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J.M. van Ede
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Beneath the Sweet Surface
A Deep Dive into Microbial Glycomolecule Precursors and Their Enzymes
The surface of microorganisms is often decorated with a wide variety of glycans and glycoconjugates. These biomolecules play key roles in structural support, cellular communication, immune recognition, and host–pathogen interactions. Despite their importance, glycan analysis still lags far behind that of DNA and proteins. This is mainly because glycans are not directly encoded in the genome but arise from the combined action of glycosyltransferases and glycosidases. Their characterization is further complicated by extensive structural diversity, as glycans consist of a large and expanding repertoire of monosaccharides. In addition, restricted access to these building blocks hinders functional studies and the exploitation of glycans as antimicrobial targets. This thesis aims to advance the discovery of microbial metabolites, glycomolecule precursors, and carbohydrate-active enzymes using mass spectrometry and bioinformatic approaches.
Chapter 2 reviews advanced mass spectrometry-based methods for the identification and quantification of monosaccharides and their nucleotide-activated forms. Key analytical challenges are highlighted, including the differentiation of stereoisomers and the limited availability of reference standards. Complementary strategies, such as high-performance separation techniques, advanced fragmentation methods, and isotope tracing, are discussed for improved structural characterization and pathway analysis.
Building on these analytical foundations, Chapter 3 presents SugarBase, a mass spectrometry and bioinformatic pipeline for untargeted exploration of microbial nucleotide sugar networks. SugarBase combines narrow-window DIA fragmentation with a theoretical sugar composition database for parent ion annotation. The platform revealed species-specific nucleotide sugar profiles, including distinct nonulosonic acid profiles in Campylobacter jejuni strains and previously unannotated compounds across diverse microbes, including a higher-carbon ulosonic acid in Magnetospirillum.
Extending this work to complex microbial systems, Chapter 4 includes a collaborative study adapting the Chapter 3 methodology into a targeted metabolomics workflow to quantify nucleotides in granular biofilms involved in enhanced biological phosphorus removal. Using fast quenching, boiling-water extraction, and high-resolution mass spectrometry with ¹³C-labeled internal standards, the approach enabled measurement of adenylate and uridylate energy charge dynamics. Applied to a lab-scale system, it revealed energy pool fluctuations during acetate uptake and polyphosphate degradation, demonstrating that energy imbalances can underlie metabolic switching in biofilms.
In Chapter 5, we demonstrate that microbial metabolite extracts can serve as effective and readily accessible substrate sources for glycosyltransferase assays, without further purification. Using a Campylobacter jejuni extract containing diverse nonulosonic acid derivatives, we show substrate promiscuity of the previously identified pseudaminyltransferase from Acinetobacter baumannii, currently the only confirmed enzyme of its kind. These findings support the production of glycans and glycoconjugates relevant for antimicrobial and vaccine development.
While glycans and glycoconjugates are central to many biological processes, carbohydrate-active enzymes are of similar interest due to their potential to replace harsh chemical processes and their ability to enable new products. A key challenge remains the targeted identification of enzymes with desired activities and characteristics. In Chapter 6, we combine enrichment cultures with metagenomics and metaproteomics to discover glycoside hydrolases. This strategy enables functional enrichment under customized conditions and yields a focused set of enzyme candidates. One of the candidates was subsequently confirmed as a pullulan-degrading enzyme using a mass spectrometry-based activity assay.
Finally, Chapter 7 summarizes some of the main findings, discusses main challenges and outlines future research directions.
...
Chapter 2 reviews advanced mass spectrometry-based methods for the identification and quantification of monosaccharides and their nucleotide-activated forms. Key analytical challenges are highlighted, including the differentiation of stereoisomers and the limited availability of reference standards. Complementary strategies, such as high-performance separation techniques, advanced fragmentation methods, and isotope tracing, are discussed for improved structural characterization and pathway analysis.
Building on these analytical foundations, Chapter 3 presents SugarBase, a mass spectrometry and bioinformatic pipeline for untargeted exploration of microbial nucleotide sugar networks. SugarBase combines narrow-window DIA fragmentation with a theoretical sugar composition database for parent ion annotation. The platform revealed species-specific nucleotide sugar profiles, including distinct nonulosonic acid profiles in Campylobacter jejuni strains and previously unannotated compounds across diverse microbes, including a higher-carbon ulosonic acid in Magnetospirillum.
