Flux balance analysis of the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC19718 unravels specific metabolic activities while degrading toxic compounds

2.5.1 Co-activation of the pentose phosphate pathway, CBB cycle, and glycolysis.

Ne can grow using organic and inorganic carbon sources. To test changes in flux distributions, we used experimentally observed growth rates of 0.03 1/h [33] and 0.1 1/h [29] as constraints to determine HCO3- uptake rates between 1.43 mmol/gDW/h and 4.35 mmol/gDW/h (Fig 2A).

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Fig 2.

Map of the metabolic flux distributions predicted under chemolithoorganotrophic and chemolithotrophic conditions.

The map shows the changes in the flux distributions under four different growth conditions. Ammonium is present under all conditions but changing the carbon source (fructose, pyruvate, and HCO3-). (A) Chemolithoorganotrophy metabolism. The fructose uptake rate was constrained to 0.746 mmol/gDW/h, and the ammonium uptake rate was 0.5mmol/gDW/h. (B) Chemolithotrophy metabolism. HCO3- uptake rates were constrained to 4.35 mmol/gDW/h for high and 1.43 mmol/gDW/h for low. Abbreviations: HCO3E, carbonic anhydrase; PYK, pyruvate kinase; PPS, phosphoenolpyruvate synthasePGK, phosphoglycerate kinase; GAPD, glyceraldehyde 3-phosphate dehydrogenase; PFK, phosphofructokinase; PGI, glucose 6-phosphate isomerase; G6PDH2r, glucose 6-phosphate dehydrogenase; PGL, 6-phosphogluconolactonase; GND, phosphogluconate dehydrogenase; RPI, ribose-5-phosphate isomerase; RPE, ribulose 5-phosphate 3-epimerase; TKT, transketolase; TPI, triose-phosphate isomerase; PRUK, phosphoribulokinase; RBPC, ribulose 1,5-bisphosphate carboxylase-oxygenase; PDH, pyruvate dehydrogenase; CS, citrate synthase; MDH, malate dehydrogenase; SUCDi, succinate dehydrogenase; SUCOAS, succinyl-CoA synthetase; AKGDH, 2-oxoglutarate dehydrogenase; ICDHyr, isocitrate dehydrogenase; ORNTAC, ornithine transacetylase; G6P, D-Glucose 6-phosphate; F6P, D-Fructose 6-phosphate; FDP, D-Fructose 1,6-bisphosphate; G3P, Glyceraldehyde 3-phosphate; 13DPG, 3-Phospho-D-glyceroyl phosphate; 3PG, 3-Phospho-D-glycerate; 2PG, D-Glycerate 2-phosphate; PEP, Phosphoenolpyruvate; Pyr, Pyruvate; AcCoA, Acetyl-CoA; Fru, D-Fructose; Cit, citrate; Acon, Aconitate; iCit, Isocitrate; AKG, 2-Oxoglutarate; SucCoA, Succinyl-CoA; Suc, Succinate; Fum, Fumarate; Mal, L-Malate; OAA,Oxaloacetate; ArgSuc, L-Argininosuccinate; L-Citr, L-Citrulline; Orn, Ornithine; AcOrn, Acetylornithine; AcGlu, Acetyl-L-glutamate; AcG5P, Acetyl-L-glutamate 5-phosphate; AcG5SA, Acetyl-L-glutamate 5-semialdehyde; L-Glu, L-Glutamate; RuBP, D-Ribose 1,5-bisphosphate; Ru5P, D-Ribulose 5-phosphate; Xu5P, D-Xylulose 5-phosphate; DHAP, Dihydroxyacetone phosphate; E4P, Erythrose 4-phosphate dehydrogenase; S7P, Sedoheptulose 7-phosphate; S17BP, Sedoheptulose 1,7-bisphosphate; R5P, Ribose 5-phosphate; 6PGC, 6-Phospho-D-gluconate; 6PGL, 6-phospho-D-glucono-1,5-lactone.

https://doi.org/10.1371/journal.pcbi.1009828.g002

Constraints for the organic carbon source fructose were set at high concentrations (0.746 mmol/gDW/h). Calculations regarding high fructose uptake rate are explained in section 2.6. The same consumption rate was established for pyruvate to ensure a high concentration of this metabolite.

When HCO3- are present in the culture media, the CBB cycle enzymes are activated. Nevertheless, the CBB cycle is incomplete because of the lack of the enzyme NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPD) [10]. This enzyme is replaced by the reversible glycolytic isoenzyme NADH-dependent GAPD in Ne metabolism [10] (Fig 2B). In other organisms, e.g., plants such as barley seedlings, spinach, pea leaves, etc., different genes encode for the glycolytic/CBB cycle enzyme phosphoglycerate kinase (PGK) [36–38]. But, Ne has only one form of PGK enzyme, which is used by the glycolytic pathway and the CCB cycle [9,29]. PGK is encoded by the gene ALW85_RS01720. The particularities of PGK and GAPD enzymes allow for the coupling of glycolysis and CBB cycle pathways, as shown in Fig 2.

In plants, the CBB cycle enzyme GAPD catalyzes the formation of glyceraldehyde-3-phosphate using NADPH as the electron donor [39]. However, Ne GAPD uses NAD/NADH as coenzymes rather than NADP/NADPH. The preference of GAPD towards NAD/NADH can be potentially associated with the differential activity of NAD and NADH in the respiratory chain (Fig 1D). Under heterotrophy, the NADH is oxidized in the electron transport chain by the NADH dehydrogenase (NADH16pp) enzyme to generate ATP. Nonetheless, when Ne is growing chemolithotrophically, NADH16pp catalyzes the reverse reaction, as shown in Fig 1D. Thus, NAD+ is reduced to NADH using ubiquinol as the electron donor. Model simulations predicted that NADH16pp and GAPD were the reactions with the highest production and consumption fluxes of NADH. This prediction suggest that the enzyme NADH16pp to produce the NADH needed by GAPD.

It has been shown that the CBB cycle is not activated under high fructose concentrations (Fig 2A). Fructose is transported into the cytosol by a phosphoenolpyruvate translocation group that forms pyruvate and fructose-6-phosphate (F6P). Then, pyruvate is used by the TCA cycle, while F6P is metabolized by glycolysis. Model simulations suggested that GAPD and NADH16pp fluxes are reversed when fructose is the carbon source. Thus, while GAPD is the largest consumer of NAD+, NADH16pp is the largest producer.

However, when pyruvate serves as the sole carbon source, GAPD produces NAD+ (Fig 2A). NAD+ production by GAPD occurs because part of the pyruvate flux that enters the organism goes to the gluconeogenesis pathway to produce G3P and F6P. These two metabolites are needed to synthesize ribulose 5-phosphate (Ru5P), a nucleotide precursor. Interestingly, NADH16pp also generates NAD+ but not NADH as during chemolithotrophic growth. A considerable amount of NAD+ is needed by the pyruvate dehydrogenase. This reaction connects the pyruvate with the TCA cycle.

PPP has an oxidative and reductive phase. The oxidative pathway (oPPP) regenerates NADPH (anabolic) that is used in the biosynthesis of the lipid. The reductive pathway (rPPP) produces glycolytic intermediaries (catabolic). Ne shares enzymes between the CBB cycle and the rPPP pathway, such as transketolase 1 (TKT1), ribulose 5-phosphate 3-epimerase (RPE), ribose-5-phosphate isomerase (RPI), transketolase 2 (TKT2), and transaldolase (TALA) (Fig 2). Under chemolithotrophy metabolism, the whole CBB/Glycolysis/rPPP superpathway was predicted to be active (except TALA) to synthesize F6P for D-ribose 1,5-biphosphate regeneration (Fig 2B). The predictions determined that the regeneration of NADPH occurs through NAD(P)+ transhydrogenase and not by oPPP. Since there is a high production rate of NADH, Ne uses this excess to synthesize NADPH.

When fructose is the carbon source, the Glycolysis/rPPP/oPPP superpathway is activated. The CBB cycle does not need to be activated since there is no presence of HCO3- in the medium. The simulation predicted that oPPP is the greatest significant generator of NADPH. The Ru5P formed is used by rPPP to produce G3P, which is utilized by glycolysis. Nonetheless, when pyruvate is in the medium, some CBB cycle enzymes are activated to produce Ru5P (Fig 2A).