ISME Communications

Quantifying the abundance and activity of bacteria within populations and communities is fundamental to systems microbiology and microbiome research. Yet direct microscopic cell counting remains low-throughput, labor-intensive, and prone to user variability, leading many researchers to rely on indirect proxies such as optical density or multicopy marker-gene quantification. These indirect approaches do not distinguish between active and inactive cells and can obscure ecological interpretation. Here, we introduce MATRIX (Microbial Activity and Total cell quantification via Rapid Imaging and eXtraction), an efficient workflow that integrates sample extraction, fluorescence staining, automated microscopy and image analysis, and Bayesian statistical inference to quantify total and redox-active cells and derive single-cell measurements for environmental microbial populations and communities. We demonstrate its reproducibility and versatility using both cultured isolates and high-diversity soil communities. The resulting quantitative, phenotypic datasets provide rapid, direct measurements of population of community size and activity, enabling well-powered analyses that strengthen mechanistic insight into microbial responses and improve the ecological grounding of microbiome studies. ImportanceMicrobiome studies commonly rely on relative abundance data, which cannot distinguish whether compositional shifts reflect true population growth, declines in total community size, or both. Without explicit measurements of population and community sizes, mechanistic interpretation of microbiome dynamics remains incomplete. Here we present a rapid, throughput workflow, MATRIX, that quantifies both total and redox-active bacterial cells from environmental samples. By integrating single-cell phenotypes with community-level metrics, this approach anchors microbiome datasets in direct ecological accounting rather than proxies. These measurements can clarify whether observed changes in community structure represent shifts in abundance, activity, or both, improving inference about microbial responses to stress or environmental change. MATRIX therefore offers an efficient way to incorporate quantitative ecology into systems-microbiology and microbiome studies and to strengthen the link between microbial cellular physiology, community dynamics, and eco-system function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/712149v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@2e5883org.highwire.dtl.DTLVardef@b5412dorg.highwire.dtl.DTLVardef@1c9fbfaorg.highwire.dtl.DTLVardef@1bdde14_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionCaves represent unique, nutrient-limited windows into the deep biosphere, yet the microbiology of the deep terrestrial subsurface remains remarkably under-explored. In this work, we took advantage of a rare expedition into Gourgouthakas Cave (Crete, Greece), one of the worlds deepest vertical systems, which had remained untouched by humans for 19 years. MethodsWe performed a high-resolution vertical profiling of the caves microbiome by sampling rock surfaces across nine different depths down to 1,100 meters. Through extensive cultivation using various media and temperatures, we established a biobank of 820 bacterial isolates. ResultsTaxonomic identification of a 362-isolate subset revealed a diverse community spanning 25 genera and 4 phyla, dominated by Pseudomonas, Bacillus, and Stenotrophomonas. Beyond characterizing diversity, we explored the biotechnological potential of these subterranean microbes against major agricultural threats. Screening 70 representative isolates against six key pathogens, including Ralstonia solanacearum, Verticillium dahliae, and Phytophthora nicotianae, uncovered a significant group of strains with potent antagonistic activity, particularly within the Pseudomonas and Brevibacillus groups. Genomic sequencing of cave-derived Actinobacteria (Streptomyces and Nocardiopsis isolates) further highlighted this potential, revealing 142 biosynthetic gene clusters (BGCs); notably, over half of these showed little to no similarity to known clusters, suggesting a hidden reservoir of novel secondary metabolites. Finally, ex vivo trials showed that the Pseudomonas sp. SRL917 isolate, significantly reduced Botrytis cinerea infections on tomato leaves, even surpassing the performance of a commercial biocontrol agent. DiscussionCollectively, our results demonstrate that deep karstic systems are not just geological wonders but vital hotspots for microbial innovation with tangible applications for sustainable agriculture.

