Biology subjects

Pabst, M.

Publications and source records attributed to Pabst, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Dark CO2 Fixation via the Ethylmalonyl-CoA Pathway Establishes Metabolic Parity: A Stoichiometric Basis for Compounding Ecosystem Shifts

A central challenge in microbial ecology is understanding how competing guilds coexist and why community structures unexpectedly drift away from theoretical steady states. While polyphosphate accumulation provides energy and redox buffering in many heterotrophs, how competing lineages occupying the same ecological niche without polyphosphate synthesis manage equivalent intracellular redox imbalances remains unposed. Using an enhanced biological phosphorus removal macrocosm, we integrated quantitative stoichiometry with metaproteomics to resolve alternative metabolic strategies that underpin cellular homeostasis in heterotrophs. We show that glycogen-accumulating organisms (GAOs) achieve baseline metabolic parity with polyphosphate-accumulating organisms (PAOs) through a parallel redox-buffering mechanism: heterotrophic CO2/HCO3- re-assimilation via the ethylmalonyl-CoA pathway. This inorganic carbon fixation couples structural carbon conservation with tight redox control, mitigating intracellular electron overflow and eliminating the long-assumed GAO bioenergetic inferiority. The bioenergetic efficiency of GAOs is further fortified by fine-tuned metabolic wiring, featuring energy-efficient high affinity acetate activation, ferredoxin-centered biochemistry, and energy-neutral polyhydroxyalkanoates (PHA) mobilization. Strikingly, minor formate co-feeding disrupted this established PAO/GAO parity. Stoichiometric simulations revealed that this formate supplementation creates an asymmetric bioenergetic niche that grants per-cycle energy gains exclusively to GAOs. Decoupled from hydraulic throughput, solids retention time control retains cells carrying accumulated intracellular inventory, translating subtle per-cycle stoichiometric edges into a multi-generational ratchet and drives a rapid community shift from PAO/GAO co-dominance to GAO dominance. By exposing limitations of traditional single-substrate and single-cycle steady-state models, our findings reveal how inorganic resource management and generational metabolic compounding govern community assembly in biomass-retaining microbial ecosystems.

bioengineering↗

bioRxiv2026

A protocol for lab-scale production of 13C yeast extract as internal standard for metabolomics and quantification of intracellular metabolites

Metabolomics enables the profiling of small-molecule metabolites and thereby captures the biochemical state of a living organism at a given moment and enables to monitor its cellular responses to stimuli. This technique has become a powerful tool in pharmaceutical research, the food industry, and microbial research. Metabolomics aims to obtain an unbiased metabolic profile; however, this is complicated by compound instability, complex and often extensive sample processing, and nonlinear responses in mass spectrometry. Therefore, correcting for metabolite loss and mass spectrometry-related artifacts is essential, typically achieved through relative quantification against an isotopically labelled internal standard for each metabolite of interest. This article describes how to produce 13C-labelled yeast extract and its use as internal standard for metabolomics. More specifically, it provides step-by-step protocols for the fed-batch fermentation, quenching, metabolite extraction, and LC-MS and GC-MS characterization of the internal standard. It also includes a protocol explaining how to use the internal standard for the quantification of metabolites in yeast samples.

biochemistry↗

bioRxiv2026

SugarBase: mapping glycomolecule precursors in microbes

Glycan biosynthesis relies on nucleotide-activated sugars, essential metabolites across all domains of life, yet their usage in microbes is poorly understood. Here we present SugarBase, a mass spectrometry and bioinformatic pipeline for untargeted exploration of microbial nucleotide sugar networks. SugarBase resolves the chemical complexity of microbial metabolism by combining narrow-window DIA fragmentation with a chemistry-informed parent ion identification algorithm. Applying SugarBase across a broad phylogenetic range of microbes revealed extensive, species-specific nucleotide sugar profiles, including many candidates with no existing annotation, generating the most comprehensive inventory of nucleotide sugars to date. SugarBase guided identification of gene clusters and allowed discrimination between pseudaminic- and legionaminic acid-producing strains, where genomic and proteomic data provided only ambiguous information. We resolved distinct nonulosonic acid profiles in several Campylobacter jejuni strains, sugars which may alter susceptibility towards distinct flagellotropic phages. We further identify previously undescribed CMP-activated higher-carbon ulosonic acids in Magnetospirillum, expanding the known chemical space in glycan biosynthesis. In summary, SugarBase supports scalable discovery of microbial nucleotide sugar pathways and enzymes, expanding access to chemically complex glycans and providing new targets for antimicrobial development.

