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.

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. Clade II NosZ populations are of particular biotechnological interest as they usually feature high N2O affinities and often lack other denitrification genes. We focus on the yet-unresolved ecological constraints selecting for different N2O-reducers strains and controlling the assembly of N2O-respiring communities. Two planktonic N2O-respiring mixed cultures were enriched at low dilution rates 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. Under N2O limitation, clade II N2O-reducers fully outcompeted clade I affiliates, a scenario previously only theorized based on pure-cultures. All enriched N2O-reducers encoded and expressed the sole clade II NosZ, while also possessing other denitrification genes. Two Azonexus and Thauera genera affiliates dominated the culture, and we hypothesize their coexistence to be explained by the genome-inferred metabolic exchange of cobalamin intermediates. Under excess N2O, clade I and II populations coexisted; yet, proteomic evidence suggests that clade II affiliates respired most of the N2O, de facto outcompeting clade I affiliates. The single dominant N2O-reducer (genus Azonexus) notably expressed most cobalamin biosynthesis marker genes, likely to contrast the continuous cobalamin inactivation by dissolved cytotoxic N2O concentrations (400 μM). Ultimately, our results strongly suggest the solids dilution rate to play a pivotal role in controlling the selection among NosZ clades, albeit the conditions selecting for genomes possessing the sole nosZ remain elusive. We furthermore highlight the potential significance of N2O-cobalamin interactions in shaping the composition of N2O-respiring microbiomes.

Enzymes are attractive catalysts due to their high chemo-, regio-, and enantioselectivity. In recent years, the application of enzymes in organic synthesis has expanded dramatically, especially for the synthesis of chiral alcohols and amines, two very important functional groups found in many active pharmaceutical ingredients (APIs). Indeed, many elegant routes employing such compounds have been described by industry. Yet, for the synthesis of chiral thiols and thioethers, likewise found in APIs albeit less ubiquitous, only very few biocatalytic syntheses have been reported, and stereocontrol has proved challenging. Here, we apply ene-reductases (EREDs), whose ability to initiate and control chemically challenging radical chemistries has recently emerged, to the synthesis of chiral thioethers from α-bromoacetophenones and pro-chiral vinyl sulfides, without requiring light. Depending on the choice of ERED either enantiomer of the product could be accessed. The highest conversion and selectivity were achieved with GluER T36A using fluorinated substrates, reaching up to 82% conversion and >99.5% ee. With α-bromoacetophenone and α-(methylthio)styrene, the reaction could be performed on a 100 mg scale, affording the product in a 46% isolated yield with a 93% ee. Finally, mechanistic studies were carried out using stopped-flow spectroscopy and protein mass spectrometry, providing insight into the preference of the enzyme for the intermolecular reaction. This work paves the way for new routes for the synthesis of thioether-containing compounds.

One Health seeks to integrate and balance the health of humans, animals, and environmental systems, which are intricately linked through microbiomes. These microbial communities exchange microbes and genes, influencing not only human and animal health but also key environmental, agricultural, and biotechnological processes. Preventing the emergence of pathogens as well as monitoring and controlling the composition of microbiomes through microbial effectors including virulence factors, toxins, antibiotics, non-ribosomal peptides, and viruses holds transformative potential. However, the mechanisms by which these microbial effectors shape microbiomes and their broader functional consequences for host and ecosystem health remain poorly understood. Metaproteomics offers a novel methodological framework as it provides insights into microbial dynamics by quantifying microbial biomass composition, metabolic functions, and detecting effectors like viruses, antimicrobial resistance proteins, and non-ribosomal peptides. Here, we highlight the potential of metaproteomics in elucidating microbial effectors and their impact on microbiomes and discuss their potential for modulating microbiomes to foster desired functions.

Nitrous oxide (N2O) is the third most important greenhouse gas and originates primarily from natural and engineered microbiomes. Effective emission mitigations are currently hindered by the largely unresolved ecophysiological controls of coexisting N2O-converting metabolisms in complex communities. To address this, we used biological wastewater treatment as a model ecosystem and combined long-term metagenome-resolved metaproteomics with ex situ kinetic and full-scale operational characterization over nearly 2 years. 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 seasonal N2O emissions has ultimately been resolved. We identified nitrifier denitrification as the dominant N2O-producing pathway and dissolved O2 as the prime operational parameter, paving the way to the design and fostering of robust emission control strategies. This work exemplifies the untapped potential of multi-meta-omics in the mechanistic understanding and ecological engineering of microbiomes towards reducing anthropogenic impacts and advancing sustainable biotechnological developments.