BackgroundBrazilian sponges of the genus Metania (phylum Porifera) are filter-feeding organisms from freshwater ecosystems. Here, we explored viral communities of Metania sp., their functional role in the sponge and how they differ from those in surrounding water. ResultsWe identified 1163 viral operational taxonomic units (vOTUs) from sponge tissue and adjacent water, with 555 vOTUs shared across habitats. Viral diversity was higher in sponges than in water, and community composition differed significantly (PERMANOVA, p = 0.037). Sponge-associated vOTUs exhibited broad phylogenetic diversity, including deep-branching and unclassified clades, and several exclusively sponge-associated Caudoviricetes. Virus-host predictions revealed 173 interactions, largely with sponge-associated bacteria, supported by CRISPR spacer matches, variant formation in multiple vOTUs across sponge individuals, and a high prevalence of microbial defence systems, particularly restriction-modification, abortive infection, and CRISPR-Cas pathways. Functionally, viral communities carried diverse auxiliary viral genes, including those involved in amino acid and central carbon metabolism, carbohydrate degradation, fatty acid biosynthesis, stress responses (e.g., metacaspase-1), and sulphur cycling. Nine sponge-associated vOTUs encoded carbonic anhydrase (CA), and phylogenomic as well as structural analyses showed strong conservation of CA active sites between sponge viruses, bacterial symbionts, and the sponge host. Protein-level homology searches revealed broad biogeographic distribution of viral CA homologs across global ocean microbiomes, despite limited nucleotide similarity, highlighting deep functional conservation. ConclusionsThese findings reveal a phylogenetically diverse and functionally rich viral community associated with freshwater Metania sp., characterized by extensive host interactions, diverse defence mechanisms, and auxiliary metabolic capacities. The structural conservation and widespread distribution of viral carbonic anhydrase genes further suggest ecologically significant roles in carbon transformation within freshwater sponges and potentially across aquatic ecosystems.

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane-spanning lipids (MSLs) resembling archaeal membrane lipids, as they form monolayers and are linked to glycerol backbones via ether bonds. Ubiquitous in soils, sediments, and aquatic environments, their distributions are widely applied as paleoclimate proxies for reconstructing past temperature and pH. Despite this, understanding of their biological origins and functional role in cells remain incomplete. While some Acidobacteria are known producers of brGDGTs, genomic evidence and environmental surveys indicate additional bacterial contributors. In this study, we report the first detection of potential brGDGT biosynthetic intermediates in Bacillota. Ultra-high pressure liquid chromatography high resolution multi-stage mass spectrometry (UHPLC-HRMSn) revealed membrane-spanning diglycerol lipids which contained iso-diabolic acid (13,16-dimethyl octacosanedioic acid)-derived alkyl chains. These diglycerol lipids displayed diverse structures, including tetraesters, mixed ester/ether combinations, and vinyl ether bonds. Additionally, open membrane spanning lipids analogous to brGTGTs were also identified. Notably, all brGDGT and brGTGT analogues were detected with a phosphatidylglycerol head group. Experiments showed that the two Bacillota strains, which produce these brGDGT biosynthetic intermediates, responded differently to changes in temperature and oxygen availability, suggesting that environmental regulation of brGDGT-related lipids is taxonomically dependent. Based on these findings, we propose a biosynthetic pathway for brGDGT formation and highlight the physiological implications for interpreting brGDGT-based paleoclimate proxies. This work expands the known diversity of bacterial sources of brGDGTs and provides new insights into the ecological and evolutionary significance of these lipids. IMPORTANCEBranched GDGTs (brGDGTs) are bacterial membrane spanning lipids which form a monolayer, linked through ether bonds to the glycerol backbone, characteristics more commonly found in archaeal membrane lipids. They are commonly used in paleoclimate proxies to assess past temperature and pH but their predictive power is hampered by the lack of information regarding their biological producers. Branched GDGTs have been detected in just a few species of the Acidobacteria but there are strong indications that other bacterial phyla also contribute to the pool of brGDGTs in the environment. Here, we report for the first time the production of potential brGDGT intermediates in Bacillota species. This study demonstrates that brGDGTs likely occur much more widespread in the bacterial domain than previously thought and opens a new chapter both in the understanding of the function of these membrane lipids and their use in paleoclimatology.

