Microbiome

The expansion of anthropogenic activities into natural ecosystems has intensified contact at wildlife–livestock interfaces, creating opportunities for viral exchange among wildlife, domestic animals, and humans. Although such interfaces are increasingly recognized as hotspots for emerging infectious diseases, viral diversity and cross-host viral connectivity involving threatened species remain poorly understood. In particular, little is known about how domestic animals, sympatric wildlife, and protected species jointly shape viral exposure risk in biodiversity hotspots. To address this gap, we conducted a metagenomic investigation of the virome associated with the vulnerable giant panda (Ailuropoda melanoleuca), sympatric wildlife, and domestic animals in Southwestern China. We identified 661,837 DNA vOTUs and 176,031 RNA vOTUs, revealing extensive viral dark matter that was particularly pronounced in the RNA virome, where 95.93

Urbanization may alter companion animal viromes in ways relevant to zoonotic surveillance, but the viral microbiome dynamics and assembly drivers of urban household/stray pets remain elusive. Challenging the conventional view that stray pets necessarily harbor greater viral richness, we characterized vertebrate-associated viromes of cats and dogs across Shanghai to compare vOTU richness and community composition, identify putative novel viral lineages, and investigate key assembly factors. Using an integrated meta-omics approach, we profiled vertebrate-associated viromes of household and stray cats/dogs in Shanghai. A total of 245 fecal or rectal swabs were processed through VLP enrichment, co-extraction of viral DNA and RNA, amplification, and Illumina paired-end sequencing. Viral contigs were identified with four complementary tools (VirSorter2, DeepVirFinder, VirFinder, geNomad), validated by CheckV, and analyzed via taxonomy, phylogenetics, and statistics (alpha/beta diversity, RDA, network co-occurrence). Our analysis identified 123 vertebrate viral operational taxonomic units (vOTUs) from families including Coronaviridae, Poxviridae, and Parvoviridae. Contrary to conventional expectations, household dogs exhibited the highest Chao1 richness among the four host groups and had significantly higher richness than household cats, stray cats, and stray dogs (BH-FDR-adjusted Wilcoxon rank-sum tests, p < 0.05). However, household and stray dogs did not differ significantly in overall viral community composition (PERMANOVA, R2 = 0.07, p = 0.115), indicating that the household-stray contrast in dogs was richness-specific rather than a broad compositional shift. In addition, household dogs harbored 73.7

Breast cancer patients often experience chronic stress disorders and gut dysbiosis. Baicalin has been demonstrated to have antitumor, antibacterial and microbiota regulatory functions and can be used for treating breast cancer and preventing metastasis; however, how baicalin regulates the gut microbiota to inhibit metastasis remains unexplored. Chronic stress promoted breast cancer lung metastasis mainly through Alistipes putredinis, which produced the tryptophan metabolite indole-3-acetic acid (IAA). Chronic stress did not affect tumor growth but enhanced metastasis and reshaped the immune environment at the metastatic site. Mechanistically, IAA promoted 4T1 cell migration through AhR-mediated oxidative stress, and the AhR inhibitor CH223191 inhibited migration and metastasis in vitro and in vivo. Notably, baicalin treatment inhibited lung metastasis and increased both the number and cytotoxicity of CD8+ T cells in the lungs. 16S rDNA sequencing and targeted tryptophan metabolomics revealed that baicalin treatment not only reduced the abundance of A. putredinis but also modulated the metabolism of tryptophan in A. putredinis, resulting in suppressing IAA production. Fecal microbiota transplantation was further used to confirm the effects of baicalin. Breast cancer patients with metastasis and severe depression showed an increased abundance of A. putredinis in feces and increased concentration of IAA in the serum. These findings reveal stress-driven microbiota and metabolite alterations, which may facilitate breast cancer metastasis, uncovering the underlying mechanism by which baicalin prevents metastasis.

