Revealing the Metabolic Flexibility of “ Candidatus Accumulibacter phosphatis” through Redox Cofactor Analysis and Metabolic Network Modeling

Abstract

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 non-availability 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. This allows re-thinking the ecological role of PHA accumulation as a fermentation product under anaerobic conditions and not just a stress response. 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, which demonstrated optima using different combinations of glycolysis, glyoxylate shunt or branches 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 NADH-related 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 storage 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 cofactor 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 expression of such metabolic potential.

Metabolic differentiation of co-occurring Accumulibacter clades revealed through genome-resolved metatranscriptomics

“…Accumulibacter’s anaerobic metabolism has also been shown to differ under varying substrate or stoichiometric conditions and possibly between the two Accumulibacter types. From recent simulations of ‘omics datasets paired with enzymatic assays, the routes of anaerobic metabolism for Accumulibacter may differ based on environmental conditions (83). Under high acetate conditions, polyphosphate accumulation arises through the glyoxylate shunt, whereas under increased glycogen concentrations, glycolysis is used in conjunction with the reductive branch of the TCA cycle (83).…”

“…From recent simulations of ‘omics datasets paired with enzymatic assays, the routes of anaerobic metabolism for Accumulibacter may differ based on environmental conditions (83). Under high acetate conditions, polyphosphate accumulation arises through the glyoxylate shunt, whereas under increased glycogen concentrations, glycolysis is used in conjunction with the reductive branch of the TCA cycle (83). Furthermore, Welles et al found that Type II Accumulibacter is able to switch to partial glycogen degradation more quickly than Type I, enabling Type II to fuel VFA uptake in polyphosphate-limited conditions (84, 85).…”

Production of nonulosonic acids in the extracellular polymeric substances of “CandidatusAccumulibacter phosphatis”

“…Accumulibacter” (approx. 95%, by proteomic investigations (Kleikamp et al 2020b) and by FISH staining (Guedes da Silva et al 2018)). Sialic acid-specific lectin staining displayed that sialic acids with α-2,3- and α-2,6-linkage to the sub-terminal monosaccharide were distributed widely on the cell surface of “ Ca .…”

“…Accumulibacter” has been identified as the dominant organisms responsible for EBPR (Zilles et al 2002). This microorganism has been well studied in the past decades: different genomic, proteomic, metabolic and modelling studies are available (Oehmen et al 2010; Barr et al 2016; Oyserman et al 2016; Guedes da Silva et al 2018; Guedes da Silva et al 2019; Rubio-Rincón et al 2019). However, most of the studies overlook the extracellular matrix.…”

“…Accumulibacter” was used (Guedes da Silva 2020) was used. The enrichment was maintained in a 1.5 L sequencing batch reactor (SBR), with slight modifications from the SBR-2 described in Guedes da Silva et al (2018). The COD-based acetate:propionate ratio in the feed was 65:35 gCOD/gCOD.…”