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Temperature is an important factor governing microbe-mediated carbon feedback from permafrost soils. The link between taxonomic and functional microbial responses to temperature change remains elusive due to the lack of studies assessing both aspects of microbial ecology. Our previous study reported microbial metabolic and trophic shifts in response to short-term temperature increases in Arctic peat soil, and linked these shifts to higher CH4 and CO2 production rates (Proceedings of the National Academy of Sciences of the United States of America, 112, E2507-E2516). Here, we studied the taxonomic composition and functional potential of samples from the same experiment. We see that along a high-resolution temperature gradient (1-30 degrees C), microbial communities change discretely, but not continuously or stochastically, in response to rising temperatures. The taxonomic variability may thus in part reflect the varied temperature responses of individual taxa and the competition between these taxa for resources. These taxonomic responses contrast the stable functional potential (metagenomic-based) across all temperatures or the previously observed metabolic or trophic shifts at key temperatures. Furthermore, with rising temperatures we observed a progressive decrease in species diversity (Shannon Index) and increased dispersion of greenhouse gas (GHG) production rates. We conclude that the taxonomic variation is decoupled from both the functional potential of the community and the previously observed temperature-dependent changes in microbial function. However, the reduced diversity at higher temperatures might help explain the higher variability in GHG production at higher temperatures.
Trade-offs between traits are present across different levels of biological systems and ultimately reflect constraints imposed by physicochemical laws and the structure of underlying biochemical networks. Yet, mechanistic explanation of how trade-offs between molecular traits arise and how they relate to optimization of fitness-related traits remains elusive. Here, we introduce the concept of relative flux trade-offs and propose a constraint-based approach, termed FluTOr, to identify metabolic reactions whose fluxes are in relative trade-off with respect to an optimized fitness-related cellular task, like growth. We then employed FluTOr to identify relative flux trade-offs in the genome-scale metabolic networks of Escherichia coli, Saccharomyces cerevisiae, and Arabidopsis thaliana. For the metabolic models of E. coli and S. cerevisiae we showed that: (i) the identified relative flux trade-offs depend on the carbon source used and that (ii) reactions that participated in relative trade-offs in both species were implicated in cofactor biosynthesis. In contrast to the two microorganisms, the relative flux trade-offs for the metabolic model of A. thaliana did not depend on the available nitrogen sources, reflecting the differences in the underlying metabolic network as well as the considered environments. Lastly, the established connection between relative flux trade-offs allowed us to identify overexpression targets that can be used to optimize fitness-related traits. Altogether, our computational approach and findings demonstrate how relative flux trade-offs can shape optimization of metabolic tasks, important in biotechnological applications.
Critical role of parasite-mediated energy pathway on community response to nutrient enrichment
(2022)
Parasites form an integral part of food webs, however, they are often ignored in classic food web theory or limited to the investigation of trophic transmission pathways. Specifically, direct consumption of parasites by nonhost predators is rarely considered, while it can contribute substantially to energy flow in food webs. In aquatic systems, chytrids constitute a major group of fungal parasites whose free-living infective stages (zoospores) form a highly nutritional food source to zooplankton. Thereby, the consumption of zoospores can create an energy pathway from otherwise inedible phytoplankton to zooplankton ( "mycoloop "). This parasite-mediated energy pathway might be of special importance during phytoplankton blooms dominated by inedible or toxic primary producers like cyanobacteria, which are on the rise with eutrophication and global warming. We theoretically investigated community dynamics and energy transfer in a food web consisting of an edible nonhost and an inedible host phytoplankton species, a parasitic fungus, and a zooplankton species grazing on edible phytoplankton and fungi. Food web dynamics were investigated along a nutrient gradient contrasting nonadaptive zooplankton species representative for filter feeders like cladocerans and zooplankton with the ability to actively adapt their feeding preferences like many copepod species. Overall, the importance of the mycoloop for zooplankton increases with nutrient availability. This increase is smooth for nonadaptive consumers. For adaptive consumers, we observe an abrupt shift from an almost exclusive preference for edible phytoplankton at low nutrient levels to a strong preference for parasitic fungi at high nutrient levels. The model predicts that parasitic fungi could contribute up to 50% of the zooplankton diet in nutrient-rich environments, which agrees with empirical observations on zooplankton gut content from eutrophic systems during blooms of inedible diatoms or cyanobacteria. Our findings highlight the role of parasite-mediated energy pathways for predictions of energy flow and community composition under current and future environmental change.
