No 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.

23Environmental fluctuations in the availability of nutrients lead to intricate 24 metabolic strategies. Candidatus Accumulibacter phosphatis, a polyphosphate 25 accumulating organism (PAO) responsible for enhanced biological phosphorus 26 removal (EBPR) from wastewater treatment systems, is prevalent in 27 aerobic/anaerobic environments. While the overall metabolic traits of these 28 bacteria are well described, the inexistence of isolates has led to controversial 29 conclusions on the metabolic pathways used. 30Here, we experimentally determined the redox cofactor preference of 31 different oxidoreductases in the central carbon metabolism of a highly enriched 32 Ca. A. phosphatis culture. Remarkably, we observed that the acetoacetyl-CoA 33 reductase engaged in polyhydroxyalkanoates (PHA) synthesis is NADH-34 preferring instead of the generally assumed NADPH dependency. Based on 35 previously published meta-omics data and the results of enzymatic assays, a 36 reduced central carbon metabolic network was constructed and used for 37 simulating different metabolic operating modes. In particular, scenarios with 38 different acetate-to-glycogen consumption ratios were simulated. For a high 39 ratio (i.e. more acetate), a polyphosphate-based metabolism arises as optimal 40 with a metabolic flux through the glyoxylate shunt. In case of a low acetate-to-41 glycogen ratio, glycolysis is used in combination with reductive branch of the 42 TCA cycle. Thus, optimal metabolic flux strategies will depend on the 43 environment (acetate uptake) and on intracellular storage compounds 44 availability (polyphosphate/glycogen). 45 This metabolic flexibility is enabled by the NADH-driven PHA synthesis. It 46 allows for maintaining metabolic activity under varying environmental substrate 47 3 conditions, with high carbon conservation and lower energetic costs compared 48 to NADPH dependent PHA synthesis. Such (flexible) metabolic redox coupling 49 can explain PAOs' competitiveness under oxygen-fluctuating environments. 50 IMPORTANCE 51 Here we demonstrate how microbial metabolism can adjust to a wide range 52 of environmental conditions. Such flexibility generates a selective advantage 53 under fluctuating environmental conditions. It can also explain the different 54 observations reported in PAO literature, including the capacity of Ca. 55Accumulibacter phosphatis to act like glycogen accumulating organisms 56 (GAO). These observations stem from slightly different experimental conditions 57 and controversy only arises when one assumes metabolism can only operate 58 in one single mode. Furthermore, we also show how the study of metabolic 59 strategies is possible when combining -omics data with functional assays and 60 modeling. Genomic information can only provide the potential of a 61 microorganism. The environmental context and other complementary 62 approaches are still needed to study and predict the functional application of 63 such metabolic potential. 64

No abstract

Metaproteomics has emerged as one of the most promising approaches for determining the composition and metabolic functions of complete microbial communities. Conventional metaproteomics approaches however, rely on the construction of protein sequence databases and efficient peptide-spectrum matching algorithms. Thereby, very large sequence databases impact on computational efforts and sensitivity. More recently, advanced de novo sequencing strategies - which annotate peptide sequences without the requirement for a database - have become (again) increasingly proposed for proteomics applications. Such approaches would vastly expand many metaproteomics applications by enabling rapid community profiling and by capturing unsequenced community members, which otherwise remain inaccessible for further interpretation. Nevertheless, because of the lack of efficient pipelines and validation procedures, those strategies have only rarely been employed for community proteomics. Here we report on a newly established de novo metaproteomics pipeline which was evaluated for its quantitative performance using synthetic and natural communities. Additionally, we introduce a novel validation strategy and investigate the actual content of community members within community proteomics data.