The ISME Journal

Bacterial species often harbor extensive strain-level diversity, raising the question of whether strains, rather than species, are the relevant ecological units. We quantified pairwise interactions among 12 strains from four prevalent Lactobacillus species of the honeybee gut microbiota using gnotobiotic bees. Microbiota-depleted bees were colonized with individual strains and all 66 pairwise combinations, with strain abundances measured by colony counts and strain-resolved amplicon sequencing. Negative interactions predominated, with most significant interactions involving mutual inhibition. Within-species interactions were stronger and more asymmetric than between-species interactions, producing less even community compositions. Across repeated cycles of colonization in microbiota-depleted bees, three of four within-species pairs lost one strain, whereas all four between-species pairs persisted. These results indicate that closely related strains experience greater niche overlap and stronger competition than strains from different species, supporting bacterial species as ecologically differentiated units while highlighting the importance of strain-specific traits in shaping community assembly and stability.

Many Patescibacteria lineages are abundant in wastewater treatment plants, yet their interaction partners and ecological roles remain poorly understood. Here, we identify a specific association between a novel Saccharimonadia genus, Candidatus Lutisaccharimonas, and the widespread polyphosphate-accumulating organism (PAO) Candidatus Phosphoribacter. Fluorescence in situ hybridization revealed close physical proximity between the two taxa, supporting an epibiotic relationship. Correlated population dynamics, CRISPR spacer matches and evidence of horizontal gene transfer further corroborated this association. Comparative genomic analyses showed that Ca. Lutisaccharimonas has greater metabolic versatility than is typical of Patescibacteria, including complete pathways for de novo nucleotide biosynthesis and genes involved in amino acid metabolism. These metabolic features may shape its interactions with Ca. Phosphoribacter. Together, our findings identify a previously undescribed host association for Patescibacteria, expand current understanding of their metabolic potential, and suggest that the interactions between Saccharimonadia and PAOs may influence phosphorus removal in engineered ecosystems.

Mixing plant functional groups can boost plant productivity, but the underlying rhizosphere microbial dynamics remain unclear. We tested when to expect divergence from additive expectations by measuring plant traits and active and total rhizosphere microbiomes (via BONCAT-FACS and 16S rRNA gene amplicon sequencing) in monocultures and mixtures of legumes, brassicas, and grasses under varying nitrogen. While shoot biomass was predictable from monocultures, legume-containing mixtures yielded greater-than-expected root biomass and N-fixation. Each plant species supported distinct active and total microbiomes, and nitrogen addition increased microbiome divergence among plant species and their mixtures. In most plant mixtures, microbiomes were additive combinations of those associated with the individual species; legume-grass combinations were a notable exception, showing non-additive shifts in microbial composition, microbial activity, and the abundance of Rhizobium sp. This divergence from additive expectations coincided with non-additive biomass gains in legume-grass mixtures, suggesting that emergent plant-microbe interactions may underlie synergistic belowground responses.

Microbial symbioses drive the evolutionary and functional diversification of eukaryotic clades, from single-celled protists to large invertebrates. However, our knowledge of host-associated assemblages (the "holobiont") is limited in microscopic animal phyla with a body size <1mm, due to practical challenges such as low biomass and difficult taxonomy of host species. Marine nematodes represent an ideal case study for rapidly advancing our knowledge of bacterial-animal symbioses, representing a globally abundant invertebrate group with strong links to terrestrial and model organism species within the same phylum. Here, we sequenced the holobionts of 220 marine nematodes and generated 815 metagenome-assembled genomes (MAGs) of host-associated bacteria/archaea. Our data indicates that 20-34% of marine nematodes harbor an obligate intracellular symbiont, often with multiple endosymbionts co-occurring within the same host. Three bacterial phyla (Pseudomonadota Bacteroidota, and Verrucomicrobiota) account for three-quarters of all nematode-associated MAGs, and the majority of these holobiont MAGs represent deeply divergent lineages in the prokaryotic tree of life. The Flavobacteriaceae (a core microbiome taxon in C. elegans and other terrestrial nematodes), were consistently recovered across phylogenetically diverse marine nematode lineages, suggesting evolutionary conservation of holobiont taxa across marine and terrestrial environments. We also report a novel chemoautotroph family (Ca. Thionematobacter) recovered from nematode hosts in both deep-sea and shallow-water habitats, and report the first confirmed instance of Cardinium endosymbionts from marine invertebrates. Finally, [~]65% of nematode-associated MAGs are able to degrade chitin, via hexosaminidase, implying that benthic invertebrate holobionts make significant contributions to global carbon cycling. These results underline the importance of evaluating symbiosis in microscopic marine invertebrates, and accelerating our understanding of animal evolution and ecosystem dynamics in vast benthic habitats.

