The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.

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The large unexplored space of microbial biodiversity forms a well of untapped potential of enzymes and bioactive compounds. Genomic methods have made significant improvements over the past decades in their analysis of microbial communities. However, several aspects of nature can’t be described by a genome alone, such as expressed functions in the proteome and complex products such as bacterial glycosylation. Mass spectrometry can provide both general and targeted methods for the analysis of phenotypes, but is facing challenges in its limited throughput and the demanding data analysis of complex samples, which necessitates new methodologies. This thesis therefore aims to compare, improve and develop now methods for the analysis of microbial communities using mass spectrometry...@en

Metaproteomics has emerged as one of the most promising approaches for determining the composition and metabolic functions of complete microbial communities. Conventional metaproteomics approaches rely on the construction of protein sequence databases and efficient peptide-spectrum-matching algorithms, an approach that is intrinsically biased towards the content of the constructed sequence database. Here, we introduce a highly efficient, database-independent de novo metaproteomics approach and systematically evaluate its quantitative performance using synthetic and natural microbial communities comprising dozens of taxonomic families. Our work demonstrates that the de novo sequencing approach can vastly expand many metaproteomics applications by enabling rapid quantitative profiling and by capturing unsequenced community members that otherwise remain inaccessible for further interpretation. Kleikamp et al., describe a novel de novo metaproteomics pipeline (NovoBridge) that enables rapid community profiling without the need for constructing protein sequence databases.

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The enormous chemical diversity and strain variability of prokaryotic protein glycosylation makes their large-scale exploration exceptionally challenging. Therefore, despite the universal relevance of protein glycosylation across all domains of life, the understanding of their biological significance and the evolutionary forces shaping oligosaccharide structures remains highly limited. Here, we report on a newly established mass binning glycoproteomics approach that establishes the chemical identity of the carbohydrate components and performs untargeted exploration of prokaryotic oligosaccharides from large-scale proteomics data directly. We demonstrate our approach by exploring an enrichment culture of the globally relevant anaerobic ammonium-oxidizing bacterium Ca. Kuenenia stuttgartiensis. By doing so we resolve a remarkable array of oligosaccharides, which are produced by two seemingly unrelated biosynthetic routes, and which modify the same surface-layer protein simultaneously. More intriguingly, the investigated strain also accomplished modulation of highly specialized sugars, supposedly in response to its energy metabolism—the anaerobic oxidation of ammonium—which depends on the acquisition of substrates of opposite charges. Ultimately, we provide a systematic approach for the compositional exploration of prokaryotic protein glycosylation, and reveal a remarkable example for the evolution of complex oligosaccharides in bacteria.

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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.

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Structural extracellular polymeric substances (structural EPS) can form stable hydrogels and are considered to be responsible for the stability of biofilms and aerobic granular sludge. Structural EPS were extracted from aerobic granular sludge and characterized for their gel-forming capacity with different alkaline earth and transition metal ions. The structural EPS hydrogels were compared to alginate hydrogels. Alginate is a well characterized polymer which is able to form stiff hydrogels with multivalent ions. The stiffness of the obtained hydrogels was measured with dynamic mechanical analysis and quantified by the Young's modulus. Furthermore the stability of structural EPS hydrogels towards disintegration in the presence of ethylenediaminetetraacetic acid (EDTA) was evaluated at pH 4.5–10.5 and compared to that of alginate, polygalacturonic acid and κ-carrageenan. The stiffness of alginate hydrogels was multiple times higher than that of structural EPS. Alkaline earth metals resulted in stiffer alginate hydrogels than transition metals. For structural EPS this trend was opposite to alginate. Independent of the pH, polysaccharide hydrogels were quickly disintegrated when being exposed to EDTA. Structural EPS hydrogels demonstrated greater stability towards EDTA and were still intact after one month at pH 4.5–8.5. It is suggested that the gelling mechanism of structural EPS is not only related to metal ion complexation of the polymers, but to a combination of interactions of multiple functional groups present in structural EPS. This study helps to further understand and characterize structural EPS from aerobic granular sludge, and therewith understand its stability and that of biofilms in general.

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Anammox (anaerobic ammonium oxidation) bacteria are important for the nitrogen cycle in both natural environments and wastewater treatment plants. These bacteria have a strong tendency to grow in aggregates like biofilms and granular sludge. To understand the formation of anammox aggregates, it is required to unravel the composition of the extracellular polymeric substances (EPS), which are produced by the bacteria to develop into aggregates and granules. Here, we investigated anionic polymers in anammox granular sludge, focussing on sialic acids and sulfated glycosaminoglycans. Quantification assays and fluorescent stains indicated that sialic acids and sulfated glycosaminoglycans were present in the anammox EPS (1.6% equivalents of sialic acids and 2.4% equivalents of sulfated glycosaminoglycans). Additionally, the potential genes for the biosynthesis of sialic acids and sulfated glycosaminoglycans were analyzed in the anammox draft genomes. The finding of these components in anammox granular sludge and previously in other nonpathogenic bacteria pointed out that sialic acids and sulfated glycosaminoglycans are worth investigating in the context of a broader function in microbial communities and biofilm systems in general.

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Nonulosonic acids, commonly referred to as sialic acids, are a highly important group of nine-carbon sugars common to all domains of life. They all share biosynthetic and structural features, but otherwise display a remarkable chemical diversity. In humans, sialic acids cover all cells which makes them important for processes such as cellular protection, immunity and brain development. On the other hand, sialic acids and other nonulosonic acids have been associated with pathological processes including cancer and viral infections. In prokaryotes, nonulosonic acids are commonly associated with pathogens, which developed through molecular mimicry a strategy to circumvent the host's immune response. However, the remarkably large chemical diversity of prokaryotic nonulosonic acids challenges their discovery, and research on molecular characteristics essential for medical applications are often not feasible. Here, we demonstrate a novel, universal large-scale discovery approach that tackles the unmapped diversity of prokaryotic nonulosonic acids. Thereby, we utilize selective chemical labelling combined with a newly established mass spectrometric all-ion-reaction scanning approach to identify nonulosonic acids and other ulosonic acid-like sugars. In doing so, we provide a first molecular-level comparative study on the frequency and diversity across different phyla. We not only illustrate their surprisingly wide-spread occurrence in non-pathogenic species, but also provide evidence of potential higher carbon variants. Many biomedical studies rely on synthetic routes for sialic acids, which are highly demanding and often of low product yields. Our approach enables large-scale exploration for alternative sources of these highly important compounds.

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