The ISME Journal

As key drivers of nitrification, ammonia-oxidizing archaea (AOA) play a central role in the global nitrogen cycle and contribute significantly to the emissions of the potent greenhouse gas nitrous oxide (N2O). However, the ecological implications of AOA growth as biofilms, remain poorly understood. Since nitrite production can be used to follow cellular activities directly we were able to compare biofilms with planktonic cells of the terrestrial model AOA Nitrososphaera viennensis at ecologically and agriculturally relevant conditions. Biofilms were more resistant across nearly all tested conditions and remained active at lower temperatures, acidic pH, and high ammonium concentrations. Collectively, activities in biofilm help reconcile discrepancies between earlier laboratory and environmental observations of soil AOA. Additionally, biofilms showed a high general resilience and lowered sensitivities to nitrification inhibitors. Although in situ biofilms grown in microrespiratory chambers exhibited activity and ammonia affinity similar to planktonic cells, biofilm cultures produced only half as much N2O. The enhanced fitness of biofilms across all tested conditions vastly expands the potential ecophysiological niche of AOA and supports the hypothesis that biofilm growth represents the in situ phenotype of AOA in soil environments.

Understanding how intra- and interspecific differentiation arises in natural microbial populations is central to explaining the processes that drive bacterial evolution. Motivated by the co-occurrence of several genospecies closely related to Shewanella baltica in Baltic Sea sediments, we investigated the genomic structure of this species complex across fine spatial scales. We analyzed 112 genome sequences from strains collected across multiple sediment cores and depths (0-6 cm) at Vaxon (Stockholm archipelago, Sweden), including sympatric isolates from this site as well as earlier isolates and allopatric strains from other locations in the Stockholm region obtained from both sediments and the water column. Using a reverse-ecology population genomics approach, we found that these strains form a species complex that resolves into three cohesive evolutionary lineages (G1, G2, and G3). Each lineage is characterized by extensive gene turnover, driven largely by horizontal gene transfer (HGT), and displays distinct genomic signatures of metabolic specialization. While G1 consists predominantly of a single species (S. baltica), G2 and G3 comprise a diverse set of divergent genospecies, many of which are repeatedly recovered from sediment samples. Patterns of homologous recombination indicate that speciation within G2 and G3 is primarily recombination-driven ( sexual), and that both groups derive from a common ancestor. Together, these results capture a snapshot of early-stage speciation within a shared ecosystem and provide insight into the mechanisms that diversify sympatric, recombining bacterial populations, with a sediment-associated lifestyle likely promoting this process.

1.Structured Abstract1.1 AbstractSoybean red crown rot, caused by the soil-borne fungus Calonectria ilicicola, causes substantial yield losses, but the response of the root-associated bacterial microbiome remains poorly understood. Here, we combined 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to characterize bacterial communities in soybean root-associated soils. 16S rRNA gene sequencing showed that diseased plants had rhizosphere and, more strikingly, rhizoplane microbiomes distinct from those of healthy plants, often with increased Enterobacterales. Shotgun metagenomics further revealed enrichment of genes associated with antibiotic resistance, particularly cationic antimicrobial peptide resistance, in diseased rhizoplane samples. Single-cell genomics recovered seven nonredundant Enterobacterales genomes and showed that plant pathogenicity-related genes were broadly distributed across these lineages. In contrast, dlt genes, which are associated with cationic antimicrobial peptide resistance, were detected only in the Enterobacterales lineages enriched in diseased rhizoplane soils. These results support a model in which soybean red crown rot is accompanied by microbiome restructuring and opportunistic enrichment of specific Enterobacterales lineages carrying putative cationic antimicrobial peptide resistance genes. More broadly, this study highlights the value of strain-resolved single-cell genomics for linking disease-associated community shifts to specific bacterial traits. 1.2 ImportanceUnderstanding crop disease requires resolving not only the primary pathogen but also the root-associated bacteria that respond to infection. Here, we used 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to examine the soybean rhizoplane microbiome under red crown rot. Diseased plants showed reproducible shifts in bacterial composition, including frequent enrichment of Enterobacterales and antimicrobial resistance-related functions. Strain-resolved genomes further revealed that the Enterobacterales lineages enriched in diseased rhizoplane soils specifically carried putative dlt-mediated resistance to cationic antimicrobial peptides, whereas general pathogenicity-related genes were broadly shared. These findings suggest that host defense-associated selection, rather than pathogenicity genes alone, may help shape disease-associated root microbiomes. This study demonstrates how single-cell genomics can uncover strain-level traits hidden within bulk community data and thereby clarify plant-pathogen-microbiome interactions.