Extending this work to complex microbial systems, Chapter 4 includes a collaborative study adapting the Chapter 3 methodology into a targeted metabolomics workflow to quantify nucleotides in granular biofilms involved in enhanced biological phosphorus removal. Using fast quenching, boiling-water extraction, and high-resolution mass spectrometry with ¹³C-labeled internal standards, the approach enabled measurement of adenylate and uridylate energy charge dynamics. Applied to a lab-scale system, it revealed energy pool fluctuations during acetate uptake and polyphosphate degradation, demonstrating that energy imbalances can underlie metabolic switching in biofilms.
In Chapter 5, we demonstrate that microbial metabolite extracts can serve as effective and readily accessible substrate sources for glycosyltransferase assays, without further purification. Using a Campylobacter jejuni extract containing diverse nonulosonic acid derivatives, we show substrate promiscuity of the previously identified pseudaminyltransferase from Acinetobacter baumannii, currently the only confirmed enzyme of its kind. These findings support the production of glycans and glycoconjugates relevant for antimicrobial and vaccine development.
While glycans and glycoconjugates are central to many biological processes, carbohydrate-active enzymes are of similar interest due to their potential to replace harsh chemical processes and their ability to enable new products. A key challenge remains the targeted identification of enzymes with desired activities and characteristics. In Chapter 6, we combine enrichment cultures with metagenomics and metaproteomics to discover glycoside hydrolases. This strategy enables functional enrichment under customized conditions and yields a focused set of enzyme candidates. One of the candidates was subsequently confirmed as a pullulan-degrading enzyme using a mass spectrometry-based activity assay.
Finally, Chapter 7 summarizes some of the main findings, discusses main challenges and outlines future research directions.
...
The surface of microorganisms is often decorated with a wide variety of glycans and glycoconjugates. These biomolecules play key roles in structural support, cellular communication, immune recognition, and host–pathogen interactions. Despite their importance, glycan analysis still lags far behind that of DNA and proteins. This is mainly because glycans are not directly encoded in the genome but arise from the combined action of glycosyltransferases and glycosidases. Their characterization is further complicated by extensive structural diversity, as glycans consist of a large and expanding repertoire of monosaccharides. In addition, restricted access to these building blocks hinders functional studies and the exploitation of glycans as antimicrobial targets. This thesis aims to advance the discovery of microbial metabolites, glycomolecule precursors, and carbohydrate-active enzymes using mass spectrometry and bioinformatic approaches.
Chapter 2 reviews advanced mass spectrometry-based methods for the identification and quantification of monosaccharides and their nucleotide-activated forms. Key analytical challenges are highlighted, including the differentiation of stereoisomers and the limited availability of reference standards. Complementary strategies, such as high-performance separation techniques, advanced fragmentation methods, and isotope tracing, are discussed for improved structural characterization and pathway analysis.
Building on these analytical foundations, Chapter 3 presents SugarBase, a mass spectrometry and bioinformatic pipeline for untargeted exploration of microbial nucleotide sugar networks. SugarBase combines narrow-window DIA fragmentation with a theoretical sugar composition database for parent ion annotation. The platform revealed species-specific nucleotide sugar profiles, including distinct nonulosonic acid profiles in Campylobacter jejuni strains and previously unannotated compounds across diverse microbes, including a higher-carbon ulosonic acid in Magnetospirillum.
Extending this work to complex microbial systems, Chapter 4 includes a collaborative study adapting the Chapter 3 methodology into a targeted metabolomics workflow to quantify nucleotides in granular biofilms involved in enhanced biological phosphorus removal. Using fast quenching, boiling-water extraction, and high-resolution mass spectrometry with ¹³C-labeled internal standards, the approach enabled measurement of adenylate and uridylate energy charge dynamics. Applied to a lab-scale system, it revealed energy pool fluctuations during acetate uptake and polyphosphate degradation, demonstrating that energy imbalances can underlie metabolic switching in biofilms.
In Chapter 5, we demonstrate that microbial metabolite extracts can serve as effective and readily accessible substrate sources for glycosyltransferase assays, without further purification. Using a Campylobacter jejuni extract containing diverse nonulosonic acid derivatives, we show substrate promiscuity of the previously identified pseudaminyltransferase from Acinetobacter baumannii, currently the only confirmed enzyme of its kind. These findings support the production of glycans and glycoconjugates relevant for antimicrobial and vaccine development.
While glycans and glycoconjugates are central to many biological processes, carbohydrate-active enzymes are of similar interest due to their potential to replace harsh chemical processes and their ability to enable new products. A key challenge remains the targeted identification of enzymes with desired activities and characteristics. In Chapter 6, we combine enrichment cultures with metagenomics and metaproteomics to discover glycoside hydrolases. This strategy enables functional enrichment under customized conditions and yields a focused set of enzyme candidates. One of the candidates was subsequently confirmed as a pullulan-degrading enzyme using a mass spectrometry-based activity assay.
Finally, Chapter 7 summarizes some of the main findings, discusses main challenges and outlines future research directions.
Chapter 2 reviews advanced mass spectrometry-based methods for the identification and quantification of monosaccharides and their nucleotide-activated forms. Key analytical challenges are highlighted, including the differentiation of stereoisomers and the limited availability of reference standards. Complementary strategies, such as high-performance separation techniques, advanced fragmentation methods, and isotope tracing, are discussed for improved structural characterization and pathway analysis.
Building on these analytical foundations, Chapter 3 presents SugarBase, a mass spectrometry and bioinformatic pipeline for untargeted exploration of microbial nucleotide sugar networks. SugarBase combines narrow-window DIA fragmentation with a theoretical sugar composition database for parent ion annotation. The platform revealed species-specific nucleotide sugar profiles, including distinct nonulosonic acid profiles in Campylobacter jejuni strains and previously unannotated compounds across diverse microbes, including a higher-carbon ulosonic acid in Magnetospirillum.
Extending this work to complex microbial systems, Chapter 4 includes a collaborative study adapting the Chapter 3 methodology into a targeted metabolomics workflow to quantify nucleotides in granular biofilms involved in enhanced biological phosphorus removal. Using fast quenching, boiling-water extraction, and high-resolution mass spectrometry with ¹³C-labeled internal standards, the approach enabled measurement of adenylate and uridylate energy charge dynamics. Applied to a lab-scale system, it revealed energy pool fluctuations during acetate uptake and polyphosphate degradation, demonstrating that energy imbalances can underlie metabolic switching in biofilms.
In Chapter 5, we demonstrate that microbial metabolite extracts can serve as effective and readily accessible substrate sources for glycosyltransferase assays, without further purification. Using a Campylobacter jejuni extract containing diverse nonulosonic acid derivatives, we show substrate promiscuity of the previously identified pseudaminyltransferase from Acinetobacter baumannii, currently the only confirmed enzyme of its kind. These findings support the production of glycans and glycoconjugates relevant for antimicrobial and vaccine development.
While glycans and glycoconjugates are central to many biological processes, carbohydrate-active enzymes are of similar interest due to their potential to replace harsh chemical processes and their ability to enable new products. A key challenge remains the targeted identification of enzymes with desired activities and characteristics. In Chapter 6, we combine enrichment cultures with metagenomics and metaproteomics to discover glycoside hydrolases. This strategy enables functional enrichment under customized conditions and yields a focused set of enzyme candidates. One of the candidates was subsequently confirmed as a pullulan-degrading enzyme using a mass spectrometry-based activity assay.
Finally, Chapter 7 summarizes some of the main findings, discusses main challenges and outlines future research directions.