Glaciers represent unique ecosystems harboring diverse microbial communities that dominate biomass and drive biogeochemical cycling across supraglacial habitats. These microbes transform deposited modern and ancient carbon/nitrogen, and these elements are subsequently transferred to downstream ecosystems via glacier melting, further affecting downstream ecological succession, functions, and stability. Despite such ecological significance, our understanding of the mechanisms underlying microbial functional adaptation to different supraglacial habitats remains limited. We integrated 16S rRNA amplicon and metagenomic sequencing to systematically compare taxonomic composition and functional potential across snow, ice, and cryoconite in the ablation zones of Longxiazai glacier on the Tibetan Plateau. Our analyses revealed pervasive functional redundancy, with 94

Lactic acid bacteria in fermented foods and the human gut are chronically exposed to environmental stressors that can trigger adaptive responses. Bacterial dormancy allows non-spore-forming bacteria to withstand adverse conditions through a metabolically suppressed, reversible state. This study aimed to investigate whether sublethal antibiotic stress unexpectedly triggers adaptive responses in Lactobacillus delbrueckii subsp. bulgaricus, a widely used probiotic and starter culture, and to elucidate the underlying mechanisms and functional consequences. Sublethal rifampicin-induced a dormant state in Lactobacillus delbrueckii subsp. bulgaricus sp1.1, characterized by reduced ATP levels and impaired cell division, with colony recovery upon stress removal. This state enhanced cross-tolerance to acid, alkali, and heat stress, prolonged intestinal retention in mice, and increased the impact on gut microbial community structure. Disruption of protein aggregates by pH adjustment or 1,6-hexanediol treatment abolished the enhanced tolerance. Rifampicin triggered a metabolic shift toward transcriptional and translational inhibition. Protein aggregates selectively enriched translation, RNA metabolism, and DNA repair proteins. Two disordered proteins, Gene1622 and Gene1909, were upregulated in whole cell and enriched in the aggregates. Single-cell RNA sequencing identified a cluster that sustained alaS and gatA expression under global translational suppression, contrasting with their downregulation in bulk RNA-seq, revealing population heterogeneity under stress. Our results support a model in which protein aggregates function as “molecular safe houses” that sequester and protect translation-and DNA-repair-related proteins, thereby locally enhancing the efficiency of key protein translation and DNA repair while also serving as a protein reservoir for subsequent growth recovery. This reversible dormancy provides a theoretical basis for engineering robust starter cultures, while also raising ecological considerations regarding stress-induced adaptation in the food chain, including potential impacts on gut microbial homeostasis. Schematic diagram of the mechanism by which sublethal rifampicin induces dormancy and enhances bacterial tolerance and intestinal retention capacity.

Neonatal calf diarrhea is a major challenge in livestock production and is often driven by pathogen-induced intestinal inflammation and gut dysbiosis. While the therapeutic potential of Bifidobacterium (B.) pseudocatenulatum is recognized, its mechanistic interplay with diarrhea-associated inflammatory pathology in calves remains poorly defined. This study investigated how B. pseudocatenulatum alleviates calf diarrhea and intestinal inflammation, with a focus on acetate metabolism and the T helper 17 (Th17)/IL-17A immune axis. Diarrheic calves exhibited pronounced gut microbial dysbiosis, metabolic perturbation, and inflammatory activation. Fecal microbiota transplantation (FMT) from healthy calves alleviated diarrhea and was accompanied by enrichment of B. pseudocatenulatum and increased fecal acetate. In vitro, live B. pseudocatenulatum increased acetate production. In complementary murine inflammatory models, B. pseudocatenulatum and acetate alleviated intestinal injury and inflammatory phenotypes. The protective effect was attenuated in the heat-killed B. pseudocatenulatum-treated group. Mechanistically, these protective effects were accompanied by attenuation of pathogen-induced Th17/IL-17A-related responses rather than broad suppression of basal Th17 activation. Pharmacological blockade of free fatty acid receptor 2 (FFAR2) attenuated the protective effect of live B. pseudocatenulatum, supporting the involvement of acetate-associated receptor signaling. This study revealed B. pseudocatenulatum as a microbiota-associated candidate linked to calf diarrhea remission and supports acetate-associated attenuation of pathogen-induced Th17/IL-17A-related inflammatory responses as one component of its protective effect. These findings establish a microbiota–metabolite–immune regulatory framework, providing novel therapeutic strategies for alleviating intestinal inflammation and calf diarrhea.

