Microorganisms carrying out denitrification in marine anoxic zones drive bioavailable nitrogen loss. Sequencing datasets have demonstrated the modularity of denitrification, with most populations having the genetic capability for only a subset of the pathway (NO3-➔NO2-➔NO➔N2O➔N2). Although previous work provided ecological explanations for this diversity among the functional modules, large trait variations exist within each functional module, and this within-module diversity and its biogeochemical implications remain unexplored. Here, we combine genomic data and modeling to explore how metabolic "lifestyle" strategies influence denitrifier community structure. We build a comprehensive genomic database of marine denitrifiers, and identify lifestyle differentiation among denitrifier functional groups. We then extend a mathematical ecosystem model by resolving two microbial functional types for each module representing a metabolic trade-off: a copiotroph, optimized for fast growth, and an oligotroph, optimized for high nutrient affinity. In the model, as the supply of organic matter relative to nitrate increases, the degree of copiotrophy among the community increases and then decreases. This suggests that oligotrophs are associated with either organic-matter- or nitrate-limiting conditions, whereas copiotrophic lifestyles are associated with an intermediate regime. Our model further associates NO2- reducers with oligotrophy and NO3- reducers with copiotrophy, particularly those producing greenhouse gas nitrous oxide (N2O), linking N2O production to substrate-replete conditions, which is consistent with our genome-based lifestyle estimates. Results provide insight into denitrifier ecological niches and thus the biogeochemical conditions that are associated with the production of intermediates, such as N2O, improving our understanding of how nitrogen cycling will change in a warming ocean.
Fungi readily colonize the inner bark (phloem) of spruce and other conifers despite these tissues having a high concentration of antifungal defense metabolites. These compounds include stilbenes, flavonoids and other phenolic substances, mostly present as glucosides. Yet the underlying biochemical mechanisms by which fungi resist conifer phenolics remain largely unresolved. Using untargeted metabolomics, structural elucidation and biological and biochemical assays, we investigated how and why fungi metabolize the major phenolics of Norway spruce (Picea abies). Various fungi, including those associated with bark beetles, were found to hydrolyze stilbene glucosides to their corresponding aglucones. Two basidiomycetes, the saprotroph Coprinellus radians and Cylindrobasidium ipidophilum, a symbiont of the Eurasian spruce bark beetle Ips typographus, then converted the stilbene aglucones into α-ribofuranosylated derivatives, revealing a previously unrecognized pathway in tree-colonizing fungi. Ribosylation markedly reduced the antifungal activity of the aglucones and stabilized them against hydrolysis by fungal and I. typographus enzymes, preventing regeneration of the toxic aglucones. Ribosylation was also correlated with increased growth on spruce bark-containing medium. Hence, the ability to overcome major conifer bark defenses by conversion of toxic stilbene aglucones to non-toxic α-ribosides may explain the successful colonization of this tissue by fungi, some of which support I. typographus attack.
Resident bacterial interactions can shape the invasion resistance of rhizosphere microbiomes, but whether interactions between weakly antagonistic resident bacteria can generate emergent antagonism against invading pathogens remains poorly understood. Here, we used a systematic pairwise interaction screening to identify Ralstonia pickettii RAL5 and Acinetobacter oleivorans ACI4 bacterial pair, that together provided a strong suppression of the phytopathogenic R. solanacearum Rs1115 strain. Although RAL5 and ACI4 monocultures only weakly inhibited pathogen growth, the RAL5-ACI4 co-cultures strongly suppressed the Rs1115, which was associated with asymmetric competition where the RAL5 dominated the ACI4 species. In line with this competitive asymmetry, broad transcriptional reprogramming was detected in RAL5 and only limited stress- and catabolism-associated responses in ACI4. The increased suppressiveness of co-cultures was associated with clear shifts in the extracellular metabolite profile, including the accumulation of candidate antimicrobial metabolites (e.g., a novobiocin-like feature and 4-aminophenol), and with the release of intracellular contents from ACI4 following RAL5-mediated lysis. The observed emergent antagonism also held in greenhouse experiments with tomato, where the RAL5-ACI4 consortium reduced R. solanacearum abundance and bacterial wilt severity relatively much more compared to when either strain was applied alone. Together, these results suggest that competitive interactions between resident bacteria can activate latent biocontrol potential in rhizosphere microbiomes, providing a new approach to harness resident bacterial interactions for enhanced pathogen suppression and biocontrol.
This study investigates the role of gut microbiota-derived hydrogen sulfide (H2S) in obesity and glucose metabolism disorders. By integrating human gut metagenomic data, intervention experiments in mouse models, and in vitro cellular assays, we identified a signature of microbial sulfur metabolism in human cohorts and provided experimental evidence for its causal role and underlying metabolic mechanisms in mice. In clinical cohorts with obesity and glucose metabolism disorders, we observed a notable enrichment of genes involved in sulfur transport and H2S production. In mouse models, administration of H2S-producing Desulfovibrio desulfuricans, engineered Escherichia coli expressing phsABC, and the H2S donor NaHS consistently induced body weight gain and impaired glucose tolerance. Transcriptome analysis and cellular experiments indicated that H2S was associated with downregulation of the PPAR signaling pathway and lipid metabolism pathways in the liver, which may contribute to the abnormal accumulation of lipids and glycogen. Furthermore, rescue experiments using a PPAR agonist and an H2S adsorbent partially reversed these metabolic abnormalities. Collectively, our work provides experimental evidence in mouse models demonstrating that gut microbial H2S promotes metabolic dysfunction through hepatic PPARα suppression, providing potential targets for microbiome-based therapeutic interventions.
