Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities.

BACKGROUND: Microbial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology. Different strategies for their uncovering such as bioprospecting, which relies mainly on metagenomic approaches in combination with sequence-based bioinformatic analyses, have emerged; yet accurate function prediction of their proteomes and deciphering the in vivo activity of an enzyme remains challenging. RESULTS: Here, we present environmental activity-based protein profiling (eABPP), a multi-omics approach that extends genome-resolved metagenomics with mass spectrometry-based ABPP. This combination allows direct profiling of environmental community samples in their native habitat and the identification of active enzymes based on their function, even without sequence or structural homologies to annotated enzyme families. eABPP thus bridges the gap between environmental genomics, correct function annotation, and in vivo enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases from eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. CONCLUSIONS: By reporting enzyme activities within an ecosystem in their native state, we anticipate that eABPP will not only advance current methodological approaches to sequence homology-guided enzyme discovery from environmental ecosystems for subsequent biocatalyst development but also contributes to the ecological investigation of microbial community interactions by dissecting their underlying molecular mechanisms.

Time-series metaproteogenomics of a high-CO2 aquifer reveals active viruses with fluctuating abundances and broad host ranges.

Ecosystems subject to mantle degassing are of particular interest for understanding global biogeochemistry, as their microbiomes are shaped by prolonged exposure to high CO2 and have recently been suggested to be highly active. While the genetic diversity of bacteria and archaea in these deep biosphere systems have been studied extensively, little is known about how viruses impact these microbial communities. Here, we show that the viral community in a high-CO2 cold-water geyser (Wallender Born, Germany) undergoes substantial fluctuations over a period of 12 days, although the corresponding prokaryotic community remains stable, indicating a newly observed "infect to keep in check" strategy that maintains prokaryotic community structure. We characterized the viral community using metagenomics and metaproteomics, revealing 8 654 viral operational taxonomic units (vOTUs). CRISPR spacer-to-protospacer matching linked 278 vOTUs to 32 hosts, with many vOTUs sharing hosts from different families. High levels of viral structural proteins present in the metaproteome (several structurally annotated based on AlphaFold models) indicate active virion production at the time of sampling. Viral genomes expressed many proteins involved in DNA metabolism and manipulation, and encoded for auxiliary metabolic genes, which likely bolster phosphate and sulfur metabolism of their hosts. The active viral community encodes genes to facilitate acquisition and transformation of host nutrients, and appears to consist of many nutrient-demanding members, based on abundant virion proteins. These findings indicate viruses are inextricably linked to the biogeochemical cycling in this high-CO2 environment and substantially contribute to prokaryotic community stability in the deep biosphere hotspots.

Marine viruses disperse bidirectionally along the natural water cycle.

Marine viruses in seawater have frequently been studied, yet their dispersal from neuston ecosystems at the air-sea interface towards the atmosphere remains a knowledge gap. Here, we show that 6.2% of the studied virus population were shared between air-sea interface ecosystems and rainwater. Virus enrichment in the 1-mm thin surface microlayer and sea foams happened selectively, and variant analysis proved virus transfer to aerosols collected at ~2 m height above sea level and rain. Viruses detected in rain and these aerosols showed a significantly higher percent G/C base content compared to marine viruses. CRISPR spacer matches of marine prokaryotes to foreign viruses from rainwater prove regular virus-host encounters at the air-sea interface. Our findings on aerosolization, adaptations, and dispersal support transmission of viruses along the natural water cycle.

Spatio-functional organization in virocells of small uncultivated archaea from the deep biosphere.