Decoding Sugars
Mass Spectrometric Advances in the Analysis of the Sugar Alphabet
Monosaccharides play a central role in metabolic networks and in the biosynthesis of glycomolecules, which perform essential functions across all domains of life. Thus, identifying and quantifying these building blocks is crucial in both research and industry. Routine methods have been established to facilitate the analysis of common monosaccharides. However, despite the presence of common metabolites, most organisms utilize distinct sets of monosaccharides and derivatives. These molecules therefore display a large diversity, potentially numbering in the hundreds or thousands, with many still unknown. This complexity presents significant challenges in the study of glycomolecules, particularly in microbes, including pathogens and those with the potential to serve as novel model organisms. This review discusses mass spectrometric techniques for the isomer-sensitive analysis of monosaccharides, their derivatives, and activated forms. Although mass spectrometry allows for untargeted analysis and sensitive detection in complex matrices, the presence of stereoisomers and extensive modifications necessitates the integration of advanced chromatographic, electrophoretic, ion mobility, or ion spectroscopic methods. Furthermore, stable-isotope incorporation studies are critical in elucidating biosynthetic routes in novel organisms.
...
Monosaccharides play a central role in metabolic networks and in the biosynthesis of glycomolecules, which perform essential functions across all domains of life. Thus, identifying and quantifying these building blocks is crucial in both research and industry. Routine methods have been established to facilitate the analysis of common monosaccharides. However, despite the presence of common metabolites, most organisms utilize distinct sets of monosaccharides and derivatives. These molecules therefore display a large diversity, potentially numbering in the hundreds or thousands, with many still unknown. This complexity presents significant challenges in the study of glycomolecules, particularly in microbes, including pathogens and those with the potential to serve as novel model organisms. This review discusses mass spectrometric techniques for the isomer-sensitive analysis of monosaccharides, their derivatives, and activated forms. Although mass spectrometry allows for untargeted analysis and sensitive detection in complex matrices, the presence of stereoisomers and extensive modifications necessitates the integration of advanced chromatographic, electrophoretic, ion mobility, or ion spectroscopic methods. Furthermore, stable-isotope incorporation studies are critical in elucidating biosynthetic routes in novel organisms.
Journal article
(2026)
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Zi Xuan Hu, Jia Yu Zhang, Jitske van Ede, Yao Yao Zhang, Yu Quan Li, Mattia Ghirardello, M. Carmen Galan, Martin Pabst, Li Liu, Josef Voglmeir
Sialic acids are a diverse family of acidic sugars typically found at the terminal positions of glycan chains, mediating key physiological and pathological processes across animals - particularly vertebrates - including cell signaling and host-pathogen interactions. The distribution of sialic acids in lower animals such as mollusks, however, remains largely unresolved. Here, we report the discovery of unconjugated 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), a deaminated analogue of N-acetylneuraminic acid, in the muscle tissue of Pacific oysters (Magallana gigas). Using UPLC-ESI-MS/MS fingerprinting, we identified naturally occurring free KDN at a concentration of 1.2 ± 0.1 nmol/100 mg of oyster muscle tissue. To investigate the biosynthetic pathway, four candidate genes were identified in the M. gigas genome, and the corresponding recombinant proteins were expressed and characterized. Enzymatic assays revealed that one putative sialic acid aldolase (MgNPL) specifically catalyzes the cleavage of KDN into mannose and pyruvate. To our knowledge, this represents the first molecular evidence of KDN metabolism in mollusks and highlights both the unexpected conservation of substrate-specific aldolase activity and distinct sialic acid utilization mechanisms compared to vertebrates.
...
Sialic acids are a diverse family of acidic sugars typically found at the terminal positions of glycan chains, mediating key physiological and pathological processes across animals - particularly vertebrates - including cell signaling and host-pathogen interactions. The distribution of sialic acids in lower animals such as mollusks, however, remains largely unresolved. Here, we report the discovery of unconjugated 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), a deaminated analogue of N-acetylneuraminic acid, in the muscle tissue of Pacific oysters (Magallana gigas). Using UPLC-ESI-MS/MS fingerprinting, we identified naturally occurring free KDN at a concentration of 1.2 ± 0.1 nmol/100 mg of oyster muscle tissue. To investigate the biosynthetic pathway, four candidate genes were identified in the M. gigas genome, and the corresponding recombinant proteins were expressed and characterized. Enzymatic assays revealed that one putative sialic acid aldolase (MgNPL) specifically catalyzes the cleavage of KDN into mannose and pyruvate. To our knowledge, this represents the first molecular evidence of KDN metabolism in mollusks and highlights both the unexpected conservation of substrate-specific aldolase activity and distinct sialic acid utilization mechanisms compared to vertebrates.