Trichodesmium is a major nitrogen fixer in the nutrient-limited tropical and subtropical ocean, contributing up to 50% of new nitrogen and driving primary productivity. Trichodesmium filaments often aggregate into colonies that host a diverse consortium of microbes that can influence their activity and physiology; however, it is unclear what factors shape community structure. To address this, we used metagenomics of Trichodesmium colonies collected across the subtropical and tropical Atlantic Ocean to examine how Trichodesmium diversity and abundance shape epibiont community composition. Heterotrophic metagenome-assembled-genomes from Trichodesmium colonies and genes specific to the major Trichodesmium subclades were read mapped against Trichodesmium metagenomes from the North Atlantic, the Western Tropical South Pacific, and the Red Sea. Using this global survey of Trichodesmium subclade distribution patterns and their enriched heterotrophs, we found unique core microbiomes that are specific to Trichodesmium subclades across ocean basins. Genome characteristics of the enriched heterotrophs reveal a genomically small, slow-growing community consistently associates with colonies. Our results suggest that Trichodesmium genotype may influence epibiont community structure, indicating a role for deterministic processes in shaping the microbiome.

Aquatic environments can maintain and express antibiotic resistance genes even without antibiotic pressure, but the conditions that govern their activity remain poorly understood. Redox oscillations are pervasive in natural and engineered waters and strongly alter microbial metabolism, yet whether their frequency shapes resistance expression is unknown. We reanalyzed paired metagenomes and metatranscriptomes from a published chemostat experiment in which a sulfidic stream community was cycled between oxic and anoxic conditions at high, medium and low frequency (HF, MF, LF), with strain-resolved population genomics of the dominant Pseudomonas lineage. Resistance genes were broadly distributed across treatments, whereas transcription concentrated under fast cycling, reaching a median 168 TPM under HF against 38 under LF although no antibiotic was present, so genetic potential and expression diverged. Fast cycling induced ROS-generating flavoproteins, antioxidant defenses and transcription-coupled DNA repair, and Pseudomonas supplied 99% of resistance-gene transcripts under HF, chiefly through its intrinsic efflux systems. Within the dominant Pseudomonas genome, however, the four resistance genes carried no sequence variants under HF, where coverage was deepest, and no fixed difference from the reference in any sample. Genome-wide selection remained purifying throughout (pooled pN/pS 0.72 under HF against 0.12 under LF, both below 1), and the relaxation trended toward redox and central metabolism. Redox frequency therefore raises resistance expression by selecting a metabolically versatile, high-efflux lineage, while the resistance sequences themselves stay conserved. Resistance activity in aquatic systems responds to an abiotic driver, which argues for weighing expression alongside gene presence in environmental surveillance.

