Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18O‐Phosphoramidites**
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ABSTRACT:
SUBMITTER: Haas T
PROVIDER: S-EPMC10947094 | biostudies-literature | 2021 Nov
REPOSITORIES: biostudies-literature
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Project description:Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for 18 O-labelled phosphates is presented, based on a family of modified 18 O2 -phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18 O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18 O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry.
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Project description:The high growth rate of Ulva seaweeds makes it a potential algal biomass resource. In particular, Ulva meridionalis grows up to fourfold a day. Here, we demonstrated strong carbon fixation by U. meridionalis using 13C stable isotope labelling and traced the 13C flux through sugar metabolites with isotope-ratio mass spectrometry (IR-MS), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), 13C-nuclear magnetic resonance spectrometry (13C-NMR), and gas chromatography-mass spectrometry (GC-MS). U. meridionalis was first cultured in 13C-labelled enriched artificial seawater for 0-12 h, and the algae were collected every 4 h. U. meridionalis grew 1.8-fold (dry weight), and the 13C ratio reached 40% in 12 h, whereas 13C incorporation hardly occurred under darkness. At the beginning of the light period, 13C was incorporated into nucleic diphosphate (NDP) sugars in 4 h, and 13C labelled peaks were identified using FT-ICR-MS spectra. Using semiquantitative 13C-NMR measurements and GC-MS, 13C was detected in starch and matrix polysaccharides after the formation of NDP sugars. Moreover, the 14:10 light:dark regime resulted into 85% of 13C labelling was achieved after 72 h of cultivation. The rapid 13C uptake by U. meridionalis shows its strong carbon fixation capacity as a promising seaweed biomass feedstock.
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Project description:BackgroundWe aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled "heavy" and the unlabelled "light" SIP fractions were sequenced.ResultsAn increase of the 16S rRNA copy numbers in the "heavy" fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) were highly abundant after two days of incubation with antibiotics. Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. In comparison, only two ARGs from the unbinned-assembled light fractions were identified.ConclusionsThe results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.
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Project description:UnlabelledMicroorganisms grow under a remarkable range of extreme conditions. Environmental transcriptomic and proteomic studies have highlighted metabolic pathways active in extremophilic communities. However, metabolites directly linked to their physiology are less well defined because metabolomics methods lag behind other omics technologies due to a wide range of experimental complexities often associated with the environmental matrix. We identified key metabolites associated with acidophilic and metal-tolerant microorganisms using stable isotope labeling coupled with untargeted, high-resolution mass spectrometry. We observed >3,500 metabolic features in biofilms growing in pH ~0.9 acid mine drainage solutions containing millimolar concentrations of iron, sulfate, zinc, copper, and arsenic. Stable isotope labeling improved chemical formula prediction by >50% for larger metabolites (>250 atomic mass units), many of which were unrepresented in metabolic databases and may represent novel compounds. Taurine and hydroxyectoine were identified and likely provide protection from osmotic stress in the biofilms. Community genomic, transcriptomic, and proteomic data implicate fungi in taurine metabolism. Leptospirillum group II bacteria decrease production of ectoine and hydroxyectoine as biofilms mature, suggesting that biofilm structure provides some resistance to high metal and proton concentrations. The combination of taurine, ectoine, and hydroxyectoine may also constitute a sulfur, nitrogen, and carbon currency in the communities.ImportanceMicrobial communities are central to many critical global processes and yet remain enigmatic largely due to their complex and distributed metabolic interactions. Metabolomics has the possibility of providing mechanistic insights into the function and ecology of microbial communities. However, our limited knowledge of microbial metabolites, the difficulty of identifying metabolites from complex samples, and the inability to link metabolites directly to community members have proven to be major limitations in developing advances in systems interactions. Here, we show that combining stable-isotope-enabled metabolomics with genomics, transcriptomics, and proteomics can illuminate the ecology of microorganisms at the community scale.
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Project description:Investigating active participants in the fixation of dinitrogen gas is vital as N is often a limiting factor for primary production. Biological nitrogen fixation is performed by a diverse guild of bacteria and archaea (diazotrophs), which can be free-living or symbionts. Free-living diazotrophs are widely distributed in the environment, yet our knowledge about their identity and ecophysiology is still limited. A major challenge in investigating this guild is inferring activity from genetic data as this process is highly regulated. To address this challenge, we evaluated and improved several 15 N-based methods for detecting N2 fixation activity (with a focus on soil samples) and studying active diazotrophs. We compared the acetylene reduction assay and the 15 N2 tracer method and demonstrated that the latter is more sensitive in samples with low activity. Additionally, tracing 15 N into microbial RNA provides much higher sensitivity compared to bulk soil analysis. Active soil diazotrophs were identified with a 15 N-RNA-SIP approach optimized for environmental samples and benchmarked to 15 N-DNA-SIP. Lastly, we investigated the feasibility of using SIP-Raman microspectroscopy for detecting 15 N-labelled cells. Taken together, these tools allow identifying and investigating active free-living diazotrophs in a highly sensitive manner in diverse environments, from bulk to the single-cell level.
