Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus 'Kuenenia stuttgartiensis' using time-series ¹³C and ²H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO₂ fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO₂ followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor's side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

Fig. 1. Predicted central metabolism of K. stuttgartiensis based on genome annotation.

Black arrows represent reactions of the Wood–Ljungdahl pathway, gluconeogenesis, the TCA cycle, and the pentose phosphate pathway. Grey arrows indicate amino acid biosynthetic pathways. Pink metabolites represent reducing equivalents. Red metabolites indicate ATP equivalents. “?” indicates gene or pathway not identified in genome. A list of all reactions can be found in Supplementary Table 1. Confirmation of gene expression via proteomic analysis can be found in Supplementary Dataset 1.

Fig. 2. ¹³C-enrichment of selected metabolites during ¹³C-bicarbonate dynamic tracing experiments.

a Mass isotopomer distributions (MID) for selected metabolites illustrating the effects of potential substrate channelling or compartmentation of the Wood–Ljungdahl Pathway and PFOR. b ¹³C enrichment of metabolites associated with (left) initial CO₂ fixation reactions (Wood–Ljungdahl Pathway, pyruvate:ferredoxin oxidoreductase) and metabolites downstream of pyruvate; (centre) TCA cycle metabolites; (right) gluconeogenesis and pentose phosphate pathway metabolites. c Selected mass isotopomer distributions for metabolites of the TCA cycle, gluconeogenesis, and the pentose phosphate pathway. All measured metabolite MIDs represent the average of 3 independent biological replicate experiments. Metabolite MIDs and standard errors can be found in Supplementary Dataset 3.

Fig. 3. Elucidating TCA cycle of K. stuttgartiensis with ¹³C-formate.

a Proposed labelling of TCA cycle metabolites with ¹³C-formate. b ¹³C-enrichment of selected metabolites during isotope tracer experiments with ¹³C-formate. c Time-series mass isotopomer distributions of selected TCA cycle metabolites during isotope tracer experiments with ¹³C-formate. All measured metabolite MIDs represent the average of 3 independent biological replicate experiments. Metabolite MIDs and standard errors can be found in Supplementary Dataset 3. Reaction carbon atom transitions are provided in Supplementary Dataset 4.

Fig. 4. K. stuttgartiensis flux map generated by ¹³C INST-MFA.

K. stuttgartiensis flux map under anaerobic, continuous flow, ammonium and nitrite medium conditions determined by fitting metabolites labelled with ¹³C-formate tracers to a single, statistically acceptable isotopomer network model. Flux values represent the net flux through a given reaction ± standard error defined at 95% confidence. All fluxes are normalized to a net CO₂ uptake rate of q = 100 mmol-C (g DW)⁻¹ h⁻¹ (actual CO₂ uptake rate was 0.186 mmol-C (g DW)⁻¹ h⁻¹). All isotopomer network model reactions are provided in Supplementary Dataset 4. INST-MFA solutions are provided in Supplementary Dataset 5.

Fig. 5. ¹³C-acetate tracing reveals acetate is oxidized by TCA cycle and not reverse Wood–Ljungdahl pathway.

a Expected dynamic labeling patterns of the TCA cycle based on either Re- or Si-citrate synthase from [2-¹³C]acetate tracing. Numbers above carbon molecules indicate how many rounds of the TCA cycle have been completed. For simplicity, only new isotopomers produced at each round of the TCA cycle are shown. b ¹³C-enrichment of selected metabolites during isotope tracer experiments with [2-¹³C]acetate (red). c Time-series mass isotopomer distributions of selected TCA cycle metabolites during isotope tracer experiments with ¹³C-acetate. All measured metabolite MIDs represent the average of 2 independent biological replicate experiments. Metabolite MIDs and standard errors can be found in Supplementary Dataset 3. Reaction carbon atom transitions are provided in Supplementary Dataset 4.

Fig. 6. ²H-acetate tracing suggests acetate oxidation by low abundance organism via Si-citrate synthase.

a Proposed pathways and deuterium labelling for acetate oxidation via the reverse Wood–Ljungdahl pathway and TCA cycle. Black circles indicate carbons with heavy (²H) hydrogen, red circles indicate carbons with ²H hydrogen produced via Re-citrate synthase, blue circles indicate carbons with ²H hydrogen produced via Si-citrate synthase, grey circles indicate carbons with ¹H (unlabelled) hydrogen. b Time-series mass isotopomer distributions of selected TCA cycle metabolites during isotope tracer experiments with sodium acetate-d3 (CD₃COOH). All measured metabolite MIDs represent the average of 2 independent biological replicate experiments. Metabolite MIDs and standard errors can be found in Supplementary Dataset 3. Reaction hydrogen atom transitions are provided in Supplementary Dataset 4.