Incorporation Of 14C Research Articles

866-P: Quantitative Analysis of Metformin-Induced Glucose Excretion in the Intestine

The enhancement of the uptake of 18F-labeled fluorodeoxyglucose (FDG) during [18F]FDG PET is a recently found metformin action in the gut. Taking advantage of PET-MRI, which provides better tissue registration and soft-tissue contrast than conventional PET-CT, we revealed that metformin enhanced FDG accumulation in the intestinal lumen. We now quantitatively analyzed the phenomenon with a newly developed protocol of PET-MRI. We also analyzed the intestinal glucose dynamics using 13C-labeled glucose and mass spectrometry in mice. We adopted imaging sequences of MR enterography to create 3D images of the intestinal tract, then superimposed the [18F]FDG PET images to obtain the 3D distribution of radioactivity. We conducted this test on type 2 diabetes subjects treated (N=5) or not (control, N=5) with metformin. Radioactivity was detected in the wall and lumen at similar extents in the control. However, in the metformin group, the radioactivity in the lumen, but not the wall, was three to four-fold greater than that in the control. We mathematically analyzed the data using a compartment model and calculated the glucose excretion rate (GER). GER of the control and metformin groups were 6.8 ± 1.2 mg/min and 26.1 ± 2.9 mg/min, respectively, the latter being comparable to glucose excretion into the urine induced by SGLT2 inhibitors. For mouse experiments, we intravenously injected 13C-glucose into C57/B6 mice and analyzed the feces 1 hour after injection with a gas chromatography-quadrupole mass spectrometry. We could not detect 13C-glucose in the feces, possibly because gut microbiota metabolizes glucose in the intestine. However, the content of 13C in short-chain fatty acids (SCFAs) was increased by the injection of 13C-glucose, and metformin augmented the incorporation of 13C into SCFAs. These results suggest that glucose in the circulation transfers to the intestinal lumen at a considerable rate and is metabolized by microbiota, at least partly, into SCFAs and that metformin augments the processes. Disclosure Y.Hosokawa: None. K.Sakaguchi: Research Support; Sumitomo Dainippon Pharma Co., Ltd. J.Ito: None. K.Sugawara: None. Y.Morita: None. W.Ogawa: Advisory Panel; Abbott Japan Co., Ltd., Research Support; Boehringer Ingelheim Japan, Inc., Eli Lilly Japan K.K., Novo Nordisk, Teijin Pharma Limited, Kowa Company, Ltd., Sumitomo Dainippon Pharma Co., Ltd., Abbott Japan Co., Ltd., Speaker's Bureau; Sumitomo Dainippon Pharma Co., Ltd. Funding Japan Society for the Promotion of Science (21K08578, 22K18393)

Purpurascenines A-C, Azepino-Indole Alkaloids from Cortinarius purpurascens: Isolation, Biosynthesis, and Activity Studies on the 5-HT2A Receptor.

Three previously undescribed azepino-indole alkaloids, named purpurascenines A-C (1-3), together with the new-to-nature 7-hydroxytryptophan (4) as well as two known compounds, adenosine (5) and riboflavin (6), were isolated from fruiting bodies of Cortinarius purpurascens Fr. (Cortinariaceae). The structures of 1-3 were elucidated based on spectroscopic analyses and ECD calculations. Furthermore, the biosynthesis of purpurascenine A (1) was investigated by in vivo experiments using 13C-labeled sodium pyruvate, alanine, and sodium acetate incubated with fruiting bodies of C. purpurascens. The incorporation of 13C into 1 was analyzed using 1D NMR and HRESIMS methods. With [3-13C]-pyruvate, a dramatic enrichment of 13C was observed, and hence a biosynthetic route via a direct Pictet-Spengler reaction between α-keto acids and 7-hydroxytryptophan (4) is suggested for the biosynthesis of purpurascenines A-C (1-3). Compound 1 exhibits no antiproliferative or cytotoxic effects against human prostate (PC-3), colorectal (HCT-116), and breast (MCF-7) cancer cells. An in silico docking study confirmed the hypothesis that purpurascenine A (1) could bind to the 5-HT2A serotonin receptor's active site. A new functional 5-HT2A receptor activation assay showed no functional agonistic but some antagonistic effects of 1 against the 5-HT-dependent 5-HT2A activation and likely antagonistic effects on putative constitutive activity of the 5-HT2A receptor.