Land-locked meromictic lakes sustain persistent redox stratification that structures microbial niches and sulfur–nitrogen–carbon cycling. Whole-lake overturns are unusual in such systems, and the temporal dynamics of microbial community re-establishment and functional recovery during holomictic restratification remain poorly understood. We tracked a mixing–re-stratification sequence in meromictic Lake Shira (Siberia) using depth-resolved sampling across oxic, chemocline, anoxic, and water–sediment interface layers in four hydrodynamic regimes: transitional holomixis, complete holomixis (CH), developing meromixis (DM), and stable meromixis (M). 16S rRNA amplicons showed that CH homogenized the water column and reduced depth structure, whereas DM–M rapidly re-established strong vertical partitioning, including recovery of Desulfobacterota and other anaerobic lineages in sulfidic deep waters and enrichment at the redox transition. Nanopore metagenomics reconstructed 401 metagenome-assembled genomes (MAGs) and revealed stage- and layer-specific functional repertoires consistent with redox zonation, oxic regimes enriched in sulfur oxidation, phototrophy-related traits and anoxic regimes enriched in sulfate reduction, and anaerobic nitrogen transformation. A conserved set of core MAGs, including Yoonia spp., persisted among regimes, suggesting a functional backbone that may promote rapid ecosystem recovery after overturn. These findings provide time-and depth-resolved, and genome-resolved evidence that microbial reassembly after holomixis is not simply a return of the former community composition, but that microbial succession is a process of functional reorganization. Persistence of core MAGs, re-establishment of redox-specific assemblages, and redistribution of shared metabolic modules contribute to the recovery of stratified ecosystem functions as meromictic stratification collapses and re-establishes.

Intensive swine production entails high antimicrobial use and close human–animal microbial exchange, with potential to remodel the human gut ecosystem and promote cross-host antimicrobial resistance (AMR) transmission. To quantify the impact of occupational exposure on the human gut microbiome and resistome, and to resolve putative livestock-to-human gene flow, we sampled feces from 103 swine farms in Sichuan, China. We generated metagenomes for 431 samples (farmers = 96, residents = 97, pigs = 238) and cultured 833 Escherichia coli isolates (farmers = 80, residents = 88, pigs = 665). Relative to unexposed residents, farmers exhibited reduced gut α-diversity and lower Gut Microbiome Health Index (GMHI). Taxonomic and functional profiles differed between farmers and residents, with farmers showing reduced abundances of several fiber-degrading and short-chain fatty acid-producing taxa, together with increased abundances of taxa and pathways related to mucin-derived glycan utilization. The resistome displayed an exposure gradient (pigs > farmers > residents). Farmers were enriched for determinants corresponding to commonly used veterinary drug classes (e.g., florfenicol, quinolones, MLS, aminoglycosides), whereas β-lactam genes were proportionally lower. Mobile genetic element (MGE) analysis revealed a pig-centered plasmid reservoir: among 157,298 nonredundant plasmid contigs, 1,415 carried ARGs, with 83.1