Despite the crucial role of the ocean microbiome for global ecosystem processes, its response to global change remains poorly understood. Global change will exert selection on microbial species through changes in the genetic composition of their populations, favouring strains that are better adapted to new conditions. Analyzing long-term genomic variation in microbial populations inhabiting climatically comparable but oceanographically distinct regions may provide insights into their responses to future environmental conditions. We analyzed coastal marine microbiomes from two distant long-term observatories with Mediterranean-type climates but contrasting oceanographic regimes: the Blanes Bay Microbial Observatory (BBMO; Northwestern Mediterranean Sea) and the Microbes in the Coastal Region of Orange County (MiCRO; California coast, Pacific Ocean). Sampling conducted at least monthly during 15 and 10 years, respectively, yielded 1,535 Metagenome-Assembled Genomes (MAGs) in BBMO and 1,068 MAGs plus 187 Single-Amplified Genomes (SAGs) in MiCRO. Among these, we found 250 genomes with intraspecific (>95% genome similarity) representatives occurring at both sites. In contrast, at the strain-level threshold ([&ge;]99% genome similarity), only 13 BBMO genomes matched 15 closely related representatives in MiCRO. As genome similarity increased, we observed a shift from cosmopolitan to more coastal distributions and a slight increase in genome size, pointing to niche adaptation. Across both time series, we observed widespread seasonal population structure, with most genomes (~70%) showing significant seasonal structuring of variant composition. Moreover, in MiCRO, ~76% of the tested genomes also showed El Nino Southern Oscillation (ENSO)-associated structure in variant composition beyond seasonal effects. Focusing on a Prochlorococcus genome with intraspecific representatives at both locations (>96% genome similarity) we further found recurrent seasonal mutations and non-synonymous to synonymous mutation ratios (pN/pS) from multiple genes, together with signatures of positive selection (higher pN/pS) coinciding with El Nino events in MiCRO. This suggests an imprint of ENSO in marine microbial populations. Thus, beyond seasonality, long-term climatic oscillations may shape microbial populations. This has implications for understanding how the ocean microbiome will respond to long-term environmental change and prolonged disturbances through population-level genomic variation.

Microbial fermentations emerge from the collective activity of interacting species whose relative abundances, metabolic functions, and environmental constraints continuously change over time. Water kefir provides a tractable model to investigate how multispecies communities coordinate their metabolic activities during fermentation. Here, we combined time-resolved community profiling, physicochemical measurements, metabolite quantification, and controlled co-culture assays to characterize the dynamics of a water kefir consortium comprising 19 detected microbial species. Our results reveal that water kefir fermentation is not adequately described as a simple succession of independently acting microbial groups. Instead, the community undergoes a reproducible reorganization of its collective metabolism around 20 h of fermentation. This transition marks a shift from an early regime characterized by the accumulation of soluble sugars and ethanol to a later regime dominated by enhanced sugar consumption and lactate production. Controlled interaction assays further showed that bacterial partners can alter carbon-use behaviour in Saccharomyces cerevisiae, highlighting the capacity of interspecies interactions to reshape metabolic phenotypes within the consortium. Together, these results support an ecological view of water kefir fermentation in which community-wide metabolic reorganization emerges from the interplay between microbial interactions, resource availability, and environmental change.

Bacteriophages (phages) are viruses that infect bacteria, often posing a threat to their survival. In recent years, research has shifted from studying bacteria-phage interactions in homogeneous, well-mixed environments to investigating more complex heterogeneous, structured communities, thereby more accurately reflecting bacterial life in natural and disease-associated settings. So far, these studies predominantly tested interactions of phages and single-species biofilms, despite the dominance of highly diverse biofilm communities in nature. Here, we investigated how different lytic phages spread in and infect a synthetic community composed of Escherichia coli, Kluyvera cryocrescens, and Vibrio anguillarum. We found that E. coli was protected from eradication by the T7-coliphage when embedded in a multispecies biofilm and covered by the two other species, where the V. anguillarum biofilm matrix was essential for protection of E. coli. The protective nature of the V. anguillarum matrix also ensured survival of K. cryocrescens exposed to the lytic kluyveraphage Lyn. V. anguillarum mutants lacking the matrix synthesis genes vpsMNOP or rbmC failed to protect E. coli despite forming biofilms. Labelling of phages indicated that these were not trapped in the matrix of the wildtype. Instead, advanced image analysis revealed that the mutants displayed biofilms with lower cell density, resulting in the E. coli clusters being less tightly surrounded by V. anguillarum cells, which opened gaps for phage entry. These findings highlight that community context and matrix composition play a major role in phage-bacteria dynamics.