Modern agriculture faces the dual challenge of increasing food production while reducing reliance on synthetic inputs that degrade soil ecosystems and compromise long-term sustainability. Algal biomasses have emerged as promising biostimulants, yet their capacity to selectively modulate soil microbiomes and plant growth-promoting bacterial (PGPB) functions remains poorly understood. Here, we evaluated 17 phylogenetically and biochemically diverse macro- and microalgal extracts to determine their effects on soil microbial communities, bacterial functional traits, and tomato (Solanum lycopersicum) performance. Algal supplementation selectively restructured microbial communities without disrupting overall diversity, promoting taxa associated with plant-beneficial functions, including Bacillus, Pseudomonas, and Actinobacteria. In soil microcosms, specific treatments increased culturable bacterial abundance by up to [~]200-fold relative to the initial soil. Functional assays revealed strong extract- and strain-dependent responses. Siderophore production and ACC-associated activity were the most consistently stimulated traits, whereas auxin production, biofilm formation, and proline synthesis showed more variable or context-dependent responses. Notably, Ulva sp. (AP11.2) enhanced siderophore production across the majority of isolates, with over four-fold increases in individual strains, while Arthrospira-derived extracts (NG4.1, N14.1) consistently promoted bacterial growth across multiple taxa. In contrast, extracts such as Nannochloropsis sp. (NG6.1) and Tetraselmis sp. (NG5.1) induced more selective or inhibitory responses, highlighting extract-dependent functional trade-offs. Integration of biochemical and biological datasets identified fatty acid composition as a key axis associated with microbial functional responses, whereas volatile organic compound profiles showed weaker and less consistent associations. These microbiome and functional shifts translated into improved plant performance, with algal treatments increasing tomato growth and reducing mortality by approximately 20% under non-sterile soil conditions characterized by pathogen-associated pressure. Together, these findings demonstrate that algal extracts act as selective modulators of soil microbiomes, enhancing specific bacterial functions and improving plant performance in a context-dependent manner. This work provides a mechanistic framework for the development of targeted algal-based biostimulants aimed at reducing agrochemical inputs and advancing microbiome-informed agriculture.

Chlorophyll is one of the most abundant pigments on Earth. Although its degradation is well understood in plants, the role of prokaryotes in this process - despite their vast metabolic capabilities - remains unknown. Recent developments in the field of AI-predicted protein structures have opened new avenues for investigating functional homologies between evolutionary-distant organisms previously inaccessible through traditional sequence- or profile-based methods. Here, we present the first evidence of Chlorophyll a (Chl a) degradation by prokaryotes, discovered through a novel bioinformatic framework which bridges the gap across the domains of life via structural alignments of functionally characterised plant proteins, followed by structure similarity graph-based clustering. Metagenomic sequencing data was assembled and binned, yielding over 70,000 medium- to high-quality genomes in total, furthermore publicly available datasets containing genomes from prokaryotic isolates, metagenome-assembled genomes, as well as single-cell genomes were then mined for prokaryotic homologues of Chl a degradation genes. Our analysis revealed over 400 genomes from diverse taxonomic groups and habitats that possess a complete pathway, more than 50% stemming from isolates. Additionally, many other genomes harbour partial pathways, suggesting that Chl a degradation capabilities are globally widespread across diverse ecosystems. We then validated our in silico findings using the model organism Shewanella acanthi and confirmed its Chl a degradation capability via growth experiments, fluorescence spectroscopy and HPLC analyses. Our findings reveal a previously unrecognised pathway in prokaryotes, highlighting the power of structure-based remote homology detection for uncovering metabolic capabilities and evolutionary relationships.

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota. The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales. Cells of strains AB-alg1T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1T (JCM 35393T=UQM 41574T) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae. ImportanceThe presented isolates are the first isolated representatives of an environmental family of Opitutales, part of the core microbiome of alkaline soda lakes. These bacteria feed on polysaccharides. We present the key enzymatic machinery for the polysaccharide breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example in washing powders and biomass waste recycling.