Soil metabolites serve as critical cues that orchestrate the assembly of microbial communities. However, the precise mechanisms by which specific chemical signals mediate plant–microbiome interactions to enhance disease resistance remain elusive. In particular, how engineered nanomaterials, such as SiO2 NPs, leverage this metabolic signaling to promote the establishment of disease-suppressive microbiomes is largely unexplored. We integrated metagenomics, metabolomics, and transcriptomics to elucidate the synergy between SiO2 NP–driven soil metabolic reprogramming and the establishment of biocontrol bacteria. We first demonstrated that SiO2 NPs inhibited potato common scab in a dose-dependent manner and drove significant shifts in soil microbial community structure and network complexity. We identified Bacillus as a core SiO2 NP–responsive taxon, and experiments showed that Bacillus velezensis strain PH3-11 inhibited pathogenic Streptomyces, with isovaleric acid emerging as a candidate antimicrobial metabolite associated with this antagonistic activity. Metabolomic and metagenomic analyses further indicated that SiO2 NPs stimulated inosine accumulation, and inosine was strongly associated with the community structure of SiO2 NP-responsive biomarkers. Mechanistically, transcriptomic analysis showed that inosine, as a SiO2 NP–responsive metabolite, upregulated genes involved in extracellular polysaccharide synthesis in strain PH3-11 (e.g., epsD, epsN, and epsO) and markedly promoted biofilm formation by PH3-11. Field trials further confirmed a synergistic effect of co-applying inosine with strain PH3-11, which was superior to single-strain inoculation in promoting biocontrol bacterial colonization, suppressing disease, and enhancing soil microbiome stability. Our findings unveil a “nano-metabolite-microbiome” cascade, suggesting that SiO2 NPs promote the enrichment of protective biofilm-forming bacteria by reprogramming the soil metabolome and promoting inosine accumulation. This study supports an inosine-associated mechanism contributing to disease-suppressive microbiome assembly and highlights the potential of nano-enabled synbiotics to manipulate chemical–biological coupling for sustainable plant health.

Tidal flats are critical transitional ecosystems in which microbial communities drive essential biogeochemical processes while adapting to strong environmental fluctuations. However, the mechanisms by which these communities maintain functional stability in highly dynamic tidal-flat environments remain poorly understood. In this study, we analyzed 276 tidal-flat samples from 92 coastal sites along the Chinese coastline to investigate the relationship between microbial diversity and community function. Our results revealed significant spatial variability in microbial communities across different regions of the Chinese coastline. Although latitude and geographic distance had detectable effects, the community turnover was governed primarily by environmental filtering and stochastic assembly, with temperature emerging as a key environmental driver. Despite the great variation in species diversity, the relatively similar functions of microbial communities across different regions were observed, indicating the existence of functional redundancy. Using a novel framework to quantify how the redundancy varies across microbial functions and communities, we illuminated the redundancy order of major biogeochemical functions in tidal flats as carbon > sulfur > nitrogen metabolism. We also identified three distinct redundancy patterns: high-, low-, and latitude-associated redundancy. By identifying the contributor composition underlying each function, we further revealed that the functional redundancy in tidal-flat ecosystems was shaped by the distribution of functional genes across microbial lineages, and that shifts in the balance between generalists and specialists helped explain the observed diversity of redundancy patterns. This study provides a quantitative framework for delineating microbial functional redundancy and its underlying contributor composition. By applying this framework to tidal flats along the Chinese coastline across four climatic zones, we revealed distinct patterns of functional redundancy and illuminated that a shift in the primary functional contributor would alter the redundancy pattern. These findings underscore the critical role of contributor composition in determining the stability and resilience of tidal-flat microbiomes.

As global temperatures continue to rise, heat stress (HS) has emerged as a major health threat of growing concern. HS triggers systemic inflammation and multi-organ damage, but so far its molecular mechanisms remain unclear. In this study, we explored the potential mechanism by which the gut microbial alterations amplify HS-associated inflammatory responses. We found that the gut microbiota was disrupted in HS mice as characterized by increased LPS levels and enhanced arginine catabolism. Transplant of fecal microbiota from HS mice aggravated inflammatory responses in recipient mice after HS. Exogenous arginine pretreatment notably suppressed inflammation in the liver and cortex of HS mice. Mechanistically, arginine reduced MyD88 protein levels by activating its ubiquitination and weakened the MyD88-TLR4 interaction, thereby inhibiting the nuclear translocation of p65 and the expression of pro-inflammatory genes. Clinically, lower arginine levels were detected in the serum of HS patients and positively related with liver injury and inflammatory indicators. An arginine-enriched oral inulin hydrogel was developed to prevent inflammatory responses exacerbated by the gut microbial alterations through maintaining the gut microbiota homeostasis to reduce LPS and providing a sustained supply of arginine. This study reveals a mechanism by which the gut microbial alterations exacerbates HS-associated inflammatory responses via disrupting the balance between LPS and arginine, thereby providing a novel target for the prevention of HS.