Mangroves persist under strong environmental constraints with support from microbes. Although microbial communities differ among mangrove compartments, their assembly mechanisms and compartment-specific functional signatures remain poorly resolved. Here, we combine peptide nucleic acid-clamping coupled with 16S rRNA gene amplicon sequencing and genome-resolved metagenomics to investigate the microbial communities associated with the leaves and roots of gray mangrove trees in the Red Sea. Our results suggest homogeneous selection and homogenizing dispersal as key processes of microbial community assembly in leaves. This phyllosphere hosts novel prokaryotic lineages, with some of its members probably synthesizing rhodopsins, plant polysaccharide-degrading enzymes, and gamma-aminobutyric acid, a metabolite that increase tolerance to salinity stress. Genomes affiliated with Desulfobacterales and Sedimenticolaceae taxa are abundant belowground, suggesting complementary sulfur- and nitrogen-cycling capacities similar as occur in other blue-carbon ecosystems. Comparison with root-derived genomes from cordgrass revealed a host-driven selection of Sedimenticolaceae species with convergent metabolic profiles. Overall, this study provides an integrative view about the microbial biology of gray mangrove trees, offering foundational insights into the diversity, ecology, and predictive functionality of their aboveground-belowground microbiomes.

Biological nitrogen fixation converts dinitrogen gas into ammonia, supplying new bioavailable nitrogen to marine ecosystems, but the evolutionary processes shaping its distribution among heterotrophic bacteria remain unresolved. Thalassolituus, a genus within the family Oceanospirillaceae (order Oceanospirillales), is best known for hydrocarbon degradation, yet nitrogen fixation has been confirmed in only one cultured isolate. We analyzed 74 quality-filtered genomes assigned to Thalassolituus within a broader dataset of 421 Oceanospirillaceae genomes to reconstruct the distribution and evolutionary history of the minimal nifHDKENB gene set. Twenty-five genomes encoded complete or near-complete nif loci and occurred in four well-supported clades interspersed with genomes lacking the pathway. Statistical topology tests rejected the species-tree topology for concatenated NifHDK and NifHDKENB protein alignments, and eleven recombination events across nif loci were supported by at least four detection methods. The core nifHDK gene order remained broadly conserved, but accessory neighborhoods differed among clades, and structural nifHDK genes showed stronger codon adaptation than biosynthesis nifENB genes. Clade 2 combined species-gene tree congruence, conserved gene neighborhoods, and comparatively high nifH codon adaptation, whereas Clades 1 and 4 showed greater phylogenetic discordance, more recombination, and weaker codon adaptation. These results support a reticulate history in Thalassolituus, in which lateral acquisition introduced nitrogen fixation into distinct lineages, vertical inheritance preserved it within some clades, and homologous recombination continued to reshape nif loci. These processes help explain why nitrogen fixation is unevenly distributed among closely related marine heterotrophic bacteria.

