van Loosdrecht, Mark C.M.

AbstractPseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that “Candidatus Accumulibacter” can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a “Ca. Accumulibacter” enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of “Ca. Accumulibacter” contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with “Ca. Accumulibcater” and used size-exclusion chromatography to separate them into different molecular weight fractions. This separation resulted in two high molecular weight (> 5,500 kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in “Ca. Accumulibacter” are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This suggest that they can serve as source for sepsis treatment drugs, although further purification needs to be evaluated.Graphical abstractHighlightsNulOs in “Ca. Accumulibacter” are likely located in high molecular weight polysaccharides.Size exclusion chromatography allows to obtain high molecular weight polysaccharide-rich fractions enriched with NulOs.EPS and the NulOs-rich fractions can serve as source for sepsis treatment drugs.

Abstract Nonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as an important compound in the extracellular matrix of virtually all microbial life and in “Candidatus Accumulibacter phosphatis”, a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of “Ca. Accumulibacter” to produce different types of NulOs. Proteomic analysis showed the ability of “Ca. Accumulibacter” to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of “Ca. Accumulibacter” in particular, and biofilms in general. Key Points •“Ca. Accumulibacter” has the potential to produce a range of nonulosonic acids. •Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids. •The role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

AbstractNonulosonic acids (NulOs) are a family of acidic carbohydrates with a nine-carbon backbone, which include different related structures, such as sialic acids. They have mainly been studied for their relevance in animal cells and pathogenic bacteria. Recently, sialic acids have been discovered as important compound in the extracellular matrix of virtually all microbial life and in “CandidatusAccumulibacter phosphatis”, a well-studied polyphosphate-accumulating organism, in particular. Here, bioaggregates highly enriched with these bacteria (approx. 95% based on proteomic data) were used to study the production of NulOs in an enrichment of this microorganism. Fluorescence lectin-binding analysis, enzymatic quantification, and mass spectrometry were used to analyze the different NulOs present, showing a wide distribution and variety of these carbohydrates, such as sialic acids and bacterial NulOs, in the bioaggregates. Phylogenetic analysis confirmed the potential of “Ca. Accumulibacter” to produce different types of NulOs. Proteomic analysis showed the ability of “Ca. Accumulibacter” to reutilize and reincorporate these carbohydrates. This investigation points out the importance of diverse NulOs in non-pathogenic bacteria, which are normally overlooked. Sialic acids and other NulOs should be further investigated for their role in the ecology of “Ca. Accumulibacter” in particular, and biofilms in general.Key Points“Ca.Accumulibacter” has the potential to produce a range of nonulosonic acids.Mass spectrometry and lectin binding can reveal the presence and location of nonulosonic acids.Role of nonulosonic acid in non-pathogenic bacteria needs to be studied in detail.

ABSTRACTEnvironmental fluctuations in the availability of nutrients lead to intricate metabolic strategies.CandidatusAccumulibacter 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 enrichedCa. 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.IMPORTANCEHere 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 ofCa. 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.