Viruses play a major role in regulating microbial community composition and activity, yet their diversity and host associations in anaerobic digestion (AD)—a complex biotechnological process converting organic waste into biogas—remain poorly characterized. Linking viral sequences to metabolically active hosts in such complex communities is particularly challenging. Here, we applied an integrative approach combining 13C-stable isotope probing (SIP) with shotgun sequencing of viromes and microbiomes to identify and characterize viruses infecting the active formatotrophic guilds in batch AD microcosms. Microcosms fed with 13C-labeled formate as sole carbon source selectively enriched two primary formate-consuming guilds: hydrogenotrophic methanogens (Methanobacteriales, predominantly Methanobacterium subterraneum) and acetogenic bacteria (family Natronincolaceae, genus Andreesenella). From cross-assembly of six viromes on the one hand, and six 13C-SIP microbiome fractions on the other, we assembled a catalogue of 2,368 vOTUs, of which 261 were selected for detailed analysis. The striking enrichment of vOTUs associated with methanogenic archaeal hosts, specifically in the heavy (13C-enriched) SIP fractions, contrasted with their near-absence in total viromes—demonstrates that DNA-SIP successfully resolved viruses of active formatotrophs at the viral community level. Novel viruses were identified for both primary guilds, including archaeal viruses of Methanobacteriales (family Anaerodiviridae and a possible novel family) and two Andreesenella viruses carrying diversity-generating retroelements. A provirus predicted to infect Methanothrix—a strictly acetoclastic methanogen—further illustrates the capacity of SIP-viromics to capture viral associations across trophic levels. Analysis of auxiliary viral genes (AVGs) revealed numerous defense-associated genes (dcm, metK, queC) and genuine metabolic AVG candidates, such as cysH (assimilatory sulfate reduction) and, a contiguous cluster including uxe, galU and serB (surface polysaccharide biosynthesis), which may influence on host metabolism. This study demonstrates the power of SIP-viromics for resolving virus-host associations in complex anaerobic communities, linking viral diversity directly to metabolically active formatotrophic and methanogenic guilds. The identification of novel viral lineages infecting key AD microorganisms, combined with AVGs with potential consequences for carbon and sulfur cycling, provides a foundation for understanding the functional role of viruses in AD process performance.

The symbiosis between corals and dinoflagellate algae is disrupted by heat stress, leading to bleaching and subsequent coral mortality, devastating reef habitats. Despite its planetary-scale impact, bleaching is a cellular-level breakdown of a multi-partner symbiosis. While much is known about the physiological and genomic responses of corals to bleaching, our understanding of it at the cellular level, specifically the interactions between the coral host, algal symbionts, and their microbial communities, remains limited. By combining single-cell transcriptomics, marker gene metabarcoding (16S rRNA + 18S rRNA + ITS2), and photochemical measurements, we provide a granular view of the microbial ecology of symbiotic breakdown. In heat-stress experiments with the coral Orbicella faveolata, which hosts two co-dominant algal symbionts, we observed distinct transcriptomic responses between Durusdinium and Breviolum, particularly in transcripts involved in nitrogen cycling. Coral cell type-specific expression was also observed, notably between gastrodermal cells hosting either algal symbiont, where we see symbiont-specific suppression of host heat stress genes, and in coral gland cells, where mucocytes appear to play an active role in the bleaching response. Furthermore, for the first time, we show the concurrent shifts in the prokaryotic and microeukaryotic microbiomes during experimental heat stress. This was marked by a decrease in suspected nitrifying bacteria, concurrent with an increase in suspected denitrifiers and nitrate reducers, early in the heat-stress response. This higher nitrogen availability, which is theorized to decouple Symbiodiniaceae population regulation from control by the coral host, also leads to a bloom of other microbes such as chlorophytes, diatoms, and labyrinthulids which may further contribute to the negative feedback loops that characterize coral bleaching. Using scRNA-seq and the latest metabarcoding methodologies, we provide a comprehensive view of the coral holobiont, in which all members are considered interconnected and important to the health of the entire microbial ecosystem. We found cell-type specific responses to bleaching, particularly within mucocytes and gastrodermal cells. Notably, a subpopulation of gland cells (mucocytes) showed a distinct increase during heat stress. We also saw an apparent symbiont-specific suppression of heat-stress-associated transcripts within coral gastrodermal cells. Other members of the holobiont also stood out during the bleaching process, including prokaryotes potentially important to nutrient cycling and previously overlooked protists. This study marks a starting point toward understanding the cellular dynamics of coral holobionts, shedding light on the mechanisms behind symbiosis breakdown, coral mortality, and ultimately, reef decline.

