Birgit Wild

ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

Malnutrition affects up to one billion people in the world and is a major cause of mortality. In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death. The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure, cardiovascular functions and SARS infections. Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea.

Host-compound foraging by intestinal microbiota revealed by single-cell stable isotope probing

The animal and human intestinal mucosa secretes an assortment of compounds to establish a physical barrier between the host tissue and intestinal contents, a separation that is vital for health. Some pathogenic microorganisms as well as members of the commensal intestinal microbiota have been shown to be able to break down these secreted compounds. Our understanding of host-compound degradation by the commensal microbiota has been limited to knowledge about simplified model systems because of the difficulty in studying the complex intestinal ecosystem in vivo. In this study, we introduce an approach that overcomes previous technical limitations and allows us to observe which microbial cells in the intestine use host-derived compounds. We added stable isotope-labeled threonine i.v. to mice and combined fluorescence in situ hybridization with high-resolution secondary ion mass spectrometry imaging to characterize utilization of host proteins by individual bacterial cells. We show that two bacterial species, Bacteroides acidifaciens and Akkermansia muciniphila, are important host-protein foragers in vivo. Using gnotobiotic mice we show that microbiota composition determines the magnitude and pattern of foraging by these organisms, demonstrating that a complex microbiota is necessary in order for this niche to be fully exploited. These results underscore the importance of in vivo studies of intestinal microbiota, and the approach presented in this study will be a powerful tool to address many other key questions in animal and human microbiome research.

Stoichiometric controls of nitrogen and phosphorus cycling in decomposing beech leaf litter

Resource stoichiometry (C:N:P) is an important determinant of litter decomposition. However, the effect of elemental stoichiometry on the gross rates of microbial N and P cycling processes during litter decomposition is unknown. In a mesocosm experiment, beech (Fagus sylvatica L.) litter with natural differences in elemental stoichiometry (C:N:P) was incubated under constant environmental conditions. After three and six months, we measured various aspects of nitrogen and phosphorus cycling. We found that gross protein depolymerization, N mineralization (ammonification), and nitrification rates were negatively related to litter C:N. Rates of P mineralization were negatively correlated with litter C:P. The negative correlations with litter C:N were stronger for inorganic N cycling processes than for gross protein depolymerization, indicating that the effect of resource stoichiometry on intracellular processes was stronger than on processes catalyzed by extracellular enzymes. Consistent with this, extracellular protein depolymerization was mainly limited by substrate availability and less so by the amount of protease. Strong positive correlations between the interconnected N and P pools and the respective production and consumption processes pointed to feed-forward control of microbial litter N and P cycling. A negative relationship between litter C:N and phosphatase activity (and between litter C:P and protease activity) demonstrated that microbes tended to allocate carbon and nutrients in ample supply into the production of extracellular enzymes to mine for the nutrient that is more limiting. Overall, the study demonstrated a strong effect of litter stoichiometry (C:N:P) on gross processes of microbial N and P cycling in decomposing litter; mineralization of N and P were tightly coupled to assist in maintaining cellular homeostasis of litter microbial communities.

Non-structural carbohydrates in woody plants compared among laboratories

Non-structural carbohydrates (NSC) in plant tissue are frequently quantified to make inferences about plant responses to environmental conditions. Laboratories publishing estimates of NSC of woody plants use many different methods to evaluate NSC. We asked whether NSC estimates in the recent literature could be quantitatively compared among studies. We also asked whether any differences among laboratories were related to the extraction and quantification methods used to determine starch and sugar concentrations. These questions were addressed by sending sub-samples collected from five woody plant tissues, which varied in NSC content and chemical composition, to 29 laboratories. Each laboratory analyzed the samples with their laboratory-specific protocols, based on recent publications, to determine concentrations of soluble sugars, starch and their sum, total NSC. Laboratory estimates differed substantially for all samples. For example, estimates for Eucalyptus globulus leaves (EGL) varied from 23 to 116 (mean = 56) mg g(-1) for soluble sugars, 6-533 (mean = 94) mg g(-1) for starch and 53-649 (mean = 153) mg g(-1) for total NSC. Mixed model analysis of variance showed that much of the variability among laboratories was unrelated to the categories we used for extraction and quantification methods (method category R(2) = 0.05-0.12 for soluble sugars, 0.10-0.33 for starch and 0.01-0.09 for total NSC). For EGL, the difference between the highest and lowest least squares means for categories in the mixed model analysis was 33 mg g(-1) for total NSC, compared with the range of laboratory estimates of 596 mg g(-1). Laboratories were reasonably consistent in their ranks of estimates among tissues for starch (r = 0.41-0.91), but less so for total NSC (r = 0.45-0.84) and soluble sugars (r = 0.11-0.83). Our results show that NSC estimates for woody plant tissues cannot be compared among laboratories. The relative changes in NSC between treatments measured within a laboratory may be comparable within and between laboratories, especially for starch. To obtain comparable NSC estimates, we suggest that users can either adopt the reference method given in this publication, or report estimates for a portion of samples using the reference method, and report estimates for a standard reference material. Researchers interested in NSC estimates should work to identify and adopt standard methods.