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.

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.

Purpose: Organic farming practices enhance soil health by the addition of bio-amendments, which improve microbial diversity and abundance. Improved soil health, due to enhanced dynamic interactions between abiotic and biotic components of the environment, impedes the progression of diseases caused by soil-borne phytopathogens. The present study aimed to characterize the critical microbial and edaphic factors in correlation with phytopathogen suppression in soil from mustard fields managed under different farming practices. Methods and results: Organic soil exhibited better suppression of phytopathogens, availability of macronutrients, and high biocontrol enzymatic activities than soil from conventionally managed field. In terms of maximal phytopathogen suppression, Fusarium solani was suppressed to 85% by fungal fraction of organic soil, whereas F. fujikori was suppressed to 77% by bacterial fraction of organic soil. However, available micronutrients were higher in conventionally managed soils. Positive correlations between enhanced biocontrol enzyme activities and organic farming practice were deciphered, highlighting improved disease suppressive potential of organic soil. Metagenomic sequencing of the rhizosphere soil from mustard plants grown using “Jeevan Jyoti” bio-amendment and conventional farming regimes revealed that microbial communities could play key roles in modulating general disease suppressiveness of soil. Functional annotation enabled the identification of dominant biological processes, impacting plant and soil health positively. Specifically, the open reading frames coding for bacterial proteins involved in transport and key cellular processes were enriched in datasets of organic farm soil. Conclusion: The evidences from this study delineate the correlation between microbial components present in rhizosphere soil and edaphic factors, in the context of general disease suppressiveness.

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 those used in biological wastewater treatment. Limited proteolysis of whole granules and metaproteome isolation from the culture's supernatant successfully classified over 50% of the identified protein biomass to be secreted. Moreover, structural and sequence-based classification identified 387 proteins, corresponding to over 50% of the secreted protein biomass, with characteristics that could aid the formation of aggregates, including filamentous, beta-barrel containing, and cell surface proteins. While various of these aggregate-forming proteins originated from Ca. Accumulibacter, some proteins associated with other taxa. 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.

The challenging task of designing biopharmaceutical downstream processes is initially to select the type of unit operations, followed by optimizing their operating conditions. For complex flowsheet optimizations, the strategy becomes crucial in terms of duration and outcome. In this study, we compared three optimization strategies, namely, simultaneous, top-to-bottom, and superstructure decomposition. Moreover, all strategies were evaluated by either using chromatographic Mechanistic Models (MMs) or Artificial Neural Networks (ANNs). An overall evaluation of 39 flowsheets was performed, including a buffer-exchange step between the chromatography operations. All strategies identified orthogonal structures to be optimal, and the weighted overall performance values were generally consistent between the MMs and ANNs. In terms of time-efficiency, the decomposition method with MMs stands out when utilizing multiple cores on a multiprocessing system for simulations. This study analyses the influence of different optimization strategies on flowsheet optimization and advices on suitable strategies and modeling techniques for specific scenarios.

Methane removal is an essential step in drinking water production from methane-rich groundwaters. Conventional aeration-based stripping results in significant direct methane emissions, contributing up to one-third of a treatment plant's total carbon footprint. To address this, a full-scale trickling filter was operated for biological methane oxidation upstream of a submerged sand filter, and its performance was compared to a conventional aeration–submerged sand filtration set-up. Full-scale data were combined with ex-situ batch assays and metagenome-resolved metaproteomics to quantify the individual contribution of the main (a)biotic processes and characterize the enriched microbial communities. Both treatment setups fully removed methane, iron, ammonium, and manganese, yet the underlying mechanisms differed significantly. Methane was completely removed from the effluent after trickling filtration, with stripping and biological oxidation each accounting for half of the removal, thereby halving overall methane emissions. Methane-oxidizing bacteria not only outcompeted nitrifiers in the trickling filter, but also likely contributed directly to ammonia oxidation. In contrast to the submerged filter preceded by methane stripping, signatures of biological iron oxidation were almost completely absent in the trickling filter, suggesting that the presence of methane directly or indirectly promotes chemical iron oxidation. All systems had similar ex-situ manganese oxidation capacities, yet removal occurred only in the submerged filters but not the trickling filter. Ultimately, our results demonstrate that trickling filtration is effective in promoting biological methane oxidation at comparable produced drinking water quality, highlighting its potential for advancing sustainable drinking water production.