Phenylketonuria (PKU) is an inherited metabolic disorder caused by phenylalanine hydroxylase (PAH) deficiency, leading to elevated L-phenylalanine and severe neurological damage if untreated. While phenylalanine-based biomarkers are diagnostic and phenylalanine levels correlate with disease severity, the clinical manifestations of PKU are heterogeneous. To identify additional reliable, potentially novel biomarkers, we used germ-free sex-specific, organ-resolved infant whole-body metabolic models (infant-WBMs) to simulate PAH deficiency and predicted elevated L-phenylalanine and its derivatives, alongside reduced L-tyrosine fluxes, as the product of phenylalanine hydroxylation. To test the reliability of these predicted biomarkers to variations in microbiome composition, we combined the infant-WBMs with gut microbiome models from 48 female and 42 male healthy infants. Upon integrating microbiome data, we found that microbial metabolism significantly increased the predicted L-tyrosine availability, obscuring its utility as a universal biomarker. In 23

The gut symbiotic microbiota plays a fundamental role in host health, exerting a widespread influence on brain function and behavior. Environmental pollutants—pervasive in modern life—pose serious threats to both ecological integrity and public health. Notably, their effects may extend to the disruption of host behavioral regulation. However, whether pollutant-induced changes in the structure and function of the gut microbiome contribute causally to behavioral disturbances remains unclear. In particular, little is known about whether core symbiotic taxa can mitigate the detrimental effects of pollutants on the host, or through which molecular mechanisms such protection might occur. In this study, we show that gut symbiotic microbes play a critical role in alleviating anxiety-like behavior in zebrafish. To this end, we first established a zebrafish model of anxiety-like behavior using niclosamide exposure. Microbial diversity analyses revealed pronounced differences in gut microbial community structure between zebrafish exhibiting anxiety-like behavior and normal controls. Subsequently, fecal microbiota transplantation experiments identified Cetobacterium as a key biomarker species associated with anxiety-like behavior. Importantly, oral reintroduction of Cetobacterium effectively mitigated anxiety-like behavior in germ-free zebrafish. Moreover, integrated metabolomic and transcriptomic analyses demonstrated that Cetobacterium modulates host tryptophan metabolism, thereby increasing serotonin (5-HT) levels in both the gut and serum. In contrast, pharmacological inhibition of tryptophan hydroxylase (TPH) reduced 5-HT levels and markedly attenuated the anxiolytic effects of Cetobacterium. Collectively, these findings provide mechanistic insights into how gut symbiotic microbiota regulate host anxiety-like behavior and underscore the beneficial role of core gut symbionts in shaping host cognitive and behavioral functions.