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Project description:[18 F]NS12137 (exo-3-[(6-[18 F]fluoro-2-pyridyl)oxy]8-azabicyclo[3.2.1]octane) is a highly selective norepinephrine transporter (NET) tracer. NETs are responsible for the reuptake of norepinephrine and dopamine and are linked to several neurodegenerative and neuropsychiatric disorders. The aim of this study was to develop a copper-mediated 18 F-fluorination method for the production of [18 F]NS12137 with straightforward synthesis conditions and high radiochemical yield and molar activity. [18 F]NS12137 was produced in two steps. Radiofluorination of [18 F]NS12137 was performed via a copper-mediated pathway starting with a stannane precursor and using [18 F]F- as the source of the fluorine-18 isotope. Deprotection was performed via acid hydrolysis. The radiofluorination reaction was nearly quantitative as was the deprotection based on HPLC analysis. The radiochemical yield of the synthesis was 15.1 ± 0.5%. Molar activity of [18 F]NS12137 was up to 300 GBq/μmol. The synthesis procedure is straightforward and can easily be automated and adapted for clinical production.
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Project description:Growing bacteria have a high concentration of ribosomes to ensure sufficient protein synthesis, which is necessary for genome replication and cellular division. To elucidate whether metabolic activity of soil microorganisms is coupled with growth, we investigated the relationship between rRNA and DNA synthesis in a soil bacterial community using quantitative stable isotope probing (qSIP) with H218O. Most soil bacterial taxa were metabolically active and grew, and there was no significant difference between the isotopic composition of DNA and RNA extracted from soil incubated with H218O. The positive correlation between 18O content of DNA and rRNA of taxa, with a slope statistically indistinguishable from 1 (slope = 0.96; 95% confidence interval [CI], 0.90 to 1.02), indicated that few taxa made new rRNA without synthesizing new DNA. There was no correlation between rRNA-to-DNA ratios obtained from sequencing libraries and the atom percent excess (APE) 18O values of DNA or rRNA, suggesting that the ratio of rRNA to DNA is a poor indicator of microbial growth or rRNA synthesis. Our results support the notion that metabolic activity is strongly coupled to cellular division and suggest that nondividing taxa do not dominate soil metabolic activity.IMPORTANCE Using quantitative stable isotope probing of microbial RNA and DNA with H218O, we show that most soil taxa are metabolically active and grow because their nucleic acids are significantly labeled with 18O. A majority of the populations that make new rRNA also grow, which argues against the common paradigm that most soil taxa are dormant. Additionally, our results indicate that relative sequence abundance-based RNA-to-DNA ratios, which are frequently used for identifying active microbial populations in the environment, underestimate the number of metabolically active taxa within soil microbial communities.
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Project description:Despite considerable experimental effort, the physiological mechanisms governing temperate tree species' water and carbon dynamics before the onset of the growing period remain poorly understood. We applied 2H-enriched water during winter dormancy to the soil of four potted European tree species. After 8 weeks of chilling, hydrogen isotopes in stem, twig and bud water were measured six times during 2 consecutive weeks of forcing conditions (Experiment 1). Additionally, we pulse-labelled above-ground plant tissues using 2H-enriched water vapour and 13C-enriched CO2 7 days after exposure to forcing conditions to trace atmospheric water and carbon uptake (Experiment 2). Experiment 1 revealed soil water incorporation into the above-ground organs of all species during the chilling phase and significant species-specific differences in water allocation during the forcing conditions, which we attributed to differences in structural traits. Experiment 2 illustrated water vapour incorporation into all above-ground tissue of all species. However, the incorporation of carbon was found for evergreen saplings only. Our results suggest that temperate trees take up and reallocate soil water and absorb atmospheric water to maintain sufficient above-ground tissue hydration during winter. Therefore, our findings provide new insights into the water allocation dynamics of temperate trees during early spring.
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Project description:Stable isotopes of hydrogen and oxygen (δ2H, δ18O and δ17O) can be used as natural tracers to improve our understanding of hydrological and meteorological processes. Studies of precipitation isotopes, especially 17O-excess observations, are extremely limited in the mid-latitudes. To fill this knowledge gap, we measured δ2H, δ18O and δ17O of event-based precipitation samples collected from Indianapolis, Indiana, USA over two years and investigated the influence of meteorological factors on precipitation isotope variations. The results showed that the daily temperature played a major role in controlling the isotope variations. Precipitation experienced kinetic fractionation associated with evaporation at the moisture source in the spring and summer and for rainfall, while snowfall, as well as precipitation in the fall and winter, were mainly affected by equilibrium fractionation. The 17O-excess of both rainfall and snowfall were not affected by local meteorological factors over the whole study period. At the seasonal scale, it was the case only for the spring. Therefore, 17O-excess of rainfall, snowfall and the spring precipitation could be considered as tracers of evaporative conditions at the moisture source. This study provides a unique precipitation isotope dataset for mid-latitudes and provides a more mechanistic understanding of precipitation formation mechanisms in this region.
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