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Investigating the Chemolithoautotrophic and Formate Metabolism of Nitrospira moscoviensis by Constraint-Based Metabolic Modeling and ¹³C-Tracer Analysis.

ABSTRACT: Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used flux balance analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and [¹³C]bicarbonate and [¹³C]formate tracer experiments coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings corroborate that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO₂ fixation, and we also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO₂ followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira's in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. IMPORTANCE Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and ¹³C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite-oxidizing bacteria.

SUBMITTER: Lawson CE

PROVIDER: S-EPMC8407350 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background13C tracer analysis is increasingly used to monitor cellular metabolism in vivo and in intact cells, but data interpretation is still the key element to unveil the complexity of metabolic activities. The distinct 13C labeling patterns (e.g., M + 1 species in vivo but not in vitro) of metabolites from [U-13C]-glucose or [U-13C]-glutamine tracing in vivo and in vitro have been previously reported by multiple groups. However, the reason for the difference in the M + 1 species between in vivo and in vitro experiments remains poorly understood.MethodsWe have performed [U-13C]-glucose and [U-13C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). 13C enrichment of metabolites in cultured cells and tissues was determined by LC coupled with high-resolution mass spectrometry (LC-HRMS). All p-values are obtained from the Student's t-test two-tailed using GraphPad Prism 8 unless otherwise noted.ResultsWe observed distinct enrichment patterns of tricarboxylic acid cycle intermediates in vivo and in vitro. As expected, citrate M + 2 or M + 4 was the dominant mass isotopologue in vitro. However, citrate M + 1 was unexpectedly the dominant isotopologue in mice receiving [U-13C]-glucose or [U-13C]-glutamine infusion, but not in cultured cells. Our results are consistent with a model where the difference in M + 1 species is due to the different sources of CO2 in vivo and in vitro, which was largely overlooked in the past. In addition, a time course study shows the generation of high abundance citrate M + 1 in plasma of mice as early as few minutes after [U-13C]-glucose infusion.ConclusionsAltogether, our results show that recycling of endogenous CO2 is substantial in vivo. The production and recycling of 13CO2 from the decarboxylation of [U-13C]-glucose or [U-13C]-glutamine is negligible in vitro partially due to dilution by the exogenous HCO3-/CO2 source, but in vivo incorporation of endogenous 13CO2 into M + 1 metabolites is substantial and should be considered. These findings provide a new paradigm to understand carbon atom transformations in vivo and should be taken into account when developing mathematical models to better reflect carbon flux.

Project description:IntroductionNaïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells.MethodsHere we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions.ResultsThe quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate.DiscussionOverall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells.

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Investigating the Chemolithoautotrophic and Formate Metabolism of Nitrospira moscoviensis by Constraint-Based Metabolic Modeling and ¹³C-Tracer Analysis.

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Investigating the Chemolithoautotrophic and Formate Metabolism of Nitrospira moscoviensis by Constraint-Based Metabolic Modeling and 13C-Tracer Analysis.

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OmicsDI is part of the ELIXIR infrastructure

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Investigating the Chemolithoautotrophic and Formate Metabolism of Nitrospira moscoviensis by Constraint-Based Metabolic Modeling and ¹³C-Tracer Analysis.