Global Isotope Metabolomics Reveals Adaptive Strategies for Nitrogen Assimilation

Abstract

Nitrogen cycling is a microbial metabolic process essential for global ecological/agricultural balance. To investigate the link between the well-established ammonium and the alternative nitrate assimilation metabolic pathways, global isotope metabolomics was employed to examine three nitrate reducing bacteria using 15NO3 as a nitrogen source. In contrast to a control (Pseudomonas stutzeri RCH2), the results show that two of the isolates from Oak Ridge, Tennessee (Pseudomonas N2A2 and N2E2) utilize nitrate and ammonia for assimilation concurrently with differential labeling observed across multiple classes of metabolites including amino acids and nucleotides. The data reveal that the N2A2 and N2E2 strains conserve nitrogen-containing metabolites, indicating that the nitrate assimilation pathway is a conservation mechanism for the assimilation of nitrogen. Co-utilization of nitrate and ammonia is likely an adaption to manage higher levels of nitrite since the denitrification pathways utilized by the N2A2 and N2E2 strains from the Oak Ridge site are predisposed to the accumulation of the toxic nitrite. The use of global isotope metabolomics allowed for this adaptive strategy to be investigated, which would otherwise not have been possible to decipher.

METLIN: A Technology Platform for Identifying Knowns and Unknowns

“…40 Although isoMETLIN has facilitated untargeted global isotope-tracer experiments, 26 the limited number of commercially available stable isotope-labeled molecules makes this approach insufficient for the absolute quantitation of many compounds. 5 To address this limitation, we have developed an approach to add metabolites from uniformly 13C-labeled microorganism extracts.…”

Defining and Evaluating Microbial Contributions to Metabolite Variation in Microbiome-Metabolome Association Studies

“…For example, it is possible that the predictive value of correlation analysis, as well as of other analytical methods, in this system differs substantially from that in true biological systems. Our rationale for using an in silico framework is discussed above, but we hope that future evaluation analyses will take advantage of ongoing technology developments in mass spectrometry and stable isotope probing to define key microbial contributors based on experimental, quantitative, species-specific community flux data (75–77). Such evaluations can also make use of data sets comparing community microbiome-metabolome data with in vitro monoculture or monocolonization data (39, 43, 44).…”

Mechanism Across Scales: A Holistic Modeling Framework Integrating Laboratory and Field Studies for Microbial Ecology

“…Respiration by-product exometabolites ( Kosina et al, 2016 , 2018 ) from sulfate-reducing bacteria stimulated DNR to ammonia, while inhibiting other DNR enzymes ( Otwell et al, 2021 ). With the use of isolates under nitrate-reducing conditions, high-throughput gene fitness assays ( Vaccaro et al, 2016 ), global stable isotope metabolomics profiling ( Kurczy et al, 2016 ), and assessing substrate and cofactor requirements of key DNR enzymes ( Vuono et al, 2019 ; Carlson et al, 2020 ) indicated highly selective controls on DNR pathway usage. Bioavailable molybdenum above a certain concentration is essential to support DNR ( Thorgersen et al, 2015 ; Ge et al, 2019 ) and regulated through controls on molybdate transporters ( Rajeev et al, 2019 ).…”