systems biology↗

bioRxiv2026

NovoGlyco: mapping protein glycosylation in prokaryotes

Protein glycosylation in prokaryotes shows extraordinary diversity including species-specific monosaccharides, non-canonical attachment sites, and variable glycan architectures that challenge existing glycoproteomics approaches. Current strategies are largely tailored to eukaryotic systems and depend on predefined glycan databases or prior biochemical knowledge, limiting their application to microbes. Here we present NovoGlyco, a modular glycoproteomics platform for untargeted characterisation of prokaryotic protein glycosylation from shotgun proteomics data. NovoGlyco integrates de novo oxonium ion discovery, sequence tag matching, and mass offset binning to identify novel glycans, their composition, and linking chemistry. An interactive dashboard allows exploration of glycan features and modified proteins. We demonstrate the NovoGlyco platform across published glycoproteomics datasets, spanning human pathogens, Asgard archaea, and environmental enrichment cultures, and identify previously unreported flagella O-glycans in the opportunistic pathogen Campylobacter fetus. In summary, NovoGlyco provides a scalable framework for unbiased exploration of microbial glycoproteomes in both single-organism and metaproteomic contexts.

systems biology↗

bioRxiv2025

Linking structure to function in high performing electrosynthetic biofilm communities

Biofilm-based microbial electrosynthesis (MES) is a promising technology that converts CO2 into industrially relevant organic compounds using renewable energy sources. The highest performing MES systems reported to date consist of biofilm-driven microbial communities. However, for a successful deployment of this technology, we still need to overcome key challenges, including the long colonization time of the biocathode and the difficulty of controlling the product profile. To address these challenges, it is crucial to better understand the key microbial components responsible for the desired metabolic products, and how they assemble and function as a community. In this study, we conducted an in-depth characterisation of three high-performing mixed MES communities using metagenomics, metaproteomics and advanced analysis of metagenome-derived metabolic networks. Our findings identified Eubacterium limosum, Sporomusa sphaeroides and Clostridium aromativorans as key contributors to the production of acetate, butyrate and caproate via the Wood-Ljungdahl and the reverse {beta}-oxidation pathways. A higher production of butyrate and caproate was observed in reactors with higher abundance of C. aromativorans, an organism only recently discovered and never reported in gas-fermenting systems before. The recovered genome of C. aromativorans, reconstructed in a single circular fragment, provides a more comprehensive genomic representation than the current reference. In addition, we found genes related to lactate and ethanol production from acetyl-CoA in the metagenomes, including proteomic evidence for lactate production. This study provides key insights into the microbial players, metabolic processes and microbial community features driving product formation in biofilm-based MES, bringing this technology closer to industrial application.

systems biology↗

bioRxiv2025

Meta-PepView: a metaproteomics performance evaluation and visualization platform

Microbial community proteomics is rapidly gaining traction as it allows exploration of functional processes in microbial ecosystems. Consequently, there is a growing need for user-friendly tools that enable performance evaluation and interactive visualization of the increasingly complex community proteomics data. We introduce meta-PepView, a web-based platform that enables performance evaluation and interactive visualization of metaproteomics data, ensuring transparent and reproducible metaproteomics experiments. Meta-PepView integrates spectral sequencing outputs and databases from common proteomics search engines and classification tools. It runs efficiently on laptop or desktop PCs and can be deployed as a Docker container or installed with pip, from which it is operated through the web browser. Its modular design allows easy expansion with new data sources and annotation databases. Meta-PepView is an open-source Python platform that is freely available under the Apache License 2.0. The code is available on GitHub: https://github.com/ramonzwaan/metapepview/. Here, we showcase the meta-PepView platform with data from synthetic communities and previously published microbiome studies.

systems biology↗

bioRxiv2025

D- and L-lactate consumers in the human gut are taxonomically, biochemically, and energetically different