Bacterivorous protists are central to aquatic food webs, mediating the transfer of carbon and nutrients to higher trophic levels through the microbial loop. In natural communities, a major challenge remains in linking protist grazing activity to environmental sequences and identifying which taxa are actively feeding at the community level. Here, we present the first application of quantitative stable isotope probing (qSIP) in a grazing experiment. By combining qSIP with 18S rRNA gene amplicon sequencing, we linked prey assimilation to the identity of active protist predators at the operational taxonomic unit (OTU) level. In a replicated 36-h bottle-experiment, live 13C, 15N-labeled Limnohabitans planktonicus cells were added to natural samples from a lake pelagic site and its main inlet stream. Although hydrologically connected, protist richness was higher in the inlet than in the lake, yet a similar number of taxa incorporated prey biomass, comprising 108 OTUs in the inlet and 107 OTUs in the lake, including both rare and abundant taxa. Of these, 26 OTUs were labeled at both sites. The most strongly labeled protist in the inlet was a putative phago-mixotrophic prasinophyte, whereas in the lake it was an uncultured chrysophyte. Across sites, prey incorporation occurred in a broad range of taxa, including heterotrophs (e.g., choanoflagellates, cercozoans, ciliates, centrohelids), putative mixotrophs (e.g., cryptophytes, chrysophytes, dictyochophytes), parasitic protists and fungi. These results demonstrate the potential of qSIP to resolve trophic interactions at fine taxonomic resolution in natural communities and highlight new opportunities to study complex microbial food webs across environmental systems.

Succession is an ecosystem building process in which a habitat and its community interact predictably by increasing diversity, habitat engineering, and ultimately reaching a climax community, where other ecological processes influence its dynamic. Key to succession is the establishment of primary producing habitat forming species, which drives niche differentiation leading to increasing diversity. Here, we use the primary colonizing and habitat forming seagrass, Halophila ovalis, to demonstrate that it drives bacterial succession in a meadow ecosystem, and its microbiome, both rhizoplane and phylloplane, are under host selection. Many of the characteristics attributed to plants for habitat modification are microbial processes such as nitrogen fixation and sulfide detoxification and succession is often extrapolated to such processes. To determine if succession (increasing diversity) or selection (reducing diversity) drives changes in diversity (16S rRNA gene) or habitat modifying processes (nifH, soxB, aprA, dsrA), molecular analysis was performed along chronosequences (as a proxy for succession) of seagrass patches. Bacterial communities were sampled within the meadow ecosystem and the microbiomes of H. ovalis (both rhizoplane and phylloplane). Genes involved in biogeochemical cycling are differentially impacted within the microbiome and meadow sediments, with only nifH under succession. All genes from all niches sampled for community analysis are under directional community trajectories, despite being subjected to distinct ecological processes, signifying that many ecological processes, including succession and host association, drive community assemblage.

Seagrasses are the only plants that flower and produce seeds in the marine environment, where they form vast meadows that fulfill important ecosystem functions. Yet, seagrass cover has declined in many coastal areas around the world and re-colonization is slow. Despite clonal growth, seed recruitment is essential for seagrass dispersal and regional genetic diversity. While the seed microbiome of several terrestrial plants has been shown to influence germination and seedling survival, the role of the seagrass seed microbiome is still unclear. We investigated the microbiome of eelgrass (Zostera marina) seeds, leaves and roots along the natural salinity gradient of the German North and Baltic Sea coasts. Despite this strong variability, Z. marina seeds harbor distinct prokaryotic and eukaryotic microbial communities compared to those on leaves and roots. Predicted microbial functions suggest roles in nutrient cycling and germination, which may be critical for recovery and restoration of seagrass ecosystems. Scientific significance statementSeagrass meadows are important but declining coastal ecosystems. Around the world, efforts are being made to restore seagrass meadows in order to halt the loss of biodiversity and maintain ecosystem function. With an evolutionary history on land, seagrasses have introduced unique features to the marine environment, such as seeds. In terrestrial plants, the seed microbiome has been shown to be important for seed germination and seedling health. Here we show that the northern hemisphere seagrass Zostera marina has a distinct seed microbiome, containing core bacterial and eukaryotic taxa and predicted functions that may play a role in seed germination and vertical microbiome transmission. Our results lay the foundation for future seagrass restoration efforts that seek to manipulate seed microbiomes to improve seedling survival. Data availability statementRaw DNA sequence data has been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB72211. Processed data and associated metadata will be made available on PANGEA via the German Federation for Biological data (GFBio.org).