Distinct microbial communities associated with buried soils in the Siberian tundra

Cryoturbation, the burial of topsoil material into deeper soil horizons by repeated freeze–thaw events, is an important storage mechanism for soil organic matter (SOM) in permafrost-affected soils. Besides abiotic conditions, microbial community structure and the accessibility of SOM to the decomposer community are hypothesized to control SOM decomposition and thus have a crucial role in SOM accumulation in buried soils. We surveyed the microbial community structure in cryoturbated soils from nine soil profiles in the northeastern Siberian tundra using high-throughput sequencing and quantification of bacterial, archaeal and fungal marker genes. We found that bacterial abundances in buried topsoils were as high as in unburied topsoils. In contrast, fungal abundances decreased with depth and were significantly lower in buried than in unburied topsoils resulting in remarkably low fungal to bacterial ratios in buried topsoils. Fungal community profiling revealed an associated decrease in presumably ectomycorrhizal (ECM) fungi. The abiotic conditions (low to subzero temperatures, anoxia) and the reduced abundance of fungi likely provide a niche for bacterial, facultative anaerobic decomposers of SOM such as members of the Actinobacteria, which were found in significantly higher relative abundances in buried than in unburied topsoils. Our study expands the knowledge on the microbial community structure in soils of Northern latitude permafrost regions, and attributes the delayed decomposition of SOM in buried soils to specific microbial taxa, and particularly to a decrease in abundance and activity of ECM fungi, and to the extent to which bacterial decomposers are able to act as their functional substitutes.

Responses of belowground carbon allocation dynamics to extended shading in mountain grassland

Carbon (C) allocation strongly influences plant and soil processes. Short-term C allocation dynamics in ecosystems and their responses to environmental changes are still poorly understood. Using in situ 13CO2 pulse labeling, we studied the effects of 1 wk of shading on the transfer of recent photoassimilates between sugars and starch of above- and belowground plant organs and to soil microbial communities of a mountain meadow. C allocation to roots and microbial communities was rapid. Shading strongly reduced sucrose and starch concentrations in shoots, but not roots, and affected tracer dynamics in sucrose and starch of shoots, but not roots: recent C was slowly incorporated into root starch irrespective of the shading treatment. Shading reduced leaf respiration more strongly than root respiration. It caused no reduction in the amount of 13C incorporated into fungi and Gram-negative bacteria, but increased its residence time. These findings suggest that, under interrupted C supply, belowground C allocation (as reflected by the amount of tracer allocated to root starch, soil microbial communities and belowground respiration) was maintained at the expense of aboveground C status, and that C source strength may affect the turnover of recent plant-derived C in soil microbial communities.

Allocation of carbon to fine root compounds and their residence times in a boreal forest depend on root size class and season

Fine roots play a key role in the forest carbon balance, but their carbon dynamics remain largely unknown. We pulse labelled 50 m(2) patches of young boreal forest by exposure to (13)CO(2) in early and late summer. Labelled photosynthates were traced into carbon compounds of < 1 and 1-3 mm diameter roots (fine roots), and into bulk tissue of these and first-order roots (root tips). Root tips were the most strongly labelled size class. Carbon allocation to all size classes was higher in late than in early summer; mean residence times (MRTs) in starch increased from 4 to 11 months. In structural compounds, MRTs were 0.8 yr in tips and 1.8 yr in fine roots. The MRT of carbon in sugars was in the range of days. Functional differences within the fine root population were indicated by carbon allocation patterns and residence times. Pronounced allocation of recent carbon and higher turnover rates in tips are associated with their role in nutrient and water acquisition. In fine roots, longer MRTs but high allocation to sugars and starch reflect their role in structural support and storage. Accounting for heterogeneity in carbon residence times will improve and most probably reduce the estimates of fine root production.