Understanding the assembly mechanism and function of membrane proteins is a fundamental problem in biochemical research. Among the membrane proteins, G protein-coupled receptors (GPCRs) represent the largest class in the human body and have long been considered to function as monomers.
Nowadays, the oligomeric assembly of GPCRs is widely accepted, although the functional importance and therapeutic intervention remain largely unexplored. This is partly due to difficulties in the heterologous production of membrane proteins.
Cell-free protein synthesis (CFPS) with its endogenous endoplasmic reticulum-derived structures has proven as a technique to address this issue.
In this study, we investigate for the first time the conceptual CFPS of a heteromeric GPCR, the gamma-aminobutyric acid receptor type B (GABA(B)), from its protomers BR1 and BR2 using a eukaryotic cell-free lysate. Using a fluorescence-based proximity ligation assay, we provide evidence for colocalization and thus suggesting heterodimerization.
We prove the heterodimeric assembly by a bioluminescence resonance energy transfer saturation assay providing the manufacturability of a heterodimeric GPCR by CFPS. Additionally, we show the binding of a fluorescent orthosteric antagonist, demonstrating the feasibility of combining the CFPS of GPCRs with pharmacological applications.
These results provide a simple and powerful experimental platform for the synthesis of heteromeric GPCRs and open new perspectives for the modelling of protein-protein interactions.
Accordingly, the presented technology enables the targeting of protein assemblies as a new interface for pharmacological intervention in disease-relevant dimers.
Theoretical and empirical studies show increased diversity in crops, supply chains, and markets helps stabilize food systems. At the same time global commodity markets and industrial agriculture have driven homogenization of local and regional production systems, and consolidated power in fewer larger specialized farms and distributers. This is a global challenge, with no obvious global solutions. An important question therefore, is how individual countries can build their own resilience through maintaining or increasing diversity within their borders. Here we show, using farm level data from Germany, that spreading production risk by growing the same crops across different farms carries stabilizing benefits by allowing for increased spatiotemporal asynchrony within crops. We also find that increasing asynchrony between the year-to-year production of different crops has stabilizing effects on food supply. Importantly, the benefits of increasing crop diversity are lower in specialized landscapes growing the same crop on large patches. Our results illustrate clear benefits of diversified crops, producers, and agricultural landscapes to buffer supply side shocks, and for incorporation in subsidies and other regulatory measures aimed at stabilizing food systems.
Riverine ecosystems provide various ecosystem services. One of these services is the biological control of eutrophication by grazing macroinvertebrates.
However, riverine ecosystems are subject to numerous stressors that affect community structure, functions, and stability properties. To manage rivers in response to these stressors, a better understanding of the ecological functions underlying services is needed.
This requires consideration of local and regional processes, which requires a metacommunity approach that links local food webs through drift and dispersal. This takes into account long-distance interactions that can compensate for local effects of stressors.
Our modular model MASTIFF (Multiple Aquatic STressors In Flowing Food webs) is stage-structured, spatially explicit, and includes coupled food webs consisting of benthic resource-consumer interactions between biofilm and three competing macroinvertebrate functional types. River segments are unidirectionally connected through organismal drift and bidirectionally connected through dispersal. Climate and land use stressors along the river can be accounted for. Biocontrol of biofilm eutrophication is used as an exemplary functional indicator.