Despite important ecological roles posited for virocells (i.e., cells infected with viruses), studying individual cells in situ is technically challenging. We introduce here a novel correlative microscopic approach to study the ecophysiology of virocells. By conducting concerted virusFISH, 16S rRNA FISH, and scanning electron microscopy interrogations of uncultivated archaea, we linked morphologies of various altiarchaeal cells to corresponding phylogenetic signals and indigenous virus infections. While uninfected cells exhibited moderate separation between fluorescence signals of ribosomes and DNA, virocells displayed complete cellular segregation of chromosomal DNA from viral DNA, the latter co-localizing with host ribosome signals. A similar spatial separation was observed in dividing cells, with viral signals congregating near ribosomes at the septum. These observations suggest that replication of these uncultivated viruses occurs alongside host ribosomes, which are used to generate the required proteins for virion assembly. Heavily infected cells sometimes displayed virus-like particles attached to their surface, which agree with virus structures in cells observed via transmission electron microscopy. Consequently, this approach is the first to link genomes of uncultivated viruses to their respective structures and host cells. Our findings shed new light on the complex ecophysiology of archaeal virocells in deep subsurface biofilms and provide a solid framework for future in situ studies of virocells.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

Seasonality and Strain Specificity Drive Rapid Co-evolution in an Ostreococcus-Virus System from the Western Baltic Sea.

Marine viruses are a major driver of phytoplankton mortality and thereby influence biogeochemical cycling of carbon and other nutrients. Phytoplankton-targeting viruses are important components of ecosystem dynamics, but broad-scale experimental investigations of host-virus interactions remain scarce. Here, we investigated in detail a picophytoplankton (size 1 µm) host's responses to infections by species-specific viruses from distinct geographical regions and different sampling seasons. Specifically, we used Ostreococcus tauri and O. mediterraneus and their viruses (size ca. 100 nm). Ostreococcus sp. is globally distributed and, like other picoplankton species, play an important role in coastal ecosystems at certain times of the year. Further, Ostreococcus sp. is a model organism, and the Ostreococcus-virus system is well-known in marine biology. However, only few studies have researched its evolutionary biology and the implications thereof for ecosystem dynamics. The Ostreococcus strains used here stem from different regions of the Southwestern Baltic Sea that vary in salinity and temperature and were obtained during several cruises spanning different sampling seasons. Using an experimental cross-infection set-up, we explicitly confirm species and strain specificity in Ostreococcus sp. from the Baltic Sea. Moreover, we found that the timing of virus-host co-existence was a driver of infection patterns as well. In combination, these findings prove that host-virus co-evolution can be rapid in natural systems.

Virus-Host Dynamics in Archaeal Groundwater Biofilms and the Associated Bacterial Community Composition.

Spatial and temporal distribution of lytic viruses in deep groundwater remains unexplored so far. Here, we tackle this gap of knowledge by studying viral infections of Altivir_1_MSI in biofilms dominated by the uncultivated host Candidatus Altiarchaeum hamiconexum sampled from deep anoxic groundwater over a period of four years. Using virus-targeted direct-geneFISH (virusFISH) whose detection efficiency for individual viral particles was 15%, we show a significant and steady increase of virus infections from 2019 to 2022. Based on fluorescence micrographs of individual biofilm flocks, we determined different stages of viral infections in biofilms for single sampling events, demonstrating the progression of infection of biofilms in deep groundwater. Biofilms associated with many host cells undergoing lysis showed a substantial accumulation of filamentous microbes around infected cells probably feeding off host cell debris. Using 16S rRNA gene sequencing across ten individual biofilm flocks from one sampling event, we determined that the associated bacterial community remains relatively constant and was dominated by sulfate-reducing members affiliated with Desulfobacterota. Given the stability of the virus-host interaction in these deep groundwater samples, we postulate that the uncultivated virus-host system described herein represents a suitable model system for studying deep biosphere virus-host interactions in future research endeavors.

uBin: A manual refining tool for genomes from metagenomes.