The human gut microbiota routinely produces D- and L-lactate during fermentation; however, the microbial fate of these stereoisomers, particularly the neurotoxic D-lactate, remains poorly understood. Given that D-lactate is an unavoidable byproduct of digestion, understanding its microbial turnover is essential for linking gut metabolism to host health. Here, we used chemostat bioreactors (pH 7.0, 37{degrees}C, and a solids retention time [SRT] of 4 d) to simulate gut-relevant, transient nutrient conditions and to enrich for lactate-consuming communities. DL-lactate-consuming consortia were enriched from a human-derived microbiota and then inoculated into duplicate bioreactors, which were fed exclusively D- or L-lactate. After steady-state was reached, the fed lactate stereoisomers were switched to assess community resilience. Regardless of the fed stereoisomer, the fermentation product spectra were consistent and dominated by acetate, propionate, and CO2. However, microbial communities and biomass yields diverged sharply, with a high relative abundance of Anaerotignum in D-lactate enrichments and Acidipropionibacterium and Propionibacterium in L-lactate enrichments. Notably, the biomass yield for D-lactate feeding was less than half that for L-lactate feeding, suggesting that the two isomers are metabolized through distinct biochemical pathways despite similar product spectra. Metagenomic and metaproteomic analyses confirmed this divergence at the pathway level. Our findings reveal how the stereoisomer identity of microbes shapes their niche specialization in the gut, with implications for understanding the ecology and clinical impact of lactate metabolism.

bioengineering↗

bioRxiv2025

Measuring Temporal Variations of Nucleotide Pools in Microbial Granular Biofilm Performing Enhanced Biological Phosphorous Removal

Microbial communities often face environmental fluctuations that occur on timescales much shorter than their growth rate or proteome turnover. In such cases, cellular responses are likely driven by rapid changes in metabolite pools, particularly energy nucleotides including ATP, ADP, and AMP. However, robust methods to quantify these metabolites in biofilm-forming microbial communities are lacking. Here, we developed and systematically evaluated a metabolomics workflow for a granular biofilm enrichment, which performs Enhanced Biological Phosphorous Removal (EBPR). We combined fast quenching in liquid nitrogen and a boiling water extraction, followed by high resolution mass spectrometry, using porous graphitic carbon chromatography and 13C-labeled internal reference standards. Among tested procedures, a boiling water extraction was most suitable for extraction of nucleotides, as indicated by stable adenylate energy charge (AEC) and isotopic ratios. Applied to an anaerobic-aerobic cycle of a lab scale EBPR system, the method revealed dynamic changes in AEC and uridylate energy charge (UEC) during acetate uptake and polyphosphate degradation. These results demonstrate that energy pool imbalances underlie rapid metabolic switching observed in EBPR systems. Moreover, the established method provides a foundation for performing metabolomic studies of microbial biofilms in general.

systems biology↗

bioRxiv2025

Exploration of mRNA-sized RNA import into Saccharomyces cerevisiae mitochondria by a combined synthetic biology and adaptive laboratory evolution approach

Efficient gene integration using RNA-guided endonucleases has not yet been achieved in the mitochondrial genome. Import of nucleic acids into mitochondria, a controversial feature, is essential for implementation of Cas9-mediated genome engineering of mitochondria. Import of short RNAs naturally occurs in mitochondria, and several putative import mechanisms and determinants have been proposed. However to date, import of gene-length RNA, required for gene integration in the mitochondrial genome, has never been described. The goal of this study was to devise and test experimental strategies to detect and improve the import of mRNA-sized RNA in mitochondria, using S. cerevisiae as model. A first fluorescence-based screening approach, relying on mitochondrial import of a fluorescent protein encoding mRNA was analyzed by fluorescence measurements, western blot and mRNA-FISH. Confounding results obtained with these different techniques made it difficult to unambiguously conclude on the occurrence of import of mRNA-sized RNAs into mitochondria. An adaptive laboratory evolution (ALE) approach, imposing a strong selection pressure for mRNA import to mitochondria, was then designed and tested to improve mitochondrial mRNA import. While the ALE approach did not improve mitochondrial mRNA import in the present study, it is a promising, unambiguous method for future studies testing different RNAs or mutants. The present study highlights remaining challenges in analytical techniques to identify RNA import to mitochondria, and introduces a novel application of ALE for studies on mitochondrial import of short and long RNA species.

synthetic biology↗

bioRxiv2025

Molecular basis for anti-jumbo phage immunity by AVAST Type 5

Jumbo phages protect their genomes from DNA-sensing bacterial defense systems by enclosing them within vesicles and nucleus-like compartments. Very little is known about defense systems specialized to counter these phages. Here, we show that AVAST Type 5 (Avs5) systems, part of the STAND superfamily and spanning three phylogenetic Avs5 clades, confer conserved immunity against jumbo phages. Using localization microscopy and biotin proximity labeling we demonstrate that Avs5 localizes to early infection vesicles, where it senses an essential, early expressed phage protein named JADA--Jumbophage AVAST5 Defense Activator. Recognition of phage infection triggers the Sir2-like effector domain of Avs5 across all three Avs5 clades, resulting in rapid NAD+ hydrolysis, disruption of phage nucleus formation, and arrest of infection. These findings reveal a spatially coordinated bacterial immune strategy that targets an early vulnerability in jumbo phage infection.