Subcuticular bacteria (SCB) are widespread symbionts of echinoderms and often dominate the body-wall microbiome, suggesting important roles in host physiology. However, their diversity, metabolic properties, and host associations remain poorly characterized. Here, we report a novel dominant SCB lineage associated with deep-sea holothurians, Scotoplanes spp. collected from the Northwest Pacific. We recovered two high-quality genomes, including a 649-kb complete circular genome, and propose a new genus and species, "Candidatus Abyssoplasma scotoplanesicola", within Mycoplasmatota. The two genomes showed a highly reduced metabolic repertoire, lacking central pathways including glycolysis. In contrast, acidic cell-surface-associated proteins, including large proteins exceeding 5,000 amino acids, accounted for 27.6% of the complete genome and clustered near defense islands. Localized genome plasticity in these regions, revealed by comparison between the two closely related genomes, suggests a possible mechanism for diversification of cell-surface proteins at the host-symbiont interface. "Candidatus Abyssoplasma scotoplanesicola" occupied 76.4-98.9% of the body-wall microbiome of the Scotoplanes specimens. Fluorescence in situ hybridization analysis confirmed that these bacteria formed aggregates on the epidermal side of the body wall. Overall, this study provides genome-and spatially resolved views of dominant SCB in holothurians and offers evolutionary insights into host-interface diversification in the deep-sea holothurian body wall.

Marine planktonic viruses play critical roles in shaping microbial communities and driving global biogeochemical cycles. However, quantitative, microbiome-wide analyses of marine prokaryoplankton-virus interactions in situ and their ecological impacts remain challenging due to the vast diversity of viral genomes and interaction modes and the limitations of existing methodologies. Here, we utilized GORG-Tropics, a global collection of 12,715 single amplified genomes (SAGs) generated from randomly sampled marine prokaryoplankton cells, to determine the frequency and modes of their interactions with viruses in the tropical surface ocean. We found 4.2% (range 1%-19% among samples) of GORG-Tropics SAGs to contain phage genomic material, with the highest frequency found in productive ocean regions. Prokaryoplankton lineages known to have high metabolic rates, including Prochlorococcus and Rhodobacterales, had a substantially larger fraction of cells associated with viruses (10-12% of SAGs) as compared to the less active but highly abundant lineages including Pelagibacterales (2.2% of SAGs). Cell-virus associations indicative of lysogeny were elevated in Alphaproteobacteria relative to other taxa. The collection of phages recovered from individual SAGs exhibited genomic diversity that bridged order-level taxonomies, indicating high diversity and genomic connectivity within wild phage populations. A substantial fraction of the observed cell-virus associations disagreed with the computationally predicted host identity of the virus, indicative of non-infective interactions. The extent of genetic exchange across tailed bacteriophages infecting different hosts, and connected to taxonomically distant phages provided further evidence for the role of non-infective phage entry in the lateral transfer between tailed phages. This study provides large-scale quantitative evidence of viral infection rates in the collective prokaryoplankton community across the global surface tropical ocean through large-scale identification and quantification of the specific phages, hosts, and modes of interaction at the resolution of individual cells. Our results confirm prior reports on the overall frequency of prokaryoplankton infections with viruses in the oligotrophic tropical surface ocean. Our findings uncover non-infective phage-cell associations that may be contributing to the lateral transfer of viral genes and the nutrition of marine prokaryoplankton.

Grassland microbial communities are central to mediating ecosystem function and stability, yet how long-term grazing reshapes root-associated microbiomes across contiguous soil-root habitats remains poorly understood. This limits our ability to identify robust microbial bioindicators for grassland health monitoring. In this study, we investigated the community assembly and functional variation of root-associated microbiomes of Stipa breviflora, a dominant perennial clonal grass in desert steppes, across a 17-year continuous grazing experiment with four grazing intensity treatments (no grazing, light, moderate, and heavy grazing). We show that grazing intensity induces niche-specific restructuring of microbial communities, with the most profound compositional and functional shifts occurring in the rhizosphere, followed by root endophytic compartments and bulk soil. Light and moderate grazing significantly enriches the phylum Bacillota in rhizosphere and endophytic compartments, whereas the genus Pseudomonas dominates ungrazed grassland soils and is markedly depleted under grazing conditions. Microbial community responses to grazing follow a unimodal intermediate disturbance pattern, with moderate grazing triggering the strongest microbial community differentiation, enhanced microbial network connectivity and modularity, and the highest abundance of grazing-responsive microbial biomarkers. Notably, grazing-induced microbial community variation is decoupled from intraspecific phenotypic changes in S. breviflora. Our findings demonstrate that long-term grazing acts as a strong selective filter partitioning core beneficial microbial taxon, establishing Bacillota and Pseudomonas as complementary bioindicators for evaluating desert steppe ecosystem health. This study advances the understanding of plant-microbe interactions under anthropogenic disturbance and provides microbiome-based insights for sustainable grassland management.