We present the model and the underlying considerations, and show in an exemplary application that explicit consideration of drift and dispersal is essential for understanding the spatiotemporal biocontrol of eutrophication.
The combination of drift and dispersal reduced eutrophication events. While dispersal events were linked to specific periods in the species life cycles and therefore had limited potential to control, drift was ubiquitous and thus responded more readily to changing habitat conditions.
This indicates that drift is an important factor for coping with stress situations.
Finally, we outline and discuss the potential and possibilities of MASTIFF as a tool for mechanistic, cross-scale analyses of multiple stressors to advance knowledge of riverine ecosystem functioning.
Liebchen, Uwe ; Weinelt, Ferdinand ; Scharf, Christina ; Schröder, Ines ; Paal, Michael ; Zoller, Michael ; Kloft, Charlotte ; Jung, Jette ; Michelet, Robin
Meropenem is one of the most frequently used antibiotics to treat life-threatening infections in critically ill patients.
This study aimed to develop a meropenem dosing algorithm for the treatment of Gram-negative infections based on intensive care unit (ICU)-specific resistance data. Antimicrobial susceptibility testing of Gram-negative bacteria obtained from critically ill patients was carried out from 2016 to 2020 at a tertiary care hospital.
Based on the observed MIC distribution, stochastic simulations (n = 1,000) of an evaluated pharmacokinetic meropenem model, and a defined pharmacokinetic/pharmacodynamic target (100%T->4xMIC while minimum concentrations were <44.5 mg/L), dosing recommendations for patients with varying renal function were derived.
Pathogen-specific MIC distributions were used to calculate the cumulative fraction of response (CFR), and the overall MIC distribution was used to calculate the local pathogen-independent mean fraction of response (LPIFR) for the investigated dosing regimens.
A CFR/LPIFR of >90% was considered adequate. The observed MIC distribution significantly differed from the EUCAST database. Based on the 6,520 MIC values included, a three-level dosing algorithm was developed.
If the pathogen causing the infection is unknown (level 1), known (level 2), known to be neither Pseudomonas aeruginosa nor Acinetobacrer baumannii, or classified as susceptible (level 3), a continuous infusion of 1.5 g daily reached sufficient target attainment independent of renal function.
In all other cases, dosing needs to be adjusted based on renal function. ICU-specific susceptibility data should be assessed regularly and integrated into dosing decisions. The presented workflow may serve as a blueprint for other antimicrobial settings.
Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal enzymes that catalyze the oxyfunctionalization of non-activated hydrocarbons, making them valuable biocatalysts. Despite the increasing interest in UPOs that has led to the identification of thousands of putative UPO genes, only a few of these have been successfully expressed and characterized.
There is currently no universal expression system in place to explore their full potential. Cell-free protein synthesis has proven to be a sophisticated technique for the synthesis of difficult-to-express proteins.
In this work, we aimed to establish an insect-based cell-free protein synthesis (CFPS) platform to produce UPOs. CFPS relies on translationally active cell lysates rather than living cells.
The system parameters can thus be directly manipulated without having to account for cell viability, thereby making it highly adaptable.
The insect-based lysate contains translocationally active, ER-derived vesicles, called microsomes.
These microsomes have been shown to allow efficient translocation of proteins into their lumen, promoting post-translational modifications such as disulfide bridge formation and N-glycosylations.
In this study the ability of a redox optimized, vesicle-based, eukaryotic CFPS system to synthesize functional UPOs was explored. The influence of different reaction parameters as well as the influence of translocation on enzyme activity was evaluated for a short UPO from Marasmius rotula and a long UPO from Agrocybe aegerita.
The capability of the CFPS system described here was demonstrated by the successful synthesis of a novel UPO from Podospora anserina, thus qualifying CFPS as a promising tool for the identification and evaluation of novel UPOs and variants thereof.
