chemodiversity analysis, multivariate statistics), visualization (e.g., Van Krevelen diagrams, elemental and molecular class composition plots), and presentation of direct injection high-resolution FT-ICR MS data sets after molecular formula assignment has been performed. When compared to other available FT-ICR MS software, MetaboDirect is superior in that it requires a single line of code to launch a fully automated framework for the generation and visualization of a wide range of plots, with minimal coding experience required. Among the tools evaluated, MetaboDirect is also uniquely able to automatically generate biochemical transformation networks (ab initio) based on mass differences (mass difference network-based approach) that provide an experimental assessment of metabolite connections within a given sample or a complex metabolic system, thereby providing important information about the nature of the samples and the set of microbial reactions or pathways that gave rise to them. Finally, for more experienced users, MetaboDirect allows users to customize plots, outputs, and analyses. CONCLUSION: Application of MetaboDirect to FT-ICR MS-based metabolomic data sets from a marine phage-bacterial infection experiment and a Sphagnum leachate microbiome incubation experiment showcase the exploration capabilities of the pipeline that will enable the research community to evaluate and interpret their data in greater depth and in less time. It will further advance our knowledge of how microbial communities influence and are influenced by the chemical makeup of the surrounding system. The source code and User's guide of MetaboDirect are freely available through ( https://github.com/Coayala/MetaboDirect ) and ( https://metabodirect.readthedocs.io/en/latest/ ), respectively. Video Abstract.

Elucidating Drought-Tolerance Mechanisms in Plant Roots through 1H NMR Metabolomics in Parallel with MALDI-MS, and NanoSIMS Imaging Techniques.

As direct mediators between plants and soil, roots play an important role in metabolic responses to environmental stresses such as drought, yet these responses are vastly uncharacterized on a plant-specific level, especially for co-occurring species. Here, we aim to examine the effects of drought on root metabolic profiles and carbon allocation pathways of three tropical rainforest species by combining cutting-edge metabolomic and imaging technologies in an in situ position-specific 13C-pyruvate root-labeling experiment. Further, washed (rhizosphere-depleted) and unwashed roots were examined to test the impact of microbial presence on root metabolic pathways. Drought had a species-specific impact on the metabolic profiles and spatial distribution in Piper sp. and Hibiscus rosa sinensis roots, signifying different defense mechanisms; Piper sp. enhanced root structural defense via recalcitrant compounds including lignin, while H. rosa sinensis enhanced biochemical defense via secretion of antioxidants and fatty acids. In contrast, Clitoria fairchildiana, a legume tree, was not influenced as much by drought but rather by rhizosphere presence where carbohydrate storage was enhanced, indicating a close association with symbiotic microbes. This study demonstrates how multiple techniques can be combined to identify how plants cope with drought through different drought-tolerance strategies and the consequences of such changes on below-ground organic matter composition.

Ecosystem fluxes during drought and recovery in an experimental forest.

Severe droughts endanger ecosystem functioning worldwide. We investigated how drought affects carbon and water fluxes as well as soil-plant-atmosphere interactions by tracing 13CO2 and deep water 2H2O label pulses and volatile organic compounds (VOCs) in an enclosed experimental rainforest. Ecosystem dynamics were driven by different plant functional group responses to drought. Drought-sensitive canopy trees dominated total fluxes but also exhibited the strongest response to topsoil drying. Although all canopy-forming trees had access to deep water, these reserves were spared until late in the drought. Belowground carbon transport was slowed, yet allocation of fresh carbon to VOCs remained high. Atmospheric VOC composition reflected increasing stress responses and dynamic soil-plant-atmosphere interactions, potentially affecting atmospheric chemistry and climate feedbacks. These interactions and distinct functional group strategies thus modulate drought impacts and ecosystem susceptibility to climate change.

Untargeted metabolomic profiling of Sphagnum fallax reveals novel antimicrobial metabolites.

Sphagnum mosses dominate peatlands by employing harsh ecosystem tactics to prevent vascular plant growth and microbial degradation of these large carbon stores. Knowledge about Sphagnum-produced metabolites, their structure and their function, is important to better understand the mechanisms, underlying this carbon sequestration phenomenon in the face of climate variability. It is currently unclear which compounds are responsible for inhibition of organic matter decomposition and the mechanisms by which this inhibition occurs. Metabolite profiling of Sphagnum fallax was performed using two types of mass spectrometry (MS) systems and 1H nuclear magnetic resonance spectroscopy (1H NMR). Lipidome profiling was performed using LC-MS/MS. A total of 655 metabolites, including one hundred fifty-two lipids, were detected by NMR and LC-MS/MS-329 of which were novel metabolites (31 unknown lipids). Sphagum fallax metabolite profile was composed mainly of acid-like and flavonoid glycoside compounds, that could be acting as potent antimicrobial compounds, allowing Sphagnum to control its environment. Sphagnum fallax metabolite composition comparison against previously known antimicrobial plant metabolites confirmed this trend, with seventeen antimicrobial compounds discovered to be present in Sphagnum fallax, the majority of which were acids and glycosides. Biological activity of these compounds needs to be further tested to confirm antimicrobial qualities. Three fungal metabolites were identified providing insights into fungal colonization that may benefit Sphagnum. Characterizing the metabolite profile of Sphagnum fallax provided a baseline to understand the mechanisms in which Sphagnum fallax acts on its environment, its relation to carbon sequestration in peatlands, and provide key biomarkers to predict peatland C store changes (sequestration, emissions) as climate shifts.