Anaerobic methanotrophic (ANME) archaea are important players in the microbial methane cycle, mitigating methane emissions from anoxic environments. ANME are found ubiquitously in methane-rich sediments, where they can couple anaerobic methane oxidation (AOM) to different electron acceptors such as sulfate, metal oxides, and natural organic matter (NOM). However, we still lack understanding of the geochemical niches and preferred metabolic pathways of most ANME subclades. Here, we investigated the genomic potential and ecophysiology of ANME-2a with respect to metal-dependent AOM in brackish metal-rich coastal sediments. We assembled several high-quality ANME MAGs from subclades with high strain heterogeneity and analyzed the genomic potential for metal-AOM. Additionally, we monitored long-term enrichments with various electron acceptors from the same sediments. Ultimately, we recovered 8 novel genomes of ANME-2a that clustered with an uncharacterized genus with only 2 representatives in public databases for which we propose the name Candidatus Methanoborealis. The analysis of the MAGs showed two different clusters within this genus; one comprising of MAGs from the Baltic Sea that showed high potential for extracellular electron transfer (EET) required for metal-AOM, and another cluster form more diverse environments with less EET potential. The Baltic Sea Ca. Methanoborealis were the only canonical methanotrophs in the incubations during active methane oxidation and metal reduction. Our results contribute to the understanding of the phylogenomic and metabolic diversity in ANME subclades, which will help to further characterize novel ANME lineages from complex sediment samples.

Microbial biodiversity is central to ecosystem function, yet mechanistic insights into the cell biology of environmental organisms remain limited. The underlying challenges are twofold: most microbes remain uncultivable, and a persistent gap exists between field sampling and laboratory analyses. Here, we introduce the Advanced Mobile Laboratory (AML), a field-deployable platform that integrates confocal microscopy, image-enabled cell sorting, and cryo-preparation for expansion and electron microscopy. This setup enables immediate, standardized processing and analysis of environmental communities directly at the sampling site. We demonstrate its capability using marine eukaryotic plankton, showing how the AML enables multiscale investigations, from live imaging of natural communities to enabling ultrastructural and single-cell omics analyses, while minimizing sample degradation and enabling on-site experimentation. By bringing high-end sample preparation and analytical capacity into the field, the AML enables studying life in its natural context to mechanistically understand lifes diversity in the environment.

Plasmid host range (PHR) plays a key role in the spread of ecologically important genes, alongside applications in microbiome engineering, and environmental biotechnology. PHR is a complex trait arising from the combination of plasmid, donor and recipient properties. Most studies of PHR use a single donor strain, leaving the role of the donor unexplored, and often require genetically tagged recipient strains for counter selection, which limits use of non-genetically tractable strains. Here we developed a PHR screening method using auxotrophic donors that bypasses the need to genetically tag recipients, thus allowing the screening of culturable environmental bacterial strains. Specifically, we used two auxotrophic donors (P. fluorescens and P. putida), and the plasmid pQBR57-tphKAB, an environmental plasmid engineered for terephthalic acid bioremediation. We screened a library of 101 soil isolates, as potential recipients, including common soil genera of soil bacteria, Pseudomonas, Bacillus and Xanthomonas. We only observed conjugation into other Pseudomonas, but donor identity affected PHR, with P. fluorescens conjugating the plasmid into more recipient strains than P. putida. Phylogenomic analysis revealed that transconjugants clustered with P. citronellosis and P. putida lineages. In strains that were close relatives of transconjugants but who were unable to acquire the plasmid, we observed 5 defence systems not present in transconjugants that may act as barriers to plasmid acquisition. Our method provides a rapid, tag-free framework for screening PHR in environmental isolates and for investigating the influence of donor identity on plasmid conjugation.

