Fire Impacts on the Soil Metabolome and Organic Matter Biodegradability.

Global wildfire activity has increased since the 1970s and is projected to intensify throughout the 21st century. Wildfires change the composition and biodegradability of soil organic matter (SOM) which contains nutrients that fuel microbial metabolism. Though persistent forms of SOM often increase postfire, the response of more biodegradable SOM remains unclear. Here we simulated severe wildfires through a controlled "pyrocosm" approach to identify biodegradable sources of SOM and characterize the soil metabolome immediately postfire. Using microbial amplicon (16S/ITS) sequencing and gas chromatography-mass spectrometry, heterotrophic microbes (Actinobacteria, Firmicutes, and Protobacteria) and specific metabolites (glycine, protocatechuate, citric cycle intermediates) were enriched in burned soils, indicating that burned soils contain a variety of substrates that support microbial metabolism. Molecular formulas assigned by 21 T Fourier transform ion cyclotron resonance mass spectrometry showed that SOM in burned soil was lower in molecular weight and featured 20 to 43% more nitrogen-containing molecular formulas than unburned soil. We also measured higher water extractable organic carbon concentrations and higher CO2 efflux in burned soils. The observed enrichment of biodegradable SOM and microbial heterotrophs demonstrates the resilience of these soils to severe burning, providing important implications for postfire soil microbial and plant recolonization and ecosystem recovery.

Identification of a Novel 2,4-D Metabolic Detoxification Pathway in 2,4-D-Resistant Waterhemp (Amaranthus tuberculatus).

A 2,4-dichlorophenoxyactic acid (2,4-D)-resistant population of Amaranthus tuberculatus (common waterhemp) from Nebraska, USA, was previously found to have rapid metabolic detoxification of the synthetic auxin herbicide 2,4-D. We purified the main 2,4-D metabolites from resistant and susceptible plants, solved their structures by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS), and synthesized the metabolites to determine their in planta toxicity. Susceptible plants conjugated 2,4-D to aspartate to form 2,4-D-aspartic acid (2,4-D-Asp), while resistant plants had a unique metabolic profile where 2,4-D was hydroxylated into 5-OH-2,4-D, followed by conjugation into a sugar metabolite (2,4-D-5-O-d-glucopyranoside) and subsequent malonylation into 2,4-D-(6'-O-malonyl)-5-O-d-glucopyranoside. Toxicological studies on waterhemp and Arabidopsis thaliana confirmed that the hydroxylated metabolite lost its auxinic action and toxicity. In contrast, the 2,4-D-Asp metabolite found in susceptible plants retained some auxinic action and toxicity. These results demonstrate that 2,4-D-resistant A. tuberculatus evolved novel detoxification reactions not present in susceptible plants to rapidly metabolize 2,4-D, potentially mediated by cytochrome P450 enzymes that perform the initial 5-hydroxylation reaction. This novel mechanism is more efficient to detoxify 2,4-D and produces metabolites with lower toxicity compared to the aspartic acid conjugation found in susceptible waterhemp.

The Effect of ProHydrolase® on the Amino Acid and Intramuscular Anabolic Signaling Response to Resistance Exercise in Trained Males.

This double-blind study examined effects of a protease enzyme blend (Prohydrolase®) added to whey protein on post-resistance exercise aminoacidemia and intramuscular anabolic signaling were investigated in ten resistance-trained males. Participants completed 4 sets of 8-10 repetitions in the leg press and leg extension exercises at 75% of 1-repetition maximum. Participants then consumed either 250 mg of Prohydrolase® + 26 g of whey protein (PW), 26 g whey alone (W), or non-nutritive control (CON) in counterbalanced order. Blood samples were obtained prior to exercise (baseline) and then immediately-post (IP), 30-, 60-, 90-, 120-, and 180-min post-exercise. Muscle biopsies were taken at baseline, 1-h (1H), and 3-h (3H) post-exercise. Phosphorylation of AKTSer437 was decreased (3H only: p < 0.001), mTORSer2448 was increased (1H: p = 0.025; 3H: p = 0.009), and p70S6KThr412 remained unchanged similarly for each condition. Plasma leucine, branch-chained amino acids, and essential amino acid concentrations for PW were significantly higher than CON (p < 0.05) at 30 min and similar to W. Compared to IP, PW was the only treatment with elevated plasma leucine levels at 30 min (p = 0.007; ∆ = 57.8 mmol/L, 95% Confidence Interval (CI): 20.0, 95.6) and EAA levels at 180 min (p = 0.003; ∆ = 179.1 mmol/L, 95% CI: 77.5, 280.7). Area under the curve amino acid analysis revealed no differences between PW and W. While no different than W, these data indicate that PW was the only group to produce elevated amino acid concentrations 30-min and 180-min post-ingestion.

Secondary Extended Mannan Side Chains and Attachment of the Arabinan in Mycobacterial Lipoarabinomannan.

Mycobacterial lipoarabinomannan (LAM) in an essential cell envelope lipopolysaccharide anchored both to the plasma and outer membranes. To understand critical biological questions such as the biosynthesis, spatial organization of LAM within the cell envelope, structural remodeling during growth, and display or lack of display of LAM-based antigenicity all requires a basic understanding of the primary structure of the mannan, arabinan and how they are attached to each other. Herein, using enzymatic digestions and high-resolution mass spectrometry, we show that the arabinan component of LAM is attached at the non-reducing end of the mannan rather than to internal regions. Further, we show the presence of secondary extended mannan side chains attached to the internal mannan region. Such findings lead to a significant revision of the structure of LAM and lead to guidance of biosynthetic studies and to hypotheses of the role of LAM both in the periplasm and outside the cell as a fundamental part of the dynamic mycobacterial cell envelope.

Plasma Amino Acid Response to Whey Protein Ingestion Following 28 Days of Probiotic (Bacillus subtilis DE111) Supplementation in Active Men and Women.

We sought to determine if 28 days of probiotic supplementation influenced the plasma amino acid (AA) response to acute whey protein feeding. METHODS: Twenty-two recreationally active men (n = 11; 24.3 ± 3.2 yrs; 89.3 ± 7.2 kg) and women (n = 11; 23.0 ± 2.8 yrs; 70.2 ± 15.2 kg) participated in this double-blind, placebo-controlled, randomized study. Before (PRE) and after 28 days of supplementation (POST), participants reported to the lab following a 10-hr fast and provided a resting blood draw (0 min), then subsequently consumed 25 g of whey protein. Blood samples were collected at 15-min intervals for 2 h post-consumption (15-120 min) and later analyzed for plasma leucine, branched-chain AA (BCAA), essential AA (EAA), and total AA (TAA). Participants received a probiotic (PROB) consisting of 1 x10-9 colony forming units (CFU) Bacillus subtilis DE111 (n = 11) or a maltodextrin placebo (PL) (n = 11) for 28 days. Plasma AA response and area under the curve (AUC) values were analyzed via repeated measures analysis of variance. RESULTS: Our analysis indicated no significant (p < 0.05) differential responses for plasma leucine, BCAA, EAA, or TAA between PROB and PL from PRE to POST. AUC analysis revealed no group × time interaction for plasma leucine (p = 0.524), BCAA (p = 0.345), EAA (p = 0.512), and TAA (p = 0.712). CONCLUSION: These data indicate that 28 days of Bacillus subtilis DE111 does not affect plasma AA appearance following acute whey protein ingestion.

Resource heterogeneity structures aquatic bacterial communities.

Microorganisms are strongly influenced by the bottom-up effects of resource supply. While many species respond to fluctuations in the concentration of resources, microbial diversity may also be affected by the heterogeneity of the resource pool, which often reflects a mixture of distinct molecules. To test this hypothesis, we examined resource-diversity relationships for bacterioplankton in a set of north temperate lakes that varied in their concentration and composition of dissolved organic matter (DOM), which is an important resource for heterotrophic bacteria. Using 16S rRNA transcript sequencing and ecosystem metabolomics, we documented strong relationships between bacterial alpha-diversity (richness and evenness) and the bulk concentration and the number of molecules in the DOM pool. Similarly, bacterial community beta-diversity was related to both DOM concentration and composition. However, in some lakes the relative abundance of resource generalists, which was inversely related to the DOM concentration, may have reduced the effect of DOM heterogeneity on community composition. Together, our results demonstrate the potential metabolic interactions between bacteria and organic matter and suggest that changes in organic matter composition may alter the structure and function of bacterial communities.

River channel connectivity shifts metabolite composition and dissolved organic matter chemistry.

Biogeochemical processing of dissolved organic matter (DOM) in headwater rivers regulates aquatic food web dynamics, water quality, and carbon storage. Although headwater rivers are critical sources of energy to downstream ecosystems, underlying mechanisms structuring DOM composition and reactivity are not well quantified. By pairing mass spectrometry and fluorescence spectroscopy, here we show that hydrology and river geomorphology interactively shape molecular patterns in DOM composition. River segments with a single channel flowing across the valley bottom export DOM with a similar chemical profile through time. In contrast, segments with multiple channels of flow store large volumes of water during peak flows, which they release downstream throughout the summer. As flows subside, losses of lateral floodplain connectivity significantly increase the heterogeneity of DOM exported downstream. By linking geomorphologic landscape-scale processes with microbial metabolism, we show DOM heterogeneity increases as a function of fluvial complexity, with implications for ecosystem function and watershed management.

Understanding how microbiomes influence the systems they inhabit.

Translating the ever-increasing wealth of information on microbiomes (environment, host or built environment) to advance our understanding of system-level processes is proving to be an exceptional research challenge. One reason for this challenge is that relationships between characteristics of microbiomes and the system-level processes that they influence are often evaluated in the absence of a robust conceptual framework and reported without elucidating the underlying causal mechanisms. The reliance on correlative approaches limits the potential to expand the inference of a single relationship to additional systems and advance the field. We propose that research focused on how microbiomes influence the systems they inhabit should work within a common framework and target known microbial processes that contribute to the system-level processes of interest. Here, we identify three distinct categories of microbiome characteristics (microbial processes, microbial community properties and microbial membership) and propose a framework to empirically link each of these categories to each other and the broader system-level processes that they affect. We posit that it is particularly important to distinguish microbial community properties that can be predicted using constituent taxa (community-aggregated traits) from those properties that cannot currently be predicted using constituent taxa (emergent properties). Existing methods in microbial ecology can be applied to more explicitly elucidate properties within each of these three categories of microbial characteristics and connect them with each other. We view this proposed framework, gleaned from a breadth of research on environmental microbiomes and ecosystem processes, as a promising pathway with the potential to advance discovery and understanding across a broad range of microbiome science.

Structural determinants in a glucose-containing lipopolysaccharide from Mycobacterium tuberculosis critical for inducing a subset of protective T cells.

Mycobacteria synthesize intracellular, 6-O-methylglucose-containing lipopolysaccharides (mGLPs) proposed to modulate bacterial fatty acid metabolism. Recently, it has been shown that Mycobacterium tuberculosis mGLP specifically induces a specific subset of protective γ9δ2 T cells. Mild base treatment, which removes all the base-labile groups, reduces the specific activity of mGLP required for induction of these T cells, suggesting that acylation of the saccharide moieties is required for γ9δ2 T-cell activation. On the basis of this premise, we used analytical LC/MS and NMR methods to identify and locate the acyl functions on the mGLP saccharides. We found that mGLP is heterogeneous with respect to acyl functions and contains acetyl, isobutyryl, succinyl, and octanoyl groups and that all acylations in mGLP, except for succinyl and octanoyl residues, reside on the glucosyl residues immediately following the terminal 3-O-methylglucose. Our analyses also indicated that the octanoyl residue resides at position 2 of an internal glucose toward the reducing end. LC/MS analysis of the residual product obtained by digesting the mGLP with pancreatic α-amylase revealed that the product is an oligosaccharide terminated by α-(1→4)-linked 6-O-methyl-d-glucosyl residues. This oligosaccharide retained none of the acyl groups, except for the octanoyl group, and was unable to induce protective γ9δ2 T cells. This observation confirmed that mGLP induces γ9δ2 T cells and indicated that the acylated glucosyl residues at the nonreducing terminus of mGLP are required for this activity.

Biosynthesis of the Methylthioxylose Capping Motif of Lipoarabinomannan in Mycobacterium tuberculosis.

Lipoarabinomannan (LAM) is a lipoglycan found in abundant quantities in the cell envelope of all mycobacteria. The nonreducing arabinan termini of LAM display species-specific structural microheterogeneity that impacts the biological activity of the entire molecule. Mycobacterium tuberculosis, for instance, produces mannoside caps made of one to three α-(1 → 2)-Manp-linked residues that may be further substituted with an α-(1 → 4)-linked methylthio-d-xylose (MTX) residue. While the biological functions and catalytic steps leading to the formation of the mannoside caps of M. tuberculosis LAM have been well established, the biosynthetic origin and biological relevance of the MTX motif remain elusive. We here report on the discovery of a five-gene cluster dedicated to the biosynthesis of the MTX capping motif of M. tuberculosis LAM, and on the functional characterization of two glycosyltransferases, MtxS and MtxT, responsible, respectively, for the production of decaprenyl-phospho-MTX (DP-MTX) and the transfer of MTX from DP-MTX to the mannoside caps of LAM. Collectively, our NMR spectroscopic and mass spectrometric analyses of mtxS and mtxT overexpressors and knockout mutants support a biosynthetic model wherein the conversion of 5'-methylthioadenosine, which is a ubiquitous byproduct of spermidine biosynthesis, into 5'-methylthioribose-1-phosphate precedes the formation of a 5'-methylthioribose nucleotide sugar, followed by the epimerization at C-3 of the ribose residue, and the transfer of MTX from the nucleotide sugar to decaprenyl-phosphate yielding the substrate for transfer onto LAM. The conservation of the MTX biosynthetic genes in a number of Actinomycetes suggests that this discrete glycosyl substituent may be more widespread in prokaryotes than originally thought.

Dietary supplementation with rice bran or navy bean alters gut bacterial metabolism in colorectal cancer survivors.

SCOPE: Heat-stabilized rice bran (SRB) and cooked navy bean powder (NBP) contain a variety of phytochemicals that are fermented by colonic microbiota and may influence intestinal health. Dietary interventions with these foods should be explored for modulating colorectal cancer risk. METHODS AND RESULTS: A randomized-controlled pilot clinical trial investigated the effects of eating SRB (30 g/day) or cooked navy bean powder (35 g/day) on gut microbiota and metabolites (NCT01929122). Twenty-nine overweight/obese volunteers with a prior history of colorectal cancer consumed a study-provided meal and snack daily for 28 days. Volunteers receiving SRB or NBP showed increased gut bacterial diversity and altered gut microbial composition at 28 days compared to baseline. Supplementation with SRB or NBP increased total dietary fiber intake similarly, yet only rice bran intake led to a decreased Firmicutes:Bacteroidetes ratio and increased SCFA (propionate and acetate) in stool after 14 days but not at 28 days. CONCLUSION: These findings support modulation of gut microbiota and fermentation byproducts by SRB and suggest that foods with similar ability to increase dietary fiber intake may not have equal effects on gut microbiota and microbial metabolism.

Linking microbial community structure and microbial processes: an empirical and conceptual overview.

A major goal of microbial ecology is to identify links between microbial community structure and microbial processes. Although this objective seems straightforward, there are conceptual and methodological challenges to designing studies that explicitly evaluate this link. Here, we analyzed literature documenting structure and process responses to manipulations to determine the frequency of structure-process links and whether experimental approaches and techniques influence link detection. We examined nine journals (published 2009-13) and retained 148 experimental studies measuring microbial community structure and processes. Many qualifying papers (112 of 148) documented structure and process responses, but few (38 of 112 papers) reported statistically testing for a link. Of these tested links, 75% were significant and typically used Spearman or Pearson's correlation analysis (68%). No particular approach for characterizing structure or processes was more likely to produce significant links. Process responses were detected earlier on average than responses in structure or both structure and process. Together, our findings suggest that few publications report statistically testing structure-process links. However, when links are tested for they often occur but share few commonalities in the processes or structures that were linked and the techniques used for measuring them.

Rhizosphere stoichiometry: are C : N : P ratios of plants, soils, and enzymes conserved at the plant species-level?

As a consequence of the tight linkages among soils, plants and microbes inhabiting the rhizosphere, we hypothesized that soil nutrient and microbial stoichiometry would differ among plant species and be correlated within plant rhizospheres. We assessed plant tissue carbon (C) : nitrogen (N) : phosphorus (P) ratios for eight species representing four different plant functional groups in a semiarid grassland during near-peak biomass. Using intact plant species-specific rhizospheres, we examined soil C : N : P, microbial biomass C : N, and soil enzyme C : N : P nutrient acquisition activities. We found that few of the plant species' rhizospheres demonstrated distinct stoichiometric properties from other plant species and unvegetated soil. Plant tissue nutrient ratios and components of below-ground rhizosphere stoichiometry predominantly differed between the C4 plant species Buchloe dactyloides and the legume Astragalus laxmannii. The rhizospheres under the C4 grass B. dactyloides exhibited relatively higher microbial C and lower soil N, indicative of distinct soil organic matter (SOM) decomposition and nutrient mineralization activities. Assessing the ecological stoichiometry among plant species' rhizospheres is a high-resolution tool useful for linking plant community composition to below-ground soil microbial and nutrient characteristics. By identifying how rhizospheres differ among plant species, we can better assess how plant-microbial interactions associated with ecosystem-level processes may be influenced by plant community shifts.

Cloud shading and fog drip influence the metabolism of a coastal pine ecosystem.

Assessing the ecological importance of clouds has substantial implications for our basic understanding of ecosystems and for predicting how they will respond to a changing climate. This study was conducted in a coastal Bishop pine forest ecosystem that experiences regular cycles of stratus cloud cover and inundation in summer. Our objective was to understand how these clouds impact ecosystem metabolism by contrasting two sites along a gradient of summer stratus cover. The site that was under cloud cover ~15% more of the summer daytime hours had lower air temperatures and evaporation rates, higher soil moisture content, and received more frequent fog drip inputs than the site with less cloud cover. These cloud-driven differences in environmental conditions translated into large differences in plant and microbial activity. Pine trees at the site with greater cloud cover exhibited less water stress in summer, larger basal area growth, and greater rates of sap velocity. The difference in basal area growth between the two sites was largely due to summer growth. Microbial metabolism was highly responsive to fog drip, illustrated by an observed ~3-fold increase in microbial biomass C with increasing summer fog drip. In addition, the site with more cloud cover had greater total soil respiration and a larger fractional contribution from heterotrophic sources. We conclude that clouds are important to the ecological functioning of these coastal forests, providing summer shading and cooling that relieve pine and microbial drought stress as well as regular moisture inputs that elevate plant and microbial metabolism. These findings are important for understanding how these and other seasonally dry coastal ecosystems will respond to predicted changes in stratus cover, rainfall, and temperature.

The Microbial Efficiency-Matrix Stabilization (MEMS) framework integrates plant litter decomposition with soil organic matter stabilization: do labile plant inputs form stable soil organic matter?

The decomposition and transformation of above- and below-ground plant detritus (litter) is the main process by which soil organic matter (SOM) is formed. Yet, research on litter decay and SOM formation has been largely uncoupled, failing to provide an effective nexus between these two fundamental processes for carbon (C) and nitrogen (N) cycling and storage. We present the current understanding of the importance of microbial substrate use efficiency and C and N allocation in controlling the proportion of plant-derived C and N that is incorporated into SOM, and of soil matrix interactions in controlling SOM stabilization. We synthesize this understanding into the Microbial Efficiency-Matrix Stabilization (MEMS) framework. This framework leads to the hypothesis that labile plant constituents are the dominant source of microbial products, relative to input rates, because they are utilized more efficiently by microbes. These microbial products of decomposition would thus become the main precursors of stable SOM by promoting aggregation and through strong chemical bonding to the mineral soil matrix.

Seasonal and episodic moisture controls on plant and microbial contributions to soil respiration.

Moisture inputs drive soil respiration (SR) dynamics in semi-arid and arid ecosystems. However, determining the contributions of root and microbial respiration to SR, and their separate temporal responses to periodic drought and water pulses, remains poorly understood. This study was conducted in a pine forest ecosystem with a Mediterranean-type climate that receives seasonally varying precipitation inputs from both rainfall (in the winter) and fog-drip (primarily in the summer). We used automated SR measurements, radiocarbon SR source partitioning, and a water addition experiment to understand how SR, and its separate root and microbial sources, respond to seasonal and episodic changes in moisture. Seasonal changes in SR were driven by surface soil water content and large changes in root respiration contributions. Superimposed on these seasonal patterns were episodic pulses of precipitation that determined the short-term SR patterns. Warm season precipitation pulses derived from fog-drip, and rainfall following extended dry periods, stimulated the largest SR responses. Microbial respiration dominated these SR responses, increasing within hours, whereas root respiration responded more slowly over days. We conclude that root and microbial respiration sources respond differently in timing and magnitude to both seasonal and episodic moisture inputs. These findings have important implications for the mechanistic representation of SR in models and the response of dry ecosystems to changes in precipitation patterns.

Pinpointing pseurotins from a marine-derived Aspergillus as tools for chemical genetics using a synthetic lethality yeast screen.

A new compound of mixed polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS) origin, 11- O-methylpseurotin A ( 1), was identified from a marine-derived Aspergillus fumigatus. Bioassay-guided fractionation using a yeast halo assay with wild-type and cell cycle-related mutant strains of Saccharomyces cerevisiae resulted in the isolation of 1, which selectively inhibited a Hof1 deletion strain. Techniques including 1D and 2D NMR, HRESIMS, optical rotation, J-based analysis, and biosynthetic parallels were used in the elucidation of the planar structure and absolute configuration of 1. A related known compound, pseurotin A ( 2), was also isolated and found to be inactive in the yeast screen.

Four Classes of Structurally Unusual Peptides from Two Marine-Derived Fungi: Structures and Bioactivities.

The structures and biological properties of peptides produced by two genera of marine-derived fungi, an atypical Acremonium sp., and a Metarrhizium sp. were explored. The Acremonium strain was isolated from a marine sponge and has previously been shown by our group to produce peptides from the efrapeptin and RHM families. The isolation and structure elucidation of the new linear pentadecapeptides efrapeptins Eα (1), H (2) and N-methylated octapeptides RHM3 (3) and RHM4 (4) were carried out through a combination of 1D and 2D NMR techniques and tandem MS. Additional known efrapeptins E, F, G and the known syctalidamides A and B were also isolated. The absolute configurations of 1 - 4 are proposed to be the same as the original compound families. The marine-sponge derived Metarrhizium sp. was shown to produce destruxin cyclic depsipeptides including A, B, B2, desmethyl B, E chlorohydrin and E2 chlorohydrin. Efrapeptins Eα (1), F and G each displayed IC(50)s of 1.3 nM against H125 cells, and destruxin E2 chlorohydrin displayed an IC(50) of 160 nM against HCT-116 cells. An initial therapeutic assessment suggested a continuous (168 h) exposure of at least 2 ng/ml, or a daily (24 h) exposure of at least 300 ng/ml for H125 cells treated with efrapeptin G, and a continuous (168 h) exposure of at least 190 ng/ml for HCT-116 cells treated with destruxin E2 chlorohydrin, will cause 90% tumor cell death in vitro.

Highly N-methylated linear peptides produced by an atypical sponge-derived Acremonium sp.

RHM1 (1) and RHM2 (2) are highly N-methylated linear octapeptides produced by an atypical strain of Acremonium sp., cultured from a marine sponge collected in Papua New Guinea. The known peptaibiotic efrapeptin G (3) was also isolated from this fungus. The planar structures of 1 and 2 were assigned based on 1D- and 2D-NMR experiments and fragmentation patterns from ESIMS. The absolute configuration of 1 was determined via Marfey's method; the absolute configuration of 2 is proposed to be identical. Efrapeptin G (3) displayed potent cytotoxicity against murine cancer cell lines, while RHM1 (1) and RHM2 (2) showed weak cytotoxicity against murine cancer cell lines; efrapeptin G (3) and RHM1 (1) also demonstrated antibacterial activity.