Microbial communities associated with plants are dynamic ecosystems whose structure and functions influence host health and fitness. While the ecological roles and beneficial function of plant microbiomes are increasingly well understood, the evolutionary dynamics that shape them over time remain unclear. Experimental evolution constitutes a powerful tool to study microbial adaptation, but so far, its application has been largely focused on free-living or single-species populations. Here, we use experimental evolution to test whether taxonomically complex synthetic bacterial communities can adapt to novel plant hosts. We show that replicate microbial communities undergo rapid and parallel genomic evolution, with measurable gains in colonization fitness. Adaptive changes involved mutations in regulatory processes, motility, and core metabolism, leading to significant fitness gains across diverse taxa. Our findings demonstrate that the plant host can drive microbiome evolution without disrupting the stability of the root microbiota.

Hydrostatic pressure is a defining feature of the deep ocean, yet how it structures microbial activity across the subseafloor biosphere is poorly understood. Here, we assess pressure effects on anabolic activity in seafloor and subseafloor communities across a margin-to-hadal-to-abyssal transect of the Puerto Rico Trench region (493-8,385 m water depth). Community-level responses to pressure reverse with burial, revealing a previously unrecognized shift toward piezophily in resident subseafloor communities down to 20 meters below seafloor (mbsf). In pressure-incubation experiments, atmospheric pressure yielded higher proportions of active cells in surficial sediments, whereas in situ pressure yielded higher proportions in the subseafloor. Pressure also structured active-community composition; in trench-axis sediments extending to 8.35 mbsf, JS1/Atribacterota populations belonging to geographically widespread hadal lineages exhibited a physiological response to in situ pressure, indicative of subseafloor piezophily. These findings suggest that hydrostatic pressure acts as an ecological filter after burial, favoring piezophilic traits that may support microbial persistence in the deep subseafloor biosphere.

Viral infection is a major yet poorly quantified driver of microbial interactions and biogeochemical fluxes in the ocean. In diatoms, which are key contributors to marine primary production, the extent to which viruses reprogram host cell metabolism and alter elemental cycling remains largely unresolved. Here, we investigated how infection by a lytic single-stranded RNA virus reshapes carbon (C) and nitrogen (N) fluxes in the ecologically relevant nanoplanktonic diatom Mediolabrus comicus. Using controlled infection experiments coupled with flow cytometry, electron microscopy, PAM fluorimetry, and stable isotope probing, we resolved infection-driven changes from the population to the cellular scale. Infection induced rapid optical shifts and cellular reorganization, including the formation of membrane-bound viral replication compartments. These changes coincided with early impairment of plastidial functions, as shown by disruption of photosystem II functionality and a concomitant decline in photosynthetic carbon fixation. In contrast, nitrogen uptake was maintained and strongly enhanced during late stages of infection, indicating sustained resource acquisition to support viral replication. This decoupling led to dynamic changes in cellular stoichiometry and, overall, to substantial reductions in population-level carbon and nitrogen assimilation due to growth inhibition. Together, these findings demonstrate that diatom RNA virus infection reshapes host carbon and nitrogen metabolism, with cascading effects on elemental cycling. Our results identify diatom RNA viruses as important drivers of marine biogeochemical processes, with implications for primary production and the fate of organic matter in the ocean.