The discovery of stress-tolerant microorganisms from complex microbiomes is frequently constrained by low screening throughput and the inability of culture-based approaches to access single-cell functional phenotypes, particularly for rare but highly resilient taxa. Here, we establish a metabolism-driven screen-before-culture strategy by integrating D2O-labelled single-cell Raman spectroscopy (SCRS) with Raman-activated cell sorting (RACS) to directly target ethanol-tolerant cells based on metabolic vitality. By quantifying carbon-deuterium (C-D) incorporation as a single-cell readout of de novo anabolic activity under ethanol stress, this platform enabled culture-independent enrichment of a rare, high-vitality subpopulation ([~]0.2% abundance) from pit mud microbiomes at a sorting throughput of [~]2,400 cells per hour. Using this approach, six highly ethanol-tolerant strains were successfully isolated, whereas parallel conventional culture-first screening of the same samples yielded predominantly low-tolerance isolates, with an overall screening efficiency of only 22.22%. Rapid single-cell ethanol tolerance assessment based on SCRS, completed within 7 h, showed that all sorted strains exhibited strong tolerance to 8% (v/v) ethanol, with Raman Tolerance Index (RTI) values exceeding 50%. Among them, Lactiplantibacillus plantarum F4 displayed the highest tolerance (RTI = 85.05 {+/-} 3.41%). Comparative transcriptomic analyses of representative strains revealed mechanistically coherent ethanol adaptation strategies, including ethanol-derived carbon recycling, dynamic membrane lipid remodeling, and reinforced redox homeostasis. These responses directly underpin the metabolic activity captured by the Raman screening signal, validating its physiological relevance. This integrated SCRS-RACS workflow achieved orders-of-magnitude higher screening throughput, a 4.5-fold improvement in sorting accuracy, and a 6.86-fold increase in assessment efficiency compared with conventional methods. This study establishes a versatile, metabolism-based paradigm for the targeted mining of rare, stress-tolerant microorganisms from complex microbiomes, with broad implications for industrial biotechnology and microbial ecology.

Endophytic microbiomes of crop wild relatives (CWRs) adapted to extreme environments, such as halophytes, are promising sources of plant-beneficial bacteria and secondary metabolites for sustainable food production. Here, we analyzed 25 Bacilli isolates obtained from CWRs, halophytes, and other plant species in Crete, Greece. Using a hybrid Illumina-PacBio sequencing approach, we generated high-quality genomes and performed comparative genomics, phylogenetic, and pangenome analyses, complemented by in vitro assays. We identified 312 biosynthetic gene clusters (BGCs), nearly 60% of which showed no similarity to known clusters, revealing extensive unexplored biosynthetic potential. These unique BGCs may constitute an adaptive feature enabling endophytic Bacilli to colonize and interact with host plants. The isolates spanned diverse genera (Bacillus, Paenibacillus, Peribacillus, Neobacillus, Cytobacillus, Rossellomorea), including three novel species. Phenotypic assays of our isolates demonstrated high salinity tolerance (up to 17.5% w/v NaCl) and strong antagonism against major bacterial and fungal phytopathogens. Genome mining further revealed a broad array of putatively plant-beneficial traits related to growth promotion, stress adaptation, host interaction and inhibition of pathogens. Together, these findings show that Bacilli endophytes from wild and halophytic plants possess exceptional phylogenetic novelty, functional diversity, and biosynthetic capacity, providing new genomic and ecological insights into Bacilli associated with plants inhabiting extreme environments.