Distinct carbon incorporation from 13C-labelled rice straw into microbial amino sugars in soils applied with manure versus mineral fertilizer

Emerging evidences have emphasized the primacy of microorganisms as executor in soil organic C (SOC) formation. Yet, whether and how microbial transformation of crop residue-derived C into necromass is affected by fertilization regimes remains poorly understood. To address this, soils subjected to 3-year applications of chemical fertilizer (NPK) vs manure plus chemical fertilizer (NPKM) were incubated with or without addition of 13C-labelled rice straw for 60 days. The 13C incorporation into amino sugars (microbial necromass biomarker) was identified by using an amino sugar-based stable isotope probing approach. The straw-C were transformed more rapidly into total amino sugars (13C-ASs) in NPK-treated soil, while more pronounced 13C-ASs accumulation occurred in NPKM soil at later stage, indicating temporal microbial C uptake to generate necromass varied depending on fertilization regimes. Moreover, straw-derived fungal C was significantly higher in NPKM over NPK while bacterial counterpart reversed during the incubation. Our results reinforced changes in microbial communities and their C utilization capacities for efficient residue production in different fertilized soils. Additionally, NPKM-treated soil sustained a prolonged but likely more efficient utilization of straw-C with increasing time as evidenced by significantly higher percentage of straw-dervied C enrichment in 13C amino sugar pools by the end of incubation. These findings reveal that fertilization regimes can alter response strategies of microbial transformation of plant C into microbial-derived C. Together with differential contributions of these newly-formed microbial C to SOC pool, our work highlights the key role of microbial anabolisms in building up SOC following crop residue return to soils under different fertilization regimes.

Daylight-driven carbon exchange through a vertically structured microbial community.

Interactions between autotrophs and heterotrophs are central to carbon (C) exchange across trophic levels in essentially all ecosystems and metabolite exchange is a frequent mechanism for distributing C within spatially structured ecosystems. Yet, despite the importance of C exchange, the timescales at which fixed C is transferred in microbial communities is poorly understood. We employed a stable isotope tracer combined with spatially resolved isotope analysis to quantify photoautotrophic uptake of bicarbonate and track subsequent exchanges across a vertical depth gradient in a stratified microbial mat over a light-driven diel cycle. We observed that C mobility, both across the vertical strata and between taxa, was highest during periods of active photoautotrophy. Parallel experiments with 13C-labeled organic substrates (acetate and glucose) showed comparably less exchange of C within the mat. Metabolite analysis showed rapid incorporation of 13C into molecules that can both comprise a portion of the extracellular polymeric substances in the system and serve to transport C between photoautotrophs and heterotrophs. Stable isotope proteomic analysis revealed rapid C exchange between cyanobacterial and associated heterotrophic community members during the day with decreased exchange at night. We observed strong diel control on the spatial exchange of freshly fixed C within tightly interacting mat communities suggesting a rapid redistribution, both spatially and taxonomically, primarily during daylight periods.