microbiology↗

bioRxiv2025

Discovery of microbial glycoside hydrolases via enrichment and metaproteomics

The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities using the carbohydrate polymer pullulan at elevated temperatures and acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by -1,6 glycosidic bonds. Along with its hydrolyzing enzymes, it has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and soil from the bank of a pond. In both cases, Alicyclobacillus emerged as the dominant microorganism. Metaproteomic analysis of the enrichment biomass and secretome identified several pullulan-degrading enzymes from this organism. Notably, these enzymes were absent in the metagenomic analysis of the initial inoculum, underscoring the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes from complex microbial environments.

ecology↗

bioRxiv2025

Wastewater metaproteomics: tracking microbial and human protein biomarkers

Wastewater-based surveillance has become a powerful tool for monitoring the spread of pathogens, antibiotic resistance genes, and measuring population-level exposure to pharmaceuticals and chemicals. While surveillance methods commonly target small molecules, DNA, or RNA, wastewater also contains a vast spectrum of proteins. However, despite recent advances in environmental proteomics, large-scale monitoring of protein biomarkers in wastewater is still far from routine. Analyzing raw wastewater presents a challenge due to its heterogeneous mixture of organic and inorganic substances, microorganisms, cellular debris, and various chemical pollutants. To overcome these obstacles, we developed a wastewater metaproteomics approach including efficient protein extraction and an optimized data-processing pipeline. The pipeline utilizes de novo sequencing to customize large public sequence databases to enable comprehensive metaproteomic coverage. Using this approach, we analyzed wastewater samples collected over three months from two urban locations. This revealed a core microbiome comprising a broad spectrum of microbes, gut bacteria and potential opportunistic pathogens. Additionally, we identified nearly 200 human proteins, including promising population-level health indicators, such as immunoglobulins, uromodulin, and cancer-associated proteins.

ecology↗

bioRxiv2024

Metaproteomic profiling of the secretome of a granule-forming Ca. Accumulibacter enrichment

Extracellular proteins are supposed to play crucial roles in the formation and structure of biofilms and aggregates. However, often little is known about these proteins, in particular for microbial communities. Here, we use two advanced metaproteomic approaches to study the extracellular proteome in a granular Candidatus Accumulibacter enrichment as a proxy for microbial communities that form solid microbial granules, such as used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the cultures supernatant successfully identified over 50% of the protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. However, while most filamentous proteins originated from Ca. Accumulibacter, among others cell surface proteins did not. This suggests that not only a range of different proteins, but also multiple organisms contribute to granular biofilm formation. Therefore, the obtained extracellular metaproteome data from the granular Ca. Accumulibacter enrichment provides a resource for exploring proteins that potentially support the formation and stability of granular biofilms, whereas the demonstrated approaches can be applied to explore biofilms of microbial communities in general. SIGNIFICANCEBiofilm-forming microbial communities are widespread and pose both challenges and opportunities in various settings in life. Structure-providing, extracellular proteins likely play a crucial role in the formation of the biofilm matrix, but these proteins are challenging to characterise due to the dynamic and complex nature of these communities. We used two advanced metaproteomic approaches to enrich for the extracellular proteins in a granule-forming Candidatus Accumulibacter enrichment culture as a proxy for granule-forming communities present in wastewater treatment plants. The extracellular proteins were additionally classified using structure and sequence-based annotation tools, which identified multiple different protein categories that potentially aid in granule formation, but also may provide structure to the biofilm matrix. Interestingly, although the granules were highly enriched for Ca. Accumulibacter, several structure-providing protein categories originated from other organisms. The obtained metaproteomic data contribute to the understanding of proteins and processes that are potentially involved in granule formation. This could further help to optimise processes involving granular biofilms and identify candidates for the recovery of novel biopolymers for biotechnological applications.

systems biology↗

bioRxiv2024

A microfluidic platform for extraction and analysis of bacterial genomic DNA

Bacterial cells organize their genomes into a compact hierarchical structure called the nucleoid. Studying the nucleoid in cells faces challenges because of the cellular complexity while in vitro assays have difficulty in handling the fragile megabase-scale DNA biopolymers that make up bacterial genomes. Here, we introduce a method that overcomes these limitations as we develop and use a microfluidic device for the sequential extraction, purification, and analysis of bacterial nucleoids in individual microchambers. Our approach avoids any transfer or pipetting of the fragile megabase-size genomes and thereby prevents their fragmentation. We show how the microfluidic system can be used to extract and analyze single chromosomes from B. subtilis cells. Upon on-chip lysis, the bacterial genome expands in size and DNA-binding proteins are flushed away. Subsequently, exogeneous proteins can be added to the trapped DNA via diffusion. We envision that integrated microfluidic platforms will become an essential tool for the bottom-up assembly of complex biomolecular systems such as artificial chromosomes.