BackgroundThe global rise of antimicrobial resistance has intensified the search for new microbial metabolites from underexplored environments and taxonomic groups. Extreme and geographically isolated habitats, such as Antarctic terrestrial ecosystems, represent promising reservoirs of novel biosynthetic diversity, particularly among rare and difficult-to-cultivate actinomycetes, where chemical mediators are thought to play key roles in microbial persistence and interaction under resource-limited conditions. ResultsHere, we report the characterization of kineochelins, a previously undescribed group of siderophores produced by an Antarctic isolate, Actinokineospora sp. UV203 representing difficult to cultivate actinomycetes. Structural elucidation revealed a set of closely related congener molecules with a mixed-ligand architecture consistent with metal-chelating activity. Genome mining combined with transcriptomic analysis identified the involvement of a dedicated nonribosomal peptide synthetase-encoding biosynthetic gene cluster responsible for kineochelin production. Comparative genomic analyses showed that, although kineochelin biosynthetic genes share limited homology with those of known mixed-ligand siderophores, their biosynthetic pathways differ substantially in gene content and organization, indicating a distinct evolutionary lineage. Functional characterization of kineochelins demonstrated strong and selective iron chelation, with pronounced affinity for ferric and ferrous iron. Crude culture extracts inhibited the growth of bacterial strains isolated from the same Antarctic environment, suggesting that kineochelin-associated chemistry contributes to iron-mediated competitive interactions within native microbial communities. In addition, kineochelin-enriched fractions exhibited selective inhibitory activity against the opportunistic yeast pathogen Nakaseomyces glabratus and a clinical isolate of Saccharomyces cerevisiae associated with invasive infection. ConclusionsTogether, these findings expand the known chemical and biosynthetic diversity of the genus Actinokineospora and demonstrate that Antarctic rare actinomycetes are a valuable source of novel natural products with potential relevance for microbial ecology and biotechnology. The ecological activities of kineochelins highlight the role of iron acquisition in shaping microbial interactions in extreme environments and underscore the biotechnological potential of metabolites derived from underexplored polar microorganisms.

Life on Earth has long been regarded as homochiral, relying almost exclusively on a single enantiomer of sugars--typically the D-form. However, recent discoveries challenge this paradigm, including the identification of L-glucose-catabolizing bacteria and microbial L-glucoside hydrolases. Despite these findings, the metabolic diversity of organisms toward a broader range of atypical sugar enantiomers and their ecological relevance remains largely unexplored. This study aimed to identify and isolate microorganisms capable of catabolizing atypical enantiomers of diverse sugars. We performed enrichment cultures with either the D- or L-forms of glucose, fructose, xylose, and sorbose, using soil and activated sludge as microbial sources. Microbial growth was observed under all tested conditions, with the dominant taxa varying depending on the sugars supplied. Six phylogenetically distinct bacterial isolates exhibited the ability to catabolize atypical sugar enantiomers, two of which exhibited growth on all tested sugars. These findings uncover a previously unrecognized diversity in microbial sugar metabolisms, providing new insights into the environmental dynamics of atypical sugar enantiomers and offering a novel perspective on the principle of biological homochirality. Furthermore, this work lays a foundation for the development of biomanufacturing processes using racemic sugar mixtures synthesized via abiotic chemical reactions.

Cyanophages infecting multicellular, heterocyst-forming cyanobacteria play diverse pivotal roles in freshwater ecosystems, yet their infection strategies and genomic adaptability remain poorly understood. Here, we isolated and characterized three Caudoviricetes cyanophages (A-Lf14, A-Alj1 and A-Hlh1) specifically infecting the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. Super-resolution microscopy revealed heterogeneous infection outcomes even among adjacent cells within the same filament, highlighting host-level defense variability. Comparative genomics placed these phages within a cluster that includes the previously isolated phages A-1(L) and N1, and revealed a conserved [~]30 kb invertible genomic region flanked by inverted repeats (IR). This region exists as two stable isomers simultaneously within phage populations, a previously unreported genomic plasticity trait in cyanophages. Consistent with such plasticity, infection kinetics were modulated by light and nitrogen availability, indicating a resource-responsive infection strategy. The novel phages encode an alkaline phosphatase of non-cyanobacterial origin, which was highly upregulated during infection indicating a role in phosphate acquisition in phosphate-limited waters. In contrast, they lack a tnpB gene present in A-1(L), which is identical in sequence to five genes in the host genome. We detected protein-coding potential for both strands of tnpB and upregulated transcription during infection, consistent with a role in this process. The 347 residues protein encoded on the tnpB reverse strand exhibited only limited similarity to other proteins or folding potential, underlining its novelty. Our work illustrates how genomic isomerism, accessory genes, and environmental sensing collectively drive functional diversification in cyanophages, providing insights into phage-host coevolution and the impact of phages on cyanobacterial blooms in dynamic freshwater ecosystems.