Antibiotic resistance genes (ARGs) are ubiquitous in marine environments, yet whether their distribution primarily reflects anthropogenic pollution or intrinsic ecological functions remains unresolved. We used genome-resolved metagenomics to characterize resistomes in 371 genomic operational taxonomic units (gOTUs) across a gradient of human impact: the heavily impacted Baltic Sea, the moderately impacted North Sea, and the minimally impacted West Greenland shelf. ARG density was distinctly elevated in the Baltic Sea (3.20 ARGs Mbp-1) relative to the North Sea (1.90) and West Greenland (1.67), which did not differ significantly from each other, suggesting a relatively uniform oceanic baseline. Variance partitioning revealed that taxonomic identity explained 20.1% of ARG density variation, with environment contributing 11.4%; critically, Baltic gOTUs carried 35.1% more ARGs than predicted from taxonomy alone, indicating environment-driven enrichment beyond baseline taxonomic carriage. Lifestyle-dependent ARG partitioning between particle-attached and free-living prokaryotes emerged only under anthropogenic pressure: free-living bacteria were enriched in multiple resistance classes in the Baltic Sea but showed no differentiation in West Greenland. Only 0.85% of detected ARGs showed [≥]70% amino acid identity to clinically characterized sequences in the CARD database, showing that marine ARGs are highly divergent from clinical resistance determinants. Virulence factor annotations were widespread but weakly coupled with ARG abundance, suggesting independent ecological selection. Our results suggest that marine resistomes integrate an intrinsic baseline of ecological functions with selective enrichment of specific resistance mechanisms under anthropogenic pressure, and that genome-resolved approaches are able to quantify the relative contributions of each.

Microbial communities are structured by environmental gradients and metabolic interactions, yet the genomic characteristics and metabolic functions of co-occurring populations remain underexplored. Here, we investigated co-occurring microbial cohorts across the Baltic Sea, a system characterized by strong salinity, temperature, and oxygen gradients. For this, we used a genomic catalog consisting of 701 species-representative genomes to recruit reads from 112 metagenomes and infer cohort structure, environmental distributions, and metabolic potential. We identified nine microbial cohorts that showed strong associations with environmental gradients, indicating deterministic assembly. Cohorts differed markedly in genomic traits, with the most abundant and prevalent taxa associated with smaller, streamlined genomes, while a low-oxygen cohort with larger genomes contributed disproportionately to nitrogen and sulfur transformations. Across cohorts, biosynthetic potential was unevenly distributed. Amino acid biosynthesis pathways were frequently complete, whereas B-vitamin pathways were typically incomplete and rarely encoded in full by individual genomes. Metabolites with low pathway completeness showed consistent taxonomic partitioning, with biosynthetic capabilities distributed across taxa rather than collectively encoded within cohorts. Together, these results show that Baltic Sea microbial cohorts are ecologically structured assemblages whose genomic repertoires reflect catabolic specialization and anabolic interdependencies. Our findings highlight microbial cohorts as a useful framework for linking environmental gradients, genome traits, and the organization of metabolic functions in natural microbial communities.

Eukaryotes originated from the symbiosis of an Asgard archaeon, the alphaproteobacterial ancestor of mitochondria, and possibly additional bacterial contributions. This transition occurred in redox-transition environments such as microbial mats or shallow sediments [~]2 billion years ago, when atmospheric oxygen was far lower than today. We investigated Asgard-enriched microbial mats from the low-oxygen, sulfidic Catherine volcano lake (Afar region, Ethiopia), mimicking early Proterozoic conditions. 16S rRNA gene metabarcoding, metagenomics, and metagenome-assembled genome analyses across redox-stratified layers of in situ and mesocosm-maintained mats revealed that Asgardarchaeota thrived in the sulfate-reduction zone, mainly co-occurring with Desulfurobacterota-Myxococcota, among others. Lokiarchaeia and Thorarchaeia preferred anoxic layers. Within Heimdallarchaeia, Heimdallarchaeales were enriched in upper layers, correlating with oxygen-tolerant hydrogenase and sulfate-reduction genes, and Hodarchaeales, in anoxic layers, correlating with methanogenesis. Although reactive-oxygen-species defense mechanisms were widespread, Asgardarchaeota lacked aerobic respiration. These results support the idea that Asgard archaea engaged primarily in syntrophic interactions with sulfate-reducers under early-Earth-like conditions.