Quantifying the influence of pollen aging on the adhesive
properties of Hypochaeris radicata pollen
(2022)
Simple Summary Pollination is the transfer of pollen from a plant's male part (anther) to the corresponding female part (stigma). It is a fundamental biological process that ensures plant reproduction. Most studies investigate pollination from a biological perspective, but the underlying physical processes are poorly understood. Many plants rely on insects to transport pollen and the forces with which pollen adhere to insects and floral surfaces are fundamental for successful pollination.
We quantified pollen adhesion by measuring the forces necessary to detach Hypochaeris radicata (catsear, a common insect-pollinated plant) pollen from glass and studied for the first time how the adhesion forces change with pollen aging.
Our results show that newly formed adhesion bonds between H. radicata pollen and glass are stronger for fresh pollen than for old ones. On the other hand, when H. radicata pollen age in contact with glass, the adhesion between pollen and glass strengthens over time. These effects are probably caused by the viscous liquid covering most pollen (pollenkitt) changing its viscoelastic properties as it dries.
Although pollination is one of the most crucial biological processes that ensures plant reproduction, its mechanisms are poorly understood. Especially in insect-mediated pollination, a pollen undergoes several attachment and detachment cycles when being transferred from anther to insect and from insect to stigma. The influence of the properties of pollen, insect and floral surfaces on the adhesion forces that mediate pollen transfer have been poorly studied.
Here, we investigate the adhesive properties of Hypochaeris radicata pollen and their dependence on pollen aging by quantifying the pull-off forces from glass slides using centrifugation and atomic force microscopy. We found that the properties of the pollenkitt-the viscous, lipid liquid on the surface of most pollen grains-influences the forces necessary to detach a pollen from hydrophilic surfaces.
Our results show that aged H. radicata pollen form weaker adhesions to hydrophilic glass than fresh ones. On the other hand, when a pollen grain ages in contact with glass, the adhesion between the two surfaces increases over time.
This study shows for the first time the pollen aging effect on the pollination mechanism.
Rybakova, Daria ; Müller, Henry ; Olimi, Expedito ; Schäfer, Angelika ; Cernava, Tomislav ; Berg, Gabriele
Volatile organic compounds (VOCs) are involved in microbial interspecies communication and in the mode of action of various antagonistic interactions. They are important for balancing host-microbe interactions and provide the basis for developing biological control strategies to control plant pathogens.
We studied the interactions between the bacterial antagonist Serratia plymuthica HRO-C48 and three fungal plant pathogens Rhizoctonia solani, Leptosphaeria maculans and Verticillium longisporum. Significant differences in fungal growth inhibition by the Serratia-emitted VOCs in pairwise dual culture assays and changes in the transcriptome of the bacterium and in the volatilomes of both interacting partners were observed. Even though the rate of fungal growth inhibition by Serratia was variable, the confrontation of the bacterium with the VOCs of all three fungi changed the levels of expression of the genes involved in stress response, biofilm formation, and the production of antimicrobial VOCs. Pairwise interacting microorganisms switched between defense (downregulation of gene expression) and attack (upregulation of gene expression and metabolism followed by growth inhibition of the interacting partner) modes, subject to the combinations of microorganisms that were interacting.
In the attack mode HRO-C48 significantly inhibited the growth of R. solani while simultaneously boosting its own metabolism; by contrast, its metabolism was downregulated when HRO-C48 went into a defense mode that was induced by the L. maculans and V. longisporum VOCs. L. maculans growth was slightly reduced by the one bacterial VOC methyl acetate that induced a strong downregulation of expression of genes involved in almost all metabolic functions in S. plymuthica.
Similarly, the interaction between S. plymuthica and V. longisporum resulted in an insignificant growth reduction of the fungus and repressed the rate of bacterial metabolism on the transcriptional level, accompanied by an intense volatile dialogue. Overall, our results indicate that VOCs substantially contribute to the highly break species-specific interactions between pathogens and their natural antagonists and thus deserving of increased consideration for pathogen control.