Kiritimatiellia are a class of Verrucomicrobiota whose few cultured representatives are known for hosting large repertoires of carbohydrate-active enzymes and sulfatases. Many families of this class are restricted to metagenome-assembled genomes not yet discussed in literature, leaving their genome organization undescribed. Here, we survey 898 Kiritimatiellia genomes using similarity-colocalization networks (SCoNes), a data structure that captures conserved synteny among homologous genes across genomes. Using this approach, we investigate four metabolic adaptations to a glycan-rich environment: complex polysaccharide degradation, nitrogen cycling, hydrogenase activity, and natural product biosynthesis. Rather than unrelated functions, these capacities envelop a single anaerobic and saccharolytic strategy where sulfated glycan hydrolysis fuels fermentation, hydrogenases balance redox, and excess carbon supports nitrogen fixation.

Winogradsky columns are a classic model ecosystem for studying microbial biogeochemistry across steep gradients of oxygen and sulfide. They also remain widely used in microbiology education, introducing generations of students to microbial diversity. Yet, the genomic potential of their microbial communities remains uncharacterized. Here, we applied shotgun metagenomic sequencing to a Winogradsky column community at multiple depths, yielding 20 metagenome-assembled genomes (MAGs) representing diverse, largely uncultivated taxa. Genome-resolved analyses revealed metabolically diverse oxygenic and anoxygenic phototrophs that could potentially contribute to carbon and nitrogen fixation across all layers of the column. Most of these phototrophs also encoded one or more pathways for sulfur oxidation, which we speculated may support both energy conservation and/or sulfide detoxification by these populations. Complex carbon degradation capacity was also widespread across the MAGs, suggestive of the potential for the transformation of the column's amended organic matter (shredded coffee filters) into smaller depolymerization products and, through fermentation, organic acids. Together, these findings reveal how distinct microbial guilds might partition interconnected carbon, sulfur, and nitrogen transformations within redox-stratified systems.

A large fraction of microbes in natural environments are auxotrophs that rely on metabolite exchange for growth. The Black Queen hypothesis and related frameworks explain gene loss and reliance on shared metabolites. Yet it is not fully understood how metabolite cross-feeding emerges and gives rise to experimentally observable community performance. Here, we propose that exchange of biosynthetic pathway intermediates provides a mechanistic route for the emergence of metabolite cross-feeding. Building on prior work demonstrating intermediate exchange in E. coli auxotrophs, we investigated co-cultures between a methionine auxotroph ({Delta}metA) and a panel of histidine auxotrophs. Among these, only {Delta}hisD exhibited efficient co-culture growth with {Delta}metA, suggesting a unique role for the final step of histidine biosynthesis, which converts histidinol to histidine. We found that {Delta}metA-{Delta}hisD co-cultures accumulated high levels of extracellular histidinol (>200 M), suggesting a mechanism for metabolite exchange. Spent media from these co-cultures supported growth of all histidine auxotrophs except {Delta}hisD, confirming that histidinol, rather than histidine, was the dominant exchanged metabolite. Furthermore, we observed that {Delta}metA efficiently converted histidinol to histidine only under methionine-limited conditions, suggesting that nutrient limitation promoted overflow of converted histidine to the extracellular environment. To quantitatively assess whether histidinol exchange could account for the observed co-culture behavior, we constructed a mechanistic model that links extracellular histidinol dynamics to co-culture growth through independently measured conversion capacity of the {Delta}metA strain. Rather than fitting growth empirically, we used experimentally derived parameters to predict co-culture growth from the measured histidinol-to-histidine conversion flux. The model accurately recapitulated the timing and magnitude of growth and population dynamics, supporting the idea that accumulation of histidinol enabled the conversion-driven supply of histidine that sustained {Delta}hisD, while coupling growth between the two strains. Taken together, these results suggest that metabolite cross-feeding in syntrophic co-cultures can emerge from the accumulation of pathway intermediates and their conditional conversion under nutrient limitation, providing a plausible mechanism for the rapid establishment of mutualistic interactions.