Journal article
(2025)
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Jitske M. van Ede, Suzanne van der Steen, Geert M. van der Kraan, Mark C.M. van Loosdrecht, Martin Pabst
The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.
...
The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.
Journal article
(2024)
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Le Min Chen, Paula Beck, Jitske van Ede, Mario Pronk, Mark C.M. van Loosdrecht, Yuemei Lin
Abstract: Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438– > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. Key points: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68–14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2 Graphical Abstract: [Figure not available: see fulltext.]
...
Abstract: Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438– > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. Key points: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68–14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2 Graphical Abstract: [Figure not available: see fulltext.]
NovoLign
Metaproteomics by sequence alignment
Journal article
(2024)
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Hugo B.C. Kleikamp, Ramon van der Zwaan, Ramon van Valderen, Jitske M. van Ede, Mario Pronk, Pim Schaasberg, Maximilienne T. Allaart, Mark C.M. van Loosdrecht, Martin Pabst
Tremendous advances in mass spectrometric and bioinformatic approaches have expanded proteomics into the field of microbial ecology. The commonly used spectral annotation method for metaproteomics data relies on database searching, which requires sample-specific databases obtained from whole metagenome sequencing experiments. However, creating these databases is complex, time-consuming, and prone to errors, potentially biasing experimental outcomes and conclusions. This asks for alternative approaches that can provide rapid and orthogonal insights into metaproteomics data. Here, we present NovoLign, a de novo metaproteomics pipeline that performs sequence alignment of de novo sequences from complete metaproteomics experiments. The pipeline enables rapid taxonomic profiling of complex communities and evaluates the taxonomic coverage of metaproteomics outcomes obtained from database searches. Furthermore, the NovoLign pipeline supports the creation of reference sequence databases for database searching to ensure comprehensive coverage. We assessed the NovoLign pipeline for taxonomic coverage and false positive annotations using a wide range of in silico and experimental data, including pure reference strains, laboratory enrichment cultures, synthetic communities, and environmental microbial communities. In summary, we present NovoLign, a de novo metaproteomics pipeline that employs large-scale sequence alignment to enable rapid taxonomic profiling, evaluation of database searching outcomes, and the creation of reference sequence databases.
...
Tremendous advances in mass spectrometric and bioinformatic approaches have expanded proteomics into the field of microbial ecology. The commonly used spectral annotation method for metaproteomics data relies on database searching, which requires sample-specific databases obtained from whole metagenome sequencing experiments. However, creating these databases is complex, time-consuming, and prone to errors, potentially biasing experimental outcomes and conclusions. This asks for alternative approaches that can provide rapid and orthogonal insights into metaproteomics data. Here, we present NovoLign, a de novo metaproteomics pipeline that performs sequence alignment of de novo sequences from complete metaproteomics experiments. The pipeline enables rapid taxonomic profiling of complex communities and evaluates the taxonomic coverage of metaproteomics outcomes obtained from database searches. Furthermore, the NovoLign pipeline supports the creation of reference sequence databases for database searching to ensure comprehensive coverage. We assessed the NovoLign pipeline for taxonomic coverage and false positive annotations using a wide range of in silico and experimental data, including pure reference strains, laboratory enrichment cultures, synthetic communities, and environmental microbial communities. In summary, we present NovoLign, a de novo metaproteomics pipeline that employs large-scale sequence alignment to enable rapid taxonomic profiling, evaluation of database searching outcomes, and the creation of reference sequence databases.
Journal article
(2024)
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Maider J. Echeveste Medrano, Andy O. Leu, Martin Pabst, Yuemei Lin, Simon J. McIlroy, Gene W. Tyson, Jitske van Ede, Irene Sánchez-Andrea, Cornelia U. Welte, More authors...
Climate change–driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12 weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-β-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-β-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress.
...
Climate change–driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12 weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-β-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-β-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress.
Journal article
(2023)
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Le Min Chen, Sunanda Keisham, Hiroaki Tateno, Jitske van Ede, Mario Pronk, Mark C.M. van Loosdrecht, Yuemei Lin
Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.
...
Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.