Members of the UBA868 group within the order Arenicellales are globally distributed marine Gammaproteobacteria predicted to participate in sulfur and carbon cycling, yet their physiology and ecological roles remain unknown due to the absence of cultured representatives. Here, we report the isolation and characterization of the first heterotrophic representative of the previously uncultured UBA868 group. Using dilution-to-extinction cultivation, we obtained four isolates from the Yellow Sea whose high-quality genomes represent a single UBA868 species. One strain, IMCC57338, maintained in axenic culture, exhibited small coccoid morphology and slow growth (doubling time [~]2.9 days), consistent with an oligotrophic lifestyle. Genome analysis revealed a predominantly aerobic chemoorganoheterotrophic lifestyle with a streamlined central carbon metabolism, including a complete glyoxylate shunt and limited carbohydrate utilization capacity, suggesting adaptation to low-nutrient conditions. The genome also encodes pathways for methylated amine oxidation coupled to formaldehyde assimilation via the serine cycle, indicating a capacity for methylotrophy. Genes encoding sulfur oxidation (Sox) and reverse dissimilatory sulfite reductase (rDsr) pathways further suggest a capacity for sulfur-based chemolithoheterotrophy. Global metagenomic and metatranscriptomic read recruitment showed that the species represented by IMCC57338 is widely distributed across ocean basins and pelagic depth layers, with higher abundance and transcriptional activity in mesopelagic waters. Our findings provide the first physiological and genomic insights into the UBA868 group and suggest that members of this lineage contribute to the cycling of organic carbon, C1 compounds, and sulfur in marine ecosystems.

Competition for a single limiting resource is expected to lead to competitive exclusion, yet diverse microbial communities persist even in nutrient-poor environments. Cross-feeding of essential metabolites is one mechanism that can promote coexistence between species, but its contribution is difficult to pinpoint experimentally. Here, we studied a prototroph-auxotroph pair growing on a single carbon source in chemostats. In minimal medium, the prototroph Comamonas testosteroni (Ct) supplied thiamine to the thiamine-auxotroph Ochrobactrum anthropi (Oa), allowing stable coexistence in agreement with consumer-resource theory. Contrary to our expectation that supplying thiamine would remove the dependency and lead to exclusion of Ct, coexistence persisted even when thiamine was supplemented. Our theoretical anlaysis showed that coexistence between competitors can be maintained by trace concentrations of an additional metabolite if it is taken up at sufficiently high affinity by the weaker competitor. Consistent with this prediction, targeted metabolomics and spent-medium assays identified growth-enhancing compounds at micromolar concentrations in Oa spent medium and as residues in fresh medium. Model analysis further showed that such weak positive effects can qualitatively change coexistence outcomes in chemostats while remaining undetected in standard batch interaction assays. Together, our results show that trace metabolites and subtle positive effects can reshape coexistence outcomes and should be incorporated into ecological models and interaction measurements.