Obesity remains a global health challenge, and responses to lifestyle-based weight-loss interventions are heterogeneous. Here, we evaluate a one-year structured lifestyle program in 50 adults with obesity (mean BMI 42 ± 7.0 kg/m2), integrating clinical, microbiome, and metabolomic profiling, to identify predictors of weight-loss success and metabolic improvement (ClinicalTrials.gov: NCT01344525). The intervention included a 3-month very low-calorie formula diet (approximately 850 kcal/day), a 3-month transition phase from a formula diet to a balanced diet (approximately 1000 kcal/day), and a 6-month maintenance period in which the participants followed a balanced diet (gradually increasing to a maximum of 2000 kcal/day). Following the intervention, the participants exhibited marked reductions in body weight, body fat percentage, C-reactive protein, and glycated hemoglobin. Longitudinal analyses revealed that shifts in the gut microbiota composition were associated with changes in clinical and anthropometric data, as well as gut barrier function. An increased abundance of Lachnospiraceae was associated with improved gut barrier function; the relationship was mediated by fecal butyrate and propionate. Multivariate analyses revealed that serum baseline levels of diacylphosphatidylcholine C40:1 predicted postintervention BMI, indicating that this metabolite could serve as a biomarker of weight loss success. A random forest model incorporating baseline microbial and clinical features was used to predict weight loss and clinical improvements with high accuracy. Our findings elucidate the interplay between the gut microbiota and host metabolism during weight loss and highlight the potential utility of baseline profiling to achieve a high success rate in personalized obesity treatment.

Indole acts as a critical interkingdom signal, promoting gastrointestinal homeostasis at basal levels while driving cytotoxicity and inflammation when accumulated at supraphysiological concentrations. Current interventions relying on broad-spectrum antibiotics or non-specific inhibitors lack the precision to distinguish pathological overproduction from homeostatic signaling, often disrupting essential commensal networks. Resolving this therapeutic dilemma requires elucidating whether indole hyperproduction arises from proportional expansion of indole-producing bacteria or from ecological enrichment of intrinsically hyperactive tryptophanase (TnaA) variants. Here, using dairy cow rumen as a model ecosystem, we integrated evolutionary genomics, computational structure modeling, and biochemical validation to dissect the structural and kinetic properties of TnaA variants and to determine their contribution to diet-induced indole hyperproduction. By integrating 462 TnaA orthologs from 21,471 reference genomes and 197 rumen metagenomes, we revealed a “conserved-fold, divergent-pocket” architecture: despite extensive sequence divergence (30–100

Understanding the interactions between diet and the gut microbiome is critical for identifying dietary interventions that support gut health. This is of particular importance for poultry where the elimination of antibiotic growth promoters has resulted in an alarming rise in enteric infections with significant economic consequences. While research has identified promising interventions including prebiotics, probiotics, and organic acids, these produce inconsistent outcomes across farms and production systems. This variability reflects a fundamental challenge: intervention efficacy depends on baseline conditions including diet composition and microbiota structure. Computational metabolic models offer powerful tools for dissecting diet-microbiome interactions, yet current approaches remain limited, largely ignoring the physiological parameters and spatial organization of the gastrointestinal tract that critically shape microbial metabolism and community dynamics. We developed the first multi-compartment, spatiotemporally resolved metabolic model of the chicken gastrointestinal tract. Our six-compartment framework integrates avian-specific physiological features including bidirectional flow through peristalsis and reverse peristalsis, feeding-fasting cycles with diurnal shifts in gut motility, and compartment-specific environmental parameters including pH gradients, oxygen levels, and transit times. The model captured distinct metabolic specialization along the gut, with upper compartments enriched for bile salt hydrolases, membrane lipid synthesis, and fatty acid biosynthesis, while cecal and colonic communities specialized in short-chain fatty acid synthesis pathways and polysaccharide degradation. In silico screening of 34 dietary supplements revealed context-dependent metabolic responses and predicted cellulose, starch, and L-threonine as robust enhancers of short-chain fatty acid production. A controlled feeding trial confirmed the model’s directional predictions for butyrate production, with cellulose and starch supplementation producing significant increases consistent with predictions. Quantitative rank-order agreement across multiple metabolites improved substantially in a trial-informed two-compartment model, demonstrating that predictive accuracy is primarily determined by the match between modeled and in vivo microbial community composition. Our findings demonstrate that microbial community composition is a primary determinant of metabolic outcomes and underscore the critical importance of context-specific modeling for precision nutrition strategies. This framework provides a mechanistic platform for rational design of dietary interventions that modulate gut microbial metabolism in poultry and is broadly adaptable to other livestock and human gastrointestinal systems.