Microbial communities of geothermal habitats are central to understanding the evolution of life on Earth. Metagenomics has provided insight into the role of viruses in shaping microbial diversity of complex environments. However, identification of novel viruses is constrained by lack of marker genes and low nucleotide similarities between related viral taxa. While microbial and viral diversity have been explored in terrestrial hot springs and hydrothermal vent systems, other volcanic features remain underexplored. Fumaroles (steam vents) are geothermal features that heat groundwater with magma, releasing steam and volcanic gases such as CO2 and H2S. Comparatively physicochemically dynamic to hot springs, fumarole temperatures and gas emissions rapidly fluctuate with volcanic activity. Here, we describe viruses identified metagenomically from microbial mats hosted near basaltic fumaroles on the Big Island of Hawai`i. To our knowledge, this is the first systematic survey of fumarole viruses. Our utilization of a sensitive profile-based approach for identification reveals high viral diversity in fumaroles, resulting in estimation of two undescribed order-level clades of Caudoviricetes (tailed phages). Viral metabolic genes provide evidence of viral-mediated adaptation of microbes to fumarole conditions. We describe patterns of viral diversity that diverge from the Bank model of viral ecology, hinting at viral dispersal between biofilms and high viral richness and evenness. Lastly, we provide a description of the first terrestrial geothermal environment dominated by Microviridae, previously only described in viral communities of deep ocean hydrothermal vents. This study offers important findings for exploration of viral ecology in extreme environments.

Nowadays, finding new sustainable ways to combat plastic pollution is a pressing challenge. Here, we provide a comprehensive genome mining analysis of 284 publicly available Stutzerimonas genomes for potential PET-active enzymes (PETases). While Stutzerimonas is a relatively newly established genus, it emerges as an interesting candidate in the search for novel biocatalysts. Hence, the first pangenome assessment of this genus based on its high-quality publicly available genomes was performed. An increasingly open pangenome was revealed, suggesting the versatility and adaptability of these strains to a variety of ecological niches. Moreover, functional characterisation of a new isolate, Stutzerimonas frequens VG-9, was carried out, confirming that enzymes found via in silico analyses may indeed display activity towards different polyesters. In summary, this study provides insights into the diversity of PETase homologues within still underexplored bacterial hosts, offering new perspectives for enzyme discovery in the Pseudomonadaceae family. Impact StatementMicrobial enzymes known as PETases have emerged as promising candidates for the biological degradation of PET. This study investigated the potential of underexplored bacterial genera by genome mining of PETase homologues. Our findings provide new insights into the distribution of PETase-like enzymes in the Pseudomonadaceae family, offering a more comprehensive view of their plastic degradation capacity. These results hold practical implications for the development of optimized enzyme discovery strategies, while also highlighting the vast genetic plasticity of Pseudomonadaceae. We also provided the first report on the Stutzerimonas pangenome and insights into the enzymatic activity towards polyesters of a newly isolated strain. Hence, the role of this genus as a highly adaptable and versatile entity was reinforced, further disclosing it as a potential source of novel biocatalysts. Data SummaryThe genome of S. frequens VG9 has been deposited in Genbank under the accession number SAMN49487720. The accession numbers of all analyzed genomes are listed in Tables S2 and S3 (available in the online Supplementary Material).