Host-associated microbiomes are typically maintained in stable configurations that support host fitness, yet the mechanisms by which metabolic perturbations destabilize these communities remain poorly understood. Using the freshwater cnidarian Hydra vulgaris AEP, we systematically assessed microbiome responses to 326 single-metabolite perturbations. Only 17 metabolites, mostly amino acid-related compounds, induced significant compositional shifts in the microbial community. Most shifts are accompanied by transitions from Curvibacter- to Pseudomonas-dominated or Legionella-dominated states, indicating the existence of three alternative community states which can be induced by metabolic triggers. Integrating 16S sequences with functional genomic information, we found that {beta}-diversity strongly predicted functional shifts, whereas reduced -diversity was associated with loss of metabolic functions. The metabolite perturbations also altered host-microbe interactions, affecting pathogenicity-, glycocalyx-, and nitrogen-related functions. In particular, nitrogen metabolism shifted from ammonia oxidation in Curvibacter-dominated communities to ammonia reduction in Pseudomonas-dominated states. Experimental validation confirmed that Pseudomonas metabolizes L-arginine and drives environmental ammonia accumulation to levels that could impair Hydras fitness and induce disease phenotypes. Conversely, Limnobacter was found to scavenge the environmental ammonia, potentially mitigating the adverse effects. These results demonstrate that metabolite-driven niche reconfiguration can destabilize host-associated microbiomes by coupling compositional shifts to functional change and host pathology, identifying metabolite-driven niche restructuring as a mechanism linking microbial community instability to host dysfunction.

Anaerobic methanotrophic archaea are key members of the biological methane filter, thereby preventing emissions of this strong greenhouse gas into the atmosphere. Previous studies on freshwater anaerobic methanotrophs targeted the activity of these microorganisms at circumneutral pH whereas molecular ecology studies identified this phylotype also in acidic environments such as peatlands; it is currently unknown whether they can adapt to low pH and remain effective in the biological methane filter in low pH environments. Here we show that a granular enrichment culture of the freshwater methanotroph Ca. M. vercellensis loses activity when experiencing pH stress but remains metabolically active down to pH 5.65 with appropriate adaptation time, indicating that adaptive changes are necessary to accommodate anaerobic methane oxidation at lower pH. Analyses of archaeal lipids revealed an increase in zwitterionic intact polar lipids over anionic lipids as an adaptation. This coincided with a change in granule structure while methane oxidation rate and enrichment state of Ca. M. vercellensis remained stable. We show that Ca. M. vercellensis remains metabolically active at lower pH values, despite increased maintenance energy demands and the need for cytoplasmic pH homeostasis. Our study demonstrates that adaptations to stress by slow-growing microorganisms may require long-term observation and is thereby instrumental for a better understanding of methane cycling in acidic ecosystems.

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.

Protistan predators are key regulators of microbial food webs, yet most are considered to occupy relatively narrow trophic niches. Here, we demonstrate that Mayorella spp. (Amoebozoa), isolated from marine and freshwater environments, exhibits exceptional trophic breadth spanning multiple trophic levels. Live-cell imaging revealed predation on bacteria, algae, dinoflagellates, diatoms, flagellates, ciliates, and multicellular prey including rotifers. Large or filamentous prey were engulfed whole or mechanically fragmented during ingestion. Notably, Mayorella consumed both trophozoites and cysts of free-living amoebae (Naegleria and Acanthamoeba), with clear digestion of cyst contents. Dense cultures showed aggregation around large prey and facultative cannibalism. Ingestion of microplastic-like particles occurred without evidence of digestion. Predator cell size and population density increased markedly when feeding on protist or mixed prey relative to bacterial diets, indicating pronounced trophic plasticity. These findings establish Mayorella as a broad-spectrum, cross-trophic predator with the capacity to exert top-down effects across microbial food webs and suggest a previously underappreciated role in the suppression of pathogenic free-living amoebae.

Parmales (Bolidophyceae), the closest relatives of diatoms, includes isolates exhibiting one of two distinct morphotypes: a silicified non-flagellated form (S-type) and a naked flagellated form (F-type). Although alternation of these two forms for a single isolate has not been formally established, previous studies hypothesized that these morphotypes represent alternating stages of a single parmalean. In this study, we investigated the global expression patterns of S- and F-type marker genes by integrating parmalean Metagenome-Assembled Genomes (MAGs) and the metatranscriptomic dataset from Tara Oceans. We detected the expression of both S- and F-type marker genes from individual environmental MAGs. This finding provides the first metatranscriptomic evidence that natural parmalean genomes possess the potential to manifest both morphotypes. Furthermore, our analysis revealed different geographical expression patterns between the two forms. The expression of F-type markers showed a broad distribution, whereas that of S-type markers was more restricted, suggesting distinct niches for the two morpho-phases. Moreover, S-type gene expression appears to require specific environmental triggers that lead to a higher population density, whereas F-type expression is rather constitutively maintained. Overall, our results support the hypothesis of a life cycle involving morphological switching and reconcile the long-standing discrepancy between the ubiquity of parmaleans in molecular surveys and the limited geographical range for the observation of silicified cells. Based on these patterns, we propose a threshold-based model in which F-type-dominated populations persist under conditions unfavorable for growth and a morphological switch to the S-type is triggered once environmental conditions exceed a critical threshold for growth.