Functional redundant soil fauna and microbial groups and processes were fairly resistant to drought in an agroecosystem

Climate change scenarios predict more frequent and intense drought periods for 2071 to 2100 for many regions of the world including Austria. Current and predicted lower precipitation scenarios were simulated at a lysimeter station containing a fertile and less fertile agricultural soil for 9 years. 13C and 15N-labeled green manure was added in year 8 with the aim to analyze how the predicted precipitation regime affects soil fauna and microbial groups and consequently nitrogen (N) and carbon (C) cycling. Among the investigated mesofauna (collembola and oribatida), the abundance and biodiversity of oribatida was significantly reduced by drought, possibly because they mainly represent K-strategist species with low mobility and consequently the need to adapt to long-term adverse environmental conditions. Microbial community composition and microbial biomass, investigated by phospholipid fatty acid (PLFA) analysis, was indistinguishable between the current and the predicted precipitation scenarios. Nonetheless, soil 13C-CO2 emissions and soil water 15N-NO3 data revealed decelerated mineralization of green manure under reduced precipitation in the first 2 weeks, but no effects were observed on soil C sequestration or on 13C incorporation into microbial PLFAs in the following 1.2 years. We found that over a 1-year time period, decomposition was rather driven by plant residue availability than water limitation of microorganisms in the investigated agroecosystem. In contrast, N2O emissions were significantly reduced under drought, and green manure derived 15N accumulated in the soil under drought, which might necessitate the adjustment of future fertilization regimes. The impacts of reduced precipitation and drought were less pronounced in the more fertile agricultural soil, due to its greater buffering capacity in terms of water storage and organic matter and nutrient availability.

Reduction in cardiolipin reduces expression of creatine transporter-1 and creatine transport in growing hCMEC/D3 human brain microvessel endothelial cells

The phospholipid cardiolipin (CL) regulates mitochondrial energy production. Endothelial cells of the blood-brain barrier (BBB) play a vital role in uptake of metabolites into the brain and are enriched in mitochondria. We examined how deficiency in BBB endothelial cell CL regulates the expression of selected drug and metabolite transporters and their function. Cardiolipin synthase-1 (hCLS1) was knocked down in a human brain microvessel endothelial cell line, hCMEC/D3, and CL levels and the mRNA expression of selected BBB drug and metabolite transporters examined. Mock transfected hCMEC/D3 cells served as controls. Incorporation of (14C)creatine and (14C)oleate into hCMEC/D3 cells was determined as a measure of solute metabolite transport. In addition, protein expression of the creatine transporter was determined. Knockdown of hCLS1 in hCMEC/D3 reduced CL and the mRNA expression of creatine transporter-1, p-glycoprotein and breast cancer resistance protein compared to controls. In contrast, mRNA expression of ATP binding cassette subfamily C members-1, -3, multidrug resistance-associated protein-4 variants 1, -2, and fatty acid transport protein-1 were unaltered. Although ATP production was unaltered by hCLS1 knockdown, incorporation of (14C)creatine into hCMEC/D3 cells was reduced compared to controls. The reduction in (14C)creatine incorporation was associated with a reduction in creatine transporter-1 protein expression. In contrast, incorporation of (14C)oleic acid into hCMEC/D3 cells and the mRNA expression of fatty acid transport protein-1 was unaltered by knockdown of hCLS1 compared to controls. Thus, knockdown of hCLS1 in hCMEC/D3, with a corresponding reduction in CL, results in alteration in expression of specific solute membrane transporters.

Long-term different fertilization regimes impact on the fate of root-derived C and microbial community structure in paddy soil

It is known that fertilization pattern could alter soil nutrient and organic matter status. However, it is still unclear how photosynthesized C is distributed in rice–rhizosphere soil-microbial system and its influencing factors due to fertilization pattern and subsequent changes in soil fertility. This study examined the allocation of rice photosynthetic C in rice–rhizosphere soil–microbial systems and the changes in microbial community under three long-term fertilizer regimes. Rice was grown in soils with 8-year history of no fertilizer, inorganic fertilizer (NPK) or NPK fertilizer plus cattle manure (NPK + CM), and was pulse–labelled with 13CO2 at rice tillering stage. The results showed that 13C incorporation in rice-rhizosphere soil system was the highest in the NPK + CM treatment, increased by 489% and 28.9% compared to unfertilized control and the NPK alone. In the >2 mm aggregate fraction, the 13C enrichment was higher in NPK + CM compared to no-fertilized control and NPK treatments. Total PLFA and PLFA groups contents in all aggregate size fractions were the greatest in the NPK + CM treatment. Partial least squares path modelling analysis revealed that microbial community in large macroaggregate (>2 mm) was significantly (P < 0.05) affected by root properties, while soil fertility had a significant (P < 0.01) impact on microbial community in microaggregate (<0.25 mm). The redundancy analysis showed that soil parameters except soil alkali-hydro nitrogen (AN) significantly impacted the soil microbial communities. Through canonical variation partitioning analysis, soil available K was the most important factor leading to variance (11.4%) in microbial community composition. On the other hand, soil pH significantly (P < 0.05) affected the distribution of 13C in microbe community. More than 20% of 13C was incorporated into G– and G+ bacteria across all treatments. Overall, it was observed the NPK fertilizer plus cattle manure treatment was most conducive to retain C from root exudates in the rice-rhizosphere soil-microbial system.

Nitrogen application influences the effect of bacteria on the belowground allocation of photosynthesized carbon under elevated CO2

Elevated carbon dioxide (eCO2) affects the translocation of photosynthesized carbon (C) from plants to soil, which is predominantly executed by soil microorganisms. However, the response strategy and mediation of the bacterial community on photosynthesized C allocation belowground remains elusive under eCO2 coupled with nitrogen (N) availability. Here, the spring wheat (Triticum aestivum L.) was continuously labeled with 13CO2 under ambient (aCO2, 350 ppm) or elevated (eCO2, 600 ppm) at three N application levels, and the incorporation of 13C into soil organic C was measured to indicate photosynthesized C accrual in soil. High-throughput MiSeq sequencing of 16S rRNA gene amplicons was used to explore soil bacterial response strategy. Compared with aCO2, eCO2 increased the relative abundance of Chloroflexi and Nitrospirae at the lowest N application level, while it increased that of Bacteroidetes at the highest N level. This shift of the response phyla from oligotrophic to copiotrophic species was closely associated with the increase in root biomass and its decomposability under eCO2 coupled with increasing N availability. The keystone taxa in the bacterial network might have strong ability to regulate such response of bacterial phyla to C and N availability. Specifically, the lower decomposability of the root-derived C caused by the intensive N deficiency under eCO2 stimulated oligotrophic bacteria, Saccharibacteria, Chloroflexi and Nitrospirae as keystone phyla, which could denote that the decomposition ability on recalcitrant substrate of bacterial phyla was enhanced before the activation of N transformation-related bacteria. Comparatively, sufficient N application under eCO2 facilitated copiotrophic bacteria as keystones for utilizing labile C derived from wheat roots, and thus the translocation efficiency of photosynthesized C belowground decreased. Our study sheds new light on the functional traits and mechanisms of the bacterial community in regulating C and N stoichiometry under eCO2.

Nitrogen addition decreases the soil cumulative priming effect and favours soil net carbon gains in Robinia pseudoacacia plantation soil

Carbon (C) and nutrient inputs regulate the soil carbon balance by affecting microbial growth and the priming effect (PE). However, we still lack a comprehensive understanding of the microbial mechanism of the soil carbon balance. To examine the regulatory effect of C or nutrient deficiency on microbial C turnover in forest systems of different ages, we collected soil from two Robinia pseudoacacia stands of different ages and conducted an incubation experiment using 13C-labelled glucose combined with nitrogen and/or phosphorus (N and/or P) addition. We determined 13C partitioning in CO2 and phospholipid fatty acids (PLFAs). Glucose and nutrient addition caused both positive and negative PEs during the 30-day incubation. The PE of the 15-year-old (15Y) stand changed from positive (0–15 days) to negative (15–30 days), while the PE of the 45-year-old (45Y) stand ranged from negative (0.5 days) to positive (after 1 day). Different microbial mechanisms play diverse roles during various decomposition stages. The 45Y stand led to a higher cumulative positive PE than the 15Y stand. For both stands, fungi assimilated a greater proportion of 13C-glucose in the glucose plus nitrogen (CN addition) treatment than in the glucose addition alone treatment, which alleviated the fungal N requirements and decreased the cumulative PE. However, the glucose plus phosphorous treatment (CP addition) had no significant influence on the cumulative PE. CN addition favoured high-yielding microbial strategists (Y-strategists) and glucose-derived SOC accumulation. This phenomenon was supported by the relatively high microbial carbon use efficiency (CUE), which increased the soil net C balance. In contrast, CP addition was conducive to resource-acquisition strategists (A-strategists), which increased soil organic carbon mineralization and decreased the soil net C balance. This was evidenced by higher 13C incorporation into the PLFAs of gram-positive bacteria and the positive PE for C and nutrient acquisition under CP addition. Consequently, CN addition decreased the cumulative PE and increased 13C-fungi and CUE, which favoured the soil net C gain. Our study highlights the importance of N fertilization for soil C sequestration in Robinia pseudoacacia plantations.

Mycelial nutrient transfer promotes bacterial co-metabolic organochlorine pesticide degradation in nutrient-deprived environments

Biotransformation of soil organochlorine pesticides (OCP) is often impeded by a lack of nutrients relevant for bacterial growth and/or co-metabolic OCP biotransformation. By providing space-filling mycelia, fungi promote contaminant biodegradation by facilitating bacterial dispersal and the mobilization and release of nutrients in the mycosphere. We here tested whether mycelial nutrient transfer from nutrient-rich to nutrient-deprived areas facilitates bacterial OCP degradation in a nutrient-deficient habitat. The legacy pesticide hexachlorocyclohexane (HCH), a non-HCH-degrading fungus (Fusarium equiseti K3), and a co-metabolically HCH-degrading bacterium (Sphingobium sp. S8) isolated from the same HCH-contaminated soil were used in spatially structured model ecosystems. Using 13C-labeled fungal biomass and protein-based stable isotope probing (protein-SIP), we traced the incorporation of 13C fungal metabolites into bacterial proteins while simultaneously determining the biotransformation of the HCH isomers. The relative isotope abundance (RIA, 7.1–14.2%), labeling ratio (LR, 0.13–0.35), and the shape of isotopic mass distribution profiles of bacterial peptides indicated the transfer of 13C-labeled fungal metabolites into bacterial proteins. Distinct 13C incorporation into the haloalkane dehalogenase (linB) and 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (LinC), as key enzymes in metabolic HCH degradation, underpin the role of mycelial nutrient transport and fungal-bacterial interactions for co-metabolic bacterial HCH degradation in heterogeneous habitats. Nutrient uptake from mycelia increased HCH removal by twofold as compared to bacterial monocultures. Fungal-bacterial interactions hence may play an important role in the co-metabolic biotransformation of OCP or recalcitrant micropollutants (MPs).

Low-dose ethanol increases aflatoxin production due to the adh1-dependent incorporation of ethanol into aflatoxin biosynthesis

Aflatoxins are toxic secondary metabolites produced by some aspergilli, including Aspergillus flavus. Recently, ethanol has attracted attention as an agent for the control of aflatoxin contamination. However, as aflatoxin biosynthesis utilizes acetyl coenzyme A, ethanol may be conversely exploited for aflatoxin production. Here, we demonstrated that not only the 13C of labeled ethanol, but also that of labeled 2-propanol, was incorporated into aflatoxin B1 and B2, and that ethanol and 2-propanol upregulated aflatoxin production at low concentrations (<1% and <0.6%, respectively). In the alcohol dehydrogenase gene adh1 deletion mutant, the 13C incorporation of labeled ethanol, but not labeled 2-propanol, into aflatoxin B1 and B2 was attenuated, indicating that the alcohols have different utilization pathways. Our results show that A.flavus utilizes ethanol and 2-propanol as carbon sources for aflatoxin biosynthesis and that adh1 indirectly controls aflatoxin production by balancing ethanol production and catabolism.