Contribution of carbon fixed by Rubisco and PEPC to phloem export in the Crassulacean acid metabolism plant Kalanchoë daigremontiana

Crassulacean acid metabolism (CAM) plants exhibit a complex interplay between CO2 fixation by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), and carbon demand for CAM maintenance and growth. This study investigated the flux of carbon from PEPC and direct Rubisco fixation to different leaf carbon pools and to phloem sap over the diurnal cycle. Concentrations and carbon isotope compositions of starch, soluble sugars, and organic acids were determined in leaves and phloem exudates of Kalanchoë daigremontiana Hamet et Perr., and related to CO2 fixation by PEPC and Rubisco. Three types of leaf carbon pools could be distinguished. (i) Starch and malate pools were dominant and showed a pattern of reciprocal mobilization and accumulation (85/54 and 13/48 mg C g−1 DW, respective, at the beginning/end of phase I). The carbon isotope composition of these pools was compatible with predominant PEPC fixation (δ13C values of –13 and –11‰ for starch and malate compared to –11‰ of PEPC fixed carbon). (ii) Isotopic composition (–17‰ and –14‰) and concentration of glucose and fructose (2 and 3 mg C g−1 DW, respectively) were not affected by diurnal metabolism, suggesting a low turnover. (iii) Sucrose (1–3 mg C g−1 DW), in contrast, exhibited large diurnal changes in δ13C values (from –17‰ in the evening to –12‰ in the morning), which were not matched by net changes in sucrose concentration. This suggests a high sucrose turnover, fed by nocturnal starch degradation and direct Rubisco fixation during the day. A detailed dissection of the carbon fixation and mobilization pattern in K. daigremontiana revealed that direct fixation of Rubisco during the light accounted for 30% of phloem sucrose, but only 15% of fixed carbon, indicating that carbon from direct Rubisco fixation was preferentially used for leaf export.

Microbial nitrogen dynamics in organic and mineral soil horizons along a latitudinal transect in western Siberia.

Soil N availability is constrained by the breakdown of N-containing polymers such as proteins to oligopeptides and amino acids that can be taken up by plants and microorganisms. Excess N is released from microbial cells as ammonium (N mineralization), which in turn can serve as substrate for nitrification. According to stoichiometric theory, N mineralization and nitrification are expected to increase in relation to protein depolymerization with decreasing N limitation, and thus from higher to lower latitudes and from topsoils to subsoils. To test these hypotheses, we compared gross rates of protein depolymerization, N mineralization and nitrification (determined using 15N pool dilution assays) in organic topsoil, mineral topsoil, and mineral subsoil of seven ecosystems along a latitudinal transect in western Siberia, from tundra (67°N) to steppe (54°N). The investigated ecosystems differed strongly in N transformation rates, with highest protein depolymerization and N mineralization rates in middle and southern taiga. All N transformation rates decreased with soil depth following the decrease in organic matter content. Related to protein depolymerization, N mineralization and nitrification were significantly higher in mineral than in organic horizons, supporting a decrease in microbial N limitation with depth. In contrast, we did not find indications for a decrease in microbial N limitation from arctic to temperate ecosystems along the transect. Our findings thus challenge the perception of ubiquitous N limitation at high latitudes, but suggest a transition from N to C limitation of microorganisms with soil depth, even in high-latitude systems such as tundra and boreal forest. Key Points We compared soil N dynamics of seven ecosystems along a latitudinal transect Shifts in N dynamics suggest a decrease in microbial N limitation with depth We found no decrease in microbial N limitation from arctic to temperate zones

Effects of Soil Organic Matter Properties and Microbial Community Composition on Enzyme Activities in Cryoturbated Arctic Soils

Enzyme-mediated decomposition of soil organic matter (SOM) is controlled, amongst other factors, by organic matter properties and by the microbial decomposer community present. Since microbial community composition and SOM properties are often interrelated and both change with soil depth, the drivers of enzymatic decomposition are hard to dissect. We investigated soils from three regions in the Siberian Arctic, where carbon rich topsoil material has been incorporated into the subsoil (cryoturbation). We took advantage of this subduction to test if SOM properties shape microbial community composition, and to identify controls of both on enzyme activities. We found that microbial community composition (estimated by phospholipid fatty acid analysis), was similar in cryoturbated material and in surrounding subsoil, although carbon and nitrogen contents were similar in cryoturbated material and topsoils. This suggests that the microbial community in cryoturbated material was not well adapted to SOM properties. We also measured three potential enzyme activities (cellobiohydrolase, leucine-amino-peptidase and phenoloxidase) and used structural equation models (SEMs) to identify direct and indirect drivers of the three enzyme activities. The models included microbial community composition, carbon and nitrogen contents, clay content, water content, and pH. Models for regular horizons, excluding cryoturbated material, showed that all enzyme activities were mainly controlled by carbon or nitrogen. Microbial community composition had no effect. In contrast, models for cryoturbated material showed that enzyme activities were also related to microbial community composition. The additional control of microbial community composition could have restrained enzyme activities and furthermore decomposition in general. The functional decoupling of SOM properties and microbial community composition might thus be one of the reasons for low decomposition rates and the persistence of 400 Gt carbon stored in cryoturbated material.