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.

Microbial habitats that receive repeated external input may not remain shaped by that input forever if local retention allows resident communities to build up over time. Here, we used a controlled bench-scale sink p-trap system to examine how community assembly unfolded during initial establishment in new, bleach-treated p-traps. Two p-traps received repeated handwashing-water input, while one received tap water as baseline. The treated p-traps, but not the control, showed clear successional change toward later resident-like states. Nested-model comparisons further showed that recent external input had its greatest influence early in succession, but the p-traps own prior state remained the stronger predictor throughout. Final-day post-flush trajectories indicated short-term displacement from pre-flush positions, with later time points tending to move back toward late-stage resident centroids. Together, these results show that repeated inoculation does not necessarily keep communities under continued outside influence. Instead, retentive microbial habitats can shift over time from early sensitivity to external input toward persistence shaped more by local history.

Nitrogen fixing legume nodules are typically viewed as the product of a bilateral mutualism between host plants and nitrogen-fixing rhizobia, yet nodules also harbor diverse non-rhizobial endophytes whose functional significance remains poorly understood, especially in wild legumes and uncultivated soil. Here, using the wild Mediterranean shrub Calicotome villosa, we performed a soil inoculation experiment to test whether plant performance is linked to the functional composition of the nodule microbiome. Soil inocula from different natural sites produced strong differences in nodulation success, plant biomass, leaf nitrogen concentration, nitrogen fixation rate, and nodule allocation under otherwise uniform conditions. Although Bradyrhizobium dominated all nodules, species composition varied among inoculation sources, and non-rhizobial endophytes reached substantial abundance in some treatments. Functional profiles of the nodule microbiome were significantly associated with plant phenotype, with the strongest coupling observed for traits related to nodule investment. Targeted and genome-wide analyses identified trait-associated genes in both symbionts and endophytes, including genes involved in nitrogen cycling, ammonium transport, denitrification, pyrimidine degradation, sulfur assimilation, and type VI secretion systems. Several of these functions were not part of the canonical symbiosis machinery, yet were strongly associated with plant nitrogen status, biomass accumulation, or nodule mass fraction. Together, our results show that legume performance is better predicted by the collective functional composition of the nodule microbiome than by the primary symbiont alone. These findings support a broader view of nodules as multipartite microbial communities.

Plant leaves harbor diverse microbial communities influenced by environmental inputs and host traits, yet it remains unclear whether leaves act as passive substrates or active ecological filters that reorganize microbial functional capacity. Phylloplane pH regulation is one hostplant trait that has been traditionally underexplored. We used metatranscriptomics to examine microbial gene expression on the phylloplane and within whole leaves of five plant species spanning the extremes of baseline phylloplane pH, including hyperalkalinizing Gossypium species, weakly buffering Beta vulgaris, and hyperacidifying Nepenthes species. Young leaves were inoculated with a common soil-derived microbial community to quantify host-associated restructuring of taxonomic and functional profiles, and short-term pH perturbations were applied to test the effect of transient abiotic stress. Across both phylloplane and whole-leaf datasets, host species identity was the primary axis structuring microbial taxonomic composition and expressed functional repertoires. Leaf-associated communities diverged from the source inoculum, but retained a substantial shared functional backbone enriched for central biosynthetic and core metabolic pathways. Host-associated differentiation reflected selective retention and redistribution of reactions within this shared environmental pool rather than acquisition of novel metabolic capacity. Enriched pathway subsets were metabolically coherent and taxonomically distributed across multiple bacterial orders, consistent with functional redundancy and trait-based assembly. Among hosts, Gossypium exhibited the strongest restructuring relative to inoculum, suggesting comparatively stronger host-associated filtering. In contrast, short-term pH manipulation did not induce consistent community-wide functional reorganization. Microbial physiological responses to the phylloplane environment and external pH were observed at the organismal level. Together, these results position leaves as active ecological filters that reorganize microbial functional landscapes through host-specific trait regimes. This work begins to implicate some role of phylloplane pH regulation in microbial assembly and function.