Resolving bacterial and archaeal genomes from metagenomes has revolutionized our understanding of Earth's biomes yet producing high-quality genomes from assembled fragments has been an ever-standing problem. While automated binning software and their combination produce prokaryotic bins in high throughput, their manual refinement has been slow, sometimes difficult or missing entirely facilitating error propagation in public databases. Here, we present uBin, a GUI-based, standalone bin refiner that runs on all major operating platforms and was additionally designed for educational purposes. When applied to the public CAMI dataset, refinement of bins using GC content, coverage and taxonomy was able to improve 78.9% of bins by decreasing their contamination. We also applied the bin refiner as a standalone binner to public metagenomes from the International Space Station and demonstrate the recovery of near-complete genomes, whose replication indices indicate the active proliferation of microbes in Earth's lower orbit. uBin is an easy to instal software for bin refinement, binning of simple metagenomes and communication of metagenomic results to other scientists and in classrooms. The software and its helper scripts are open source and available under https://github.com/ProbstLab/uBin.

Label-Free Raman Microspectroscopy for Identifying Prokaryotic Virocells.

Raman microspectroscopy has been used to thoroughly assess growth dynamics and heterogeneity of prokaryotic cells, yet little is known about how the chemistry of individual cells changes during infection with virulent viruses, resulting in so-called virocells. Here, we investigate biochemical changes of bacterial and archaeal cells of three different species in laboratory cultures before and after addition of their respective viruses using single-cell Raman microspectroscopy. By applying multivariate statistics, we identified significant differences in the spectra of single cells with/without addition of virulent dsRNA phage (phi6) for Pseudomonas syringae. A general ratio of wavenumbers that contributed the greatest differences in the recorded spectra was defined as an indicator for virocells. Based on reference spectra, this difference is likely attributable to an increase in nucleic acid versus protein ratio of virocells. This method also proved successful for identification of Bacillus subtilis cells infected with the double-stranded DNA (dsDNA) phage phi29, displaying a decrease in respective ratio, but failed for archaeal virocells (Methanosarcina mazei with the dsDNA methanosarcina spherical virus) due to autofluorescence. Multivariate and univariate analyses suggest that Raman spectral data of infected cells can also be used to explore the complex biology behind viral infections of bacteria. Using this method, we confirmed the previously described two-stage infection of P. syringae's phi6 and that infection of B. subtilis with phi29 results in a stress response within single cells. We conclude that Raman microspectroscopy is a promising tool for chemical identification of Gram-positive and Gram-negative virocells undergoing infection with virulent DNA or RNA viruses. IMPORTANCE Viruses are highly diverse biological entities shaping many ecosystems across Earth. However, understanding the infection of individual microbial cells and the related biochemical changes remains limited. Using Raman microspectroscopy in conjunction with univariate and multivariate statistics, we established a marker for identification of infected Gram-positive and Gram-negative bacteria. This nondestructive, label-free analytical method at single-cell resolution paves the way for future studies geared towards analyzing virus-host systems of prokaryotes to further understand the complex chemistry and function of virocells.

Virus-associated organosulfur metabolism in human and environmental systems.

Viruses influence the fate of nutrients and human health by killing microorganisms and altering metabolic processes. Organosulfur metabolism and biologically derived hydrogen sulfide play dynamic roles in manifestation of diseases, infrastructure degradation, and essential biological processes. Although microbial organosulfur metabolism is well studied, the role of viruses in organosulfur metabolism is unknown. Here, we report the discovery of 39 gene families involved in organosulfur metabolism encoded by 3,749 viruses from diverse ecosystems, including human microbiomes. The viruses infect organisms from all three domains of life. Six gene families encode for enzymes that degrade organosulfur compounds into sulfide, whereas others manipulate organosulfur compounds and may influence sulfide production. We show that viral metabolic genes encode key enzymatic domains, are translated into protein, and are maintained after recombination, and sulfide provides a fitness advantage to viruses. Our results reveal viruses as drivers of organosulfur metabolism with important implications for human and environmental health.

Lytic archaeal viruses infect abundant primary producers in Earth's crust.

The continental subsurface houses a major portion of life's abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.