Influence of Sink Size on 15N and 13C Allocation during Different Phenological Phases of Spring Wheat Cultivars

The scientific objective of this study was to answer the question of whether sink limitation is also true for high quality wheat varieties. We examined the incorporation of 15N and 13C during phenological phases into vegetative parts and grains of Elite wheat Triso (E) and Quality wheat Naxos (A) when the spike is halved. Three splits of fertilizer were applied at EC 11, EC 30, EC 59, whereby 10% at EC 30 and EC 59 was 15N, and plants were also labelled with 13CO2. The application of only the third split as 15N, combined with spike-halving, resulted in a significantly higher 15N-content (+11%) of 0.486 mg 15N/g DM, compared to the control (0.437 mg15N/g DM). Labelling whole plants with 13CO2 at EC 59 resulted in a significantly higher 13C-content—40%—(0.223 mg 13C/g DM) of the grains of the control for Triso at the fully-ripe stage (EC 89), compared to Naxos (0.160 mg 13C/g DM). This superiority was reduced to 34%, and was also demonstrated by spike-halving (0.226 mg 13C/g DM, 0.169 mg 13C/g DM). Remobilization of 15N for control and spike-halving treatments were 68.2% and 61.1%, respectively. This clearly demonstrates that the reduction of the sink size by spike-halving leads to a 7% reduction in the remobilization of 15N from vegetative to reproductive tissues.

Seasonal and Spatial Variability of Phytoplankton Primary Production in a Shallow Temperate Coastal Lagoon (Ria Formosa, Portugal).

Coastal lagoons are among the most productive ecosystems in the world, and they provide a wide range of ecosystem services and resources. In the Ria Formosa (southern Portugal), phytoplankton production has rarely been addressed. The main goal of this study is thus to evaluate the variability of phytoplankton production and photosynthetic characteristics over the seasonal cycle and in different locations (landward, urban, intermediate, and seaward boundaries) of the Ria Formosa coastal lagoon, subjected to distinct natural and anthropogenic stressors. Primary production was evaluated using the 14C incorporation technique, and photosynthetic parameters were estimated by fitting photosynthesis-irradiance curves. Primary production showed significant seasonal variations, with higher values in the summer associated with lower euphotic depths, higher water temperatures, and higher nutrient concentrations. No spatial differences were found for primary production or photosynthetic parameters. Primary production values were lower than previous estimates, which reflects an improvement in water quality in the Ria Formosa, but values are higher than primary production estimates for other temperate coastal ecosystems, which reflects the highly productive nature of this coastal lagoon.

Labelling of eicosapentaenoic acid with stable isotope 13C in the marine bacterium Shewanella marinintestina

Eicosapentaenoic acid (EPA) is an essential omega-3 polyunsaturated fatty acid that plays a critical role in marine life. It is present in several marine animals, including fish, but the primary producers of EPA are phytoplankton and specific marine bacteria. Although most of the EPA present in marine animals come from phytoplankton, the bacterial input into the marine EPA food web is still unknown. The labelling of EPA within a bacterial strain could be a viable strategy to help revealing this contribution. In this work, Shewanella marinintestina IRL 567, a marine bacterium isolated from fish guts and known to produce EPA, was labelled with the stable isotope 13C at small (250-mL shake flask), bench (2.5-L shake flask), and pilot scale (50-L stirred tank bioreactor). Growing the bacterium with 13C-acetate in the culture medium demonstrated that EPA was de-novo synthesized utilizing acetate as precursor. 13C incorporation into the EPA molecule resulted in values as high as 95.5% of the synthesized EPA being labelled in small scale, 95.9% in bench scale and 91.5% in pilot scale. This simple method to label EPA proved to be effective and therefore it could be a valuable tool to follow the fate of bacterial EPA into higher trophic levels.

Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO2-derived 13C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of 13C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of 13C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in 13C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.

Characterization and DNA Stable-Isotope Probing of Methanotrophic Bioaerosols.

The growth and activity of bacteria have been extensively studied in nearly every environment on Earth, but there have been limited studies focusing on the air. Suspended bacteria (outside of water droplets) may stay in the atmosphere for time frames that could allow for growth on volatile compounds, including the potent greenhouse gas methane. We investigated the ability of aerosolized methanotrophic bacteria to grow on methane in the airborne state in rotating gas-phase bioreactors. The physical half-life of the aerial bacterium-sized particles was 3 days. To assess the potential for airborne growth, gas-phase bioreactors containing the aerosolized cultures were amended with 1,500 ppmv 13CH4 or 12CH4. Three of seven experiments demonstrated 13C incorporation into DNA, indicating growth in air. Bacteria associated with the genera Methylocystis and Methylocaldum were detected in 13C-DNA fractions, thus indicating that they were synthesizing new DNA, suggesting growth in air. We conclude that methanotrophs outside of water droplets in the air can potentially grow under certain conditions. Based on our data, humidity seems to be a major limitation to bacterial growth in air. Furthermore, low biomass levels can pose problems for detecting 13C-DNA synthesis in our experimental system. IMPORTANCE Currently, the cellular activities of bacteria in the airborne state outside of water droplets have not been heavily studied. Evidence suggests that these airborne bacteria produce ribosomes and metabolize gaseous compounds. Despite having a potentially important impact on atmospheric chemistry, the ability of bacteria in the air to metabolize substrates such as methane is not well understood. Demonstrating that bacteria in the air can metabolize and grow on substrates will expand knowledge about the potential activities and functions of the atmospheric microbiome. This study provides evidence for DNA synthesis and, ultimately, growth of airborne methanotrophs.

Size matters: biochemical mineralization and microbial incorporation of dicarboxylic acids in soil

The transformation and turnover time of medium- to long-chain dicarboxylic acids (DCA) in soil is regulated by microbial uptake and mineralization. However, the chain length of n-alkyl lipids may have a remarkable influence on its microbial utilization and mineralization and therefore on the formation of stable soil organic carbon from e.g. leave- needle- and root-derived organic matter during decomposition. To investigate their size dependent mineralization and microbial incorporation, four DCA of different chain lengths (12–30 carbon atoms), that were 13C labeled at each of their terminal carboxylic groups, were applied to the Ah horizon of a Fluvic Gleysol. Incorporation of 13C into CO2 and in distinct microbial groups classified by phospholipid fatty acid (PLFA) analysis was investigated. Mineralization of DCA and incorporation into PLFA decreased with increasing chain length, and the mineralization rate was highest during the first days of incubation. Half-life time of DCA carbon in soil increased from 7.6 days for C12 DCA to 86.6 days for C18 DCA and decreased again to 46.2 days for C22 DCA, whereas C30 DCA had the longest half-life time. Rapid and efficient uptake of C12 DCA as an intact molecule was observable. Gram-negative bacteria incorporated higher amounts of DCA-derived 13C compared to other microbial groups, especially compared to actinomycetes and fungi during the first phase of incubation. However, the incorporation of C12 DCA derived 13C into the PLFA of actinomycetes, and fungi increased steadily during the entire incubation time, suggesting that those groups take up the 13C label from necromass of bacteria that used the C12 DCA for formation of their lipids before.

In situ diversity of metabolism and carbon use efficiency among soil bacteria.

The central carbon (C) metabolic network harvests energy to power the cell and feed biosynthesis for growth. In pure cultures, bacteria use some but not all of the network's major pathways, such as glycolysis and pentose phosphate and Entner-Doudoroff pathways. However, how these pathways are used in microorganisms in intact soil communities is unknown. Here, we analyzed the incorporation of 13C from glucose isotopomers into phospholipid fatty acids. We showed that groups of Gram-positive and Gram-negative bacteria in an intact agricultural soil used different pathways to metabolize glucose. They also differed in C use efficiency (CUE), the efficiency with which a substrate is used for biosynthesis. Our results provide experimental evidence for diversity among microbes in the organization of their central carbon metabolic network and CUE under in situ conditions. These results have important implications for our understanding of how community composition affects soil C cycling and organic matter formation.

Ultrahigh resolution MS1/MS2-based reconstruction of metabolic networks in mammalian cells reveals changes for selenite and arsenite action

Metabolic networks are complex, intersecting, and composed of numerous enzyme-catalyzed biochemical reactions that transfer various molecular moieties among metabolites. Thus, robust reconstruction of metabolic networks requires metabolite moieties to be tracked, which cannot be readily achieved with mass spectrometry (MS) alone. We previously developed an Ion Chromatography-ultrahigh resolution-MS1/data independent-MS2 method to track the simultaneous incorporation of the heavy isotopes 13C and 15N into the moieties of purine/pyrimidine nucleotides in mammalian cells. Ultrahigh resolution-MS1 resolves and counts multiple tracer atoms in intact metabolites, while data independent-tandem MS (MS2) determines isotopic enrichment in their moieties without concern for the numerous mass isotopologue source ions to be fragmented. Together, they enabled rigorous MS-based reconstruction of metabolic networks at specific enzyme levels. We have expanded this approach to trace the labeled atom fate of [13C6]-glucose in 3D A549 spheroids in response to the anticancer agent selenite and that of [13C5,15N2]-glutamine in 2D BEAS-2B cells in response to arsenite transformation. We deduced altered activities of specific enzymes in the Krebs cycle, pentose phosphate pathway, gluconeogenesis, and UDP-GlcNAc synthesis pathways elicited by the stressors. These metabolic details help elucidate the resistance mechanism of 3D versus 2D A549 cultures to selenite and metabolic reprogramming that can mediate the transformation of BEAS-2B cells by arsenite.

Microcosm test for pesticide fate assessment in planted water filters: 13C,15N-labeled glyphosate as an example

Planted filters are often used to remove pesticides from runoff water. However, the detailed fate of pesticides in the planted filters still remains elusive. This hampers an accurate assessment of environmental risks of the pesticides related to their fate and thereby development of proper mitigation strategies. In addition, a test system for the chemical fate analysis including plants and in particular for planted filters is not well established yet. Therefore, we developed a microcosm test to simulate the fate of pesticide in planted filters, and applied 2-13C,15N-glyphosate as a model pesticide. The fate of 2-13C,15N-glyphosate in the planted microcosms over 31 day-incubation period was balanced and compared with that in the unplanted microcosms. The mass balance of 2-13C,15N-glyphosate turnover included 13C mineralization, degradation products, and the 13C and 15N incorporation into the rhizosphere microbial biomass and plants. We observed high removal of glyphosate (> 88%) from the water mainly due to adsorption on gravel in both microcosms. More glyphosate was degraded in the planted microcosms with 4.1% of 13C being mineralized, 1.5% of 13C and 3.8% of 15N being incorporated into microbial biomass. In the unplanted microcosms, 1.1% of 13C from 2-13C,15N-glyphosate was mineralized, and only 0.2% of 13C and 0.1% of 15N were assimilated into microbial biomass. The total recovery of 13C and 15N was 81% and 85% in planted microcosms, and 91% and 93% in unplanted counterparts, respectively. The microcosm test was thus proven to be feasible for mass balance assessments of the fate of non-volatile chemicals in planted filters. The results of such studies could help better manage and design planted filters for pesticide removal.