Using shotgun metagenomics of largetooth sawfish (Pristis pristis) skin and paired pool water samples, we provide the first taxonomic and functional characterization of a sawfish skin microbiome and assess its ecological distinctiveness, assembly, and resilience relative to the surrounding water column. The skin microbiome was strongly host-associated and compositionally distinct, with lower taxonomic richness than the water column but dominated by Bacillota, particularly spore-forming Bacilli. Functional profiling revealed enrichment of genes associated with sporulation, dormancy, anaerobic metabolism, and peptide transport, consistent with adaptation to low-oxygen, low-flow pool conditions. In contrast, water-column microbiomes were more taxonomically diverse and enriched in phototrophic and polysaccharide-utilization pathways. Despite reduced taxonomic diversity, sawfish skin communities exhibited higher functional redundancy, with gene functions accumulating more rapidly per taxon. This pattern supports a host-filtered, lottery-like assembly process that produces taxonomically variable yet functionally conserved communities. The enrichment of dormancy and anaerobic pathways suggests the skin microbiome persists through periods of host quiescence and environmental stress while maintaining metabolic potential. Together, these results demonstrate that the sawfish skin supports a resilient, functionally robust microbiome distinct from ambient aquatic communities, highlighting the potential for integrating microbial functional data into conservation strategies for Critically Endangered vertebrates.

Cross-feeding interactions are pervasive in microbial communities and profoundly shape community structure, stability, and function. While previous studies have explored how cross-feeding affects evolvability, this work has predominantly focused on bidirectional mutualistic interactions in engineered auxotrophic systems where both partners reciprocally exchange essential metabolites. However, most metabolic interactions in natural microbial communities are unidirectional, with organisms feeding on the metabolic waste products of other species. Our study addresses this gap by examining how a unidirectional cross-feeding interaction affects the evolutionary dynamics of both the producer (Acinetobacter johnsonii) and consumer (Pseudomonas putida) over 800 generations of experimental evolution. We found that co-culture constrained adaptive evolution in both species. Co-cultures exhibited lower {pi}N/{pi}S ratios (0.75 for P. putida; 1.04 for A. johnsonii) than monocultures (1.44 and 2.02, respectively) indicating stronger purifying selection against nonsynonymous mutations in the community context. Lineage tracking through whole genome sequencing of populations and clones revealed greater lineage diversity and complexity in monocultures, with more mutations showing significant parallelism across replicate populations. Additionally, P. putida evolved increased dependence on its partner; co-culture-evolved P. putida grew significantly worse than its ancestor when A. johnsonii was removed. These findings demonstrate that ecological interactions fundamentally reshape fitness landscapes and constrain adaptive evolution even when fitness benefits are unidirectional, with implications for understanding microbial community stability and predicting evolutionary dynamics in complex communities.

Viruses regulate microbial mortality and biogeochemical cycling in marine sediments; however, the ecological drivers of sediment viral communities remain unclear. Infauna, including sediment-dwelling meiofauna and macrofauna, are major ecosystem engineers that reshape sediment structures and microbial processes, but their influence on viruses is unknown. We combined infaunal gradient incubations with metagenomic and metatranscriptomic analyses to assess viral DNA and RNA responses. DNA viruses showed increased abundance (3-fold), diversity, richness, and transcriptional activity under higher infauna abundance conditions, whereas RNA viruses remained unaffected, revealing striking selectivity. This selectivity reflects an infauna-dependent component mediated by bacterial activity that cannot be explained by host abundance alone. Infection profiling revealed increased transcription of viral replication and structural genes, and lytic viruses under high infauna conditions. These findings establish infauna as a previously overlooked regulator of DNA virus dynamics, integrating viral ecology into faunal-microbial frameworks in benthic ecosystems and suggesting potential influences on geochemical cycles. TeaserInfauna selectively shape viral communities in marine sediments, revealing an overlooked effect on DNA viruses.