Identifying structural proteins within the extracellular polymeric substances (EPS) will provide a better understanding of the stability of aerobic granular sludge (AGS) and biofilms in general. In this work, an abundant surface protein was identified and localized in the extracellular matrix of seawater-adapted AGS. Granules with good phosphate removal were cultivated in a sequencing batch bubble column reactor with acetate as a carbon source dissolved in seawater. “Candidatus Accumulibacter” was observed as the most dominant community member through fluorescent in-situ hybridization. A surface protein of 74.5 kDa was identified in the EPS extract of the seawater-adapted AGS by SDS-PAGE and mass spectrometry. The surface protein was produced by an Accumulibacter species and showed homology to S-layer proteins. A type 1 secretion system was found adjacent to the gene encoding for the surface protein, suggesting a possible export system. Antibodies generated from a unique peptide of the surface protein confirmed the extracellular location of the surface protein. Microscopy observations with antibody staining showed the surface protein forms dense structures within the Accumulibacter microcolonies and larger fiber structures around the microcolonies. These observations highlight the importance of the protein for the structural properties of the granule. To detect more structural proteins in the EPS, optimization of the EPS extraction and in situ imaging validation methods are essential.

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.

Nitrous oxide (N2O) is a potent greenhouse gas of primarily microbial origin. Aerobic and anoxic emissions are commonly ascribed to nitrification and denitrification, respectively. Beyond this established dichotomy, we quantitatively prove that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N2O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Planktonic, nitrification-inhibited denitrifying enrichments respired over a third of the influent organic substrate with nitrate at high oxygen concentrations. N2O accounted for up to one quarter of the aerobically respired nitrate. The constitutive detection of all denitrification enzymes in both anoxic and oxic periods highlight the selective advantage offered by metabolic preparedness in dynamic environments. We posit that aerobic denitrification and associated N2O formation is currently underestimated in dynamic microbial ecosystems.

Industrial wastewater often has high levels of salt, either due to seawater or e.g. sodium chloride (NaCl) usage in the processing. Previous work indicated that aerobic granular sludge (AGS) is differently affected by seawater or saline water at similar osmotic strength. Here we investigate in more detail the impact of NaCl concentrations and seawater on the granulation and conversion processes for AGS wastewater treatment. Glycerol was used as the carbon source since it is regularly present in industrial wastewaters, and to allow the evaluation of microbial interactions that better reflect real conditions. Long-term experiments were performed to evaluate and compare the effect of salinity on granulation, anaerobic conversions, phosphate removal, and the microbial community. Smooth and stable granules as well as enhanced biological phosphorus removal (EBPR) were achieved up to 20 g/L NaCl or when using seawater. However, at NaCl levels comparable to seawater strength (30 g/L) incomplete anaerobic glycerol uptake and aerobic phosphate uptake were observed, the effluent turbidity increased, and filamentous granules began to appear. The latter is likely due to the direct aerobic growth on the leftover substrate after the anaerobic feeding period. In all reactor conditions, except the reactor with 30 g/L NaCl, Ca. Accumulibacter was the dominant microorganism. In the reactor with 30 g/L NaCl, the relative abundance of Ca. Accumulibacter decreased to ≤1 % and an increase in the genus Zoogloea was observed. Throughout all reactor conditions, Tessaracoccus and Micropruina, both actinobacteria, were present which were likely responsible for the anaerobic conversion of glycerol into volatile fatty acids. None of the glycerol metabolizing proteins were detected in Ca. Accumulibacter which supports previous findings that glycerol can not be directly utilized by Ca. Accumulibacter. The proteome profile of the dominant taxa was analysed and the results are further discussed. The exposure of salt-adapted biomass to hypo-osmotic conditions led to significant trehalose and PO₄³⁻-P release which can be related to the osmoregulation of the cells. Overall, this study provides insights into the effect of salt on the operation and stability of the EBPR and AGS processes. The findings suggest that maintaining a balanced cation ratio is likely to be more important for the operational stability of EBPR and AGS systems than absolute salt concentrations.

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.

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.

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived.