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Revealing metabolic flexibility of Candidatus Accumulibacter phosphatis through redox cofactor analysis and metabolic network modeling

Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategies. Candidatus Accumulibacter phosphatis, a polyphosphate accumulating organism (PAO) responsible for enhanced biological phosphorus removal (EBPR) from wastewater treatment systems, is prevalent in aerobic/anaerobic environments. While the overall metabolic traits of these bacteria are well described, the inexistence of isolates has led to controversial conclusions on the metabolic pathways used. Here, we experimentally determined the redox cofactor preference of different oxidoreductases in the central carbon metabolism of a highly enriched Ca. A. phosphatis culture. Remarkably, we observed that the acetoacetyl-CoA reductase engaged in polyhydroxyalkanoates (PHA) synthesis is NADH-preferring instead of the generally assumed NADPH dependency. Based on previously published meta-omics data and the results of enzymatic assays, a reduced central carbon metabolic network was constructed and used for simulating different metabolic operating modes. In particular, scenarios with different acetate-to-glycogen consumption ratios were simulated. For a high ratio (i.e. more acetate), a polyphosphate-based metabolism arises as optimal with a metabolic flux through the glyoxylate shunt. In case of a low acetate-to-glycogen ratio, glycolysis is used in combination with reductive branch of the TCA cycle. Thus, optimal metabolic flux strategies will depend on the environment (acetate uptake) and on intracellular storage compounds availability (polyphosphate/glycogen). This metabolic flexibility is enabled by the NADH-driven PHA synthesis. It allows for maintaining metabolic activity under varying environmental substrate conditions, with high carbon conservation and lower energetic costs compared to NADPH dependent PHA synthesis. Such (flexible) metabolic redox coupling can explain PAOs’ competitiveness under oxygen-fluctuating environments. IMPORTANCE Here we demonstrate how microbial metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of Ca. Accumulibacter phosphatis to act like glycogen accumulating organisms (GAO). These observations stem from slightly different experimental conditions and controversy only arises when one assumes metabolism can only operate in one single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining-omics data with functional assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional application of such metabolic potential.