biophysics↗

bioRxiv2024

Long-term multi-meta-omics resolves the ecophysiological controls of seasonal N2O emissions

The potent greenhouse gas nitrous oxide (N2O) originates primarily from natural and engineered microbiomes. Emission seasonality is widely reported while the underlying metabolic controls remain largely unresolved, hindering effective mitigation. We use biological wastewater treatment as tractable model ecosystem over nearly two years. Long-term metagenomic-resolved metaproteomics is combined with ex situ kinetic and full-scale operational characterization. By leveraging the evidence independently obtained at multiple ecophysiological levels, from individual genetic potential to actual metabolism and emergent community phenotype, the cascade of environmental and operational triggers driving N2O emissions is resolved. We explain the dynamics in nitrite accumulation with the kinetic unbalance between ammonia and nitrite oxidisers, and identify nitrifier denitrification as the prime N2O-producing pathway. The dissolved O2 emerged as the key actionable parameter for emission control. This work exemplifies the yet-to-be-realized potential of multi-meta-omics approaches for the mechanistic understanding and ecological engineering of microbiomes, ultimately advancing sustainable biotechnological developments.

microbiology↗

bioRxiv2024

NovoLign: metaproteomics by sequence alignment

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. The NovoLign pipeline is publicly available via: https://github.com/hbckleikamp/NovoLign.

systems biology↗

bioRxiv2024

Selective enrichment of high-affinity clade II N2O-reducers in a mixed culture

Microorganisms encoding for the N2O reductase (NosZ) are the only known biological sink of the potent greenhouse gas N2O, and are central to global N2O mitigation efforts. Yet, the ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities remain largely unknown. Of particular biotechnological interest are clade II NosZ populations, which usually feature high N2O affinities and often lack other denitrification genes. Two planktonic N2O-respiring mixed cultures were enriched under limiting and excess dissolved N2O availability to assess the impact of substrate affinity and N2O cytotoxicity, respectively. Genome-resolved metaproteomics was used to infer the metabolism of the enriched populations. We show that clade II N2O-reducers outcompete clade I affiliates for N2O at sufficiently low sludge dilution rates (0.006 h-1), a scenario previously only theorized based on pure-cultures. Under N2O limitation, all enriched N2O-reducers encoded and expressed only clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture. We explain their coexistence with the genome-inferred metabolic exchange of cobalamin intermediates. Conversely, under excess N2O, clade I and II populations coexisted. Notably, the single dominant N2O-reducer (genus Azonexus) expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 {micro}M). Ultimately, we demonstrate that the solids dilution rate controls the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. Additionally, we suggest the significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.

systems biology↗

bioRxiv2024

Exploring the metabolic potential of Aeromonas to utilise the carbohydrate polymer chitin

Members of the Aeromonas genus are commonly found in natural aquatic ecosystems. However, they are also frequently present in non-chlorinated drinking water distribution systems. High densities of these bacteria indicate favorable conditions for microbial regrowth, which is considered undesirable. Studies have indicated that the presence of Aeromonas is associated with loose deposits and the presence of invertebrates, specifically Asellus aquaticus. Therefore, a potential source of nutrients in these nutrient poor environments is chitin, the structural shell component in these invertebrates. In this study, we demonstrate the ability of two Aeromonas strains, commonly encountered in drinking water distribution systems, to effectively degrade and utilize chitin as a sole carbon and nitrogen source. We conducted a quantitative proteomics study on the cell biomass and secretome of both strains, revealing a dedicated and diverse spectrum of hydrolytic enzymes and pathways for the uptake and metabolism of chitin. Furthermore, when the primary nutrient source was switched from glucose to chitin, more than half of the Aeromonas proteome showed significant changes. Additionally, a genomic analysis of Aeromonas species found in drinking water distribution systems suggests a general potential ability of this genus to degrade and utilize a variety of carbohydrate biopolymers. This study indicates the relation between the utilization of chitin by Aeromonas and their association with invertebrates such as A. aquaticus in loose deposits in drinking water distribution systems. This knowledge provides the foundation for the development of more effective water sanitation strategies.

microbiology↗