Free ammonia (FA) is a prevalent chemical inhibitor in wastewater ecosystems, yet its ecological impacts on microbial communication and cooperation remain poorly understood. Here, we demonstrated that FA stress restructured the community function of oxygenic photogranules (OPGs) by rewiring signaling-associated regulatory networks and redirecting cellular energy allocation. Temporal profiling of extracellular signaling molecules and intracellular regulatory molecules, integrated with metagenomic and metatranscriptomic analyses, revealed a concentration-dependent reconfiguration of microbial signaling. Under low FA exposure (≤ 1.0 mg/L), diffusible signal factor (DSF)- and indole-3-acetic acid (IAA)-associated pathways were more active, coinciding with photogranules consolidation and efficient nitrogen removal. At moderate FA stress (5.0 mg/L), the regulatory landscape shifted toward acyl-homoserine lactone (AHL)-associated signaling and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)-mediated intracellular regulation, consistent with enhanced aggregation and stress adaptation. In contrast, severe FA stress (25.0 mg/L) broadly attenuated signaling-associated pathways, weakened metabolite cross-feeding networks, impaired energy generation, and increased maintenance-related energetic demands. Under these constraints, microbial populations appeared to shift from cooperative metabolism toward self-maintenance-oriented carbon metabolism, evidenced by activation of the carbon-efficient glyoxylate shunt. Collectively, these findings suggest that FA-induced signaling disruption constrains microbial cooperation through cellular energy limitation, highlighting energy allocation as a key determinant of microbial social stability in phototrophic wastewater microbiomes.
The plastisphere is a unique ecosystem with microbes colonizing and potentially degrading plastic debris in the environment, provided that the polymers are enzymatically accessible as substrates to drive microbial growth. It also harbors an unusually high occurrence of antibiotic resistance genes, suggesting plastic debris as a potential vector for antibiotic-resistant microorganisms. In this study, we investigated microbial communities in forest soil degrading an emerging type of bioplastics, aliphatic long-chain polyesters (LCAPs). Sequencing analysis revealed a family-VIII esterase strongly associated with LCAP depolymerization that showed high structural similarity to type C β-lactamases. Structural modeling and substrate docking analysis indicated catalytically favorable binding of both LCAP and β-lactam antibiotics. Furthermore, the active site appeared to be located in a large, wide-open groove, rather than in a tunnel, resulting in a protein with a striking "pac-man"-like structure. Heterologous expression and in vitro activity testing confirmed its dual functionality as plastic depolymerase and β-lactam hydrolase. Sequence analysis indicated the enzyme as membrane-associated lipoprotein likely to be directed to the outer membrane. The membrane anchoring of the enzyme may offer striking microbial-ecological benefits, by preventing enzyme loss especially in aqueous environments, by increased catalytic efficiency through high enzyme concentration at the cell-plastic interface, and by spatially linking catalysis with membrane transport, thereby limiting monomer loss to non-producing plastisphere-community members (cheaters). Hence, our study highlighted a plastic depolymerizing enzyme with a striking substrate spectrum, bridging plastics and antibiotics degradation, and provides intriguing perspectives for understanding the microbial physiology, ecology, and evolution of (bio)plastic degradation in the environment.
Microbial carbon use efficiency (CUE) governs soil carbon persistence under warming, with its magnitude highly dependent on how microbial metabolism adapts to varying substrates. How agricultural management mediates this response in agroecosystems remains poorly understood. To address this, we coupled 18O-H2O tracing of community-level CUE with 13C-probing of substrate utilization efficiency (SUE) from substrates differing in apparent bioavailability after 11 years of experimental warming under conventional and conservation agriculture, using glucose as a readily available carbohydrate and vanillin as a less accessible lignin-derived aromatic substrate. Warming stimulated glucose SUE under both management regimes, whereas vanillin SUE declined by 20% exclusively under conservation agriculture. This shift in relative anabolic efficiency (RAE, SUEglucose/SUEvanillin) was associated with warming-induced changes in CUE, primarily through changes in vanillin rather than glucose SUE. Concurrently, conservation agriculture improved substrate quality under warming, reflected by a reduced litter lignocellulose index. In response, microbial communities exhibited distinct functional adaptations, shifting toward copiotrophic taxa with higher rRNA operon copy numbers and a 16% increase in the anabolic-to-catabolic gene ratio, indicating enhanced microbial growth potential and greater investment for labile carbon processing. Structural equation modeling further showed that shifts in substrate quality were a primary driver of RAE, mediated by changes in microbial life-history strategies under conservation agriculture. Together, these results provide a genome-resolved mechanistic framework showing that conservation agriculture enhances soil carbon persistence under warming by reshaping microbial relative anabolic efficiency and metabolic strategies, highlighting metabolic adaptation as a key mechanism regulating soil carbon stability in warming agroecosystems.
Soils are among the most diverse but least understood environments on the planet. Just below our feet, secluded from our sight, we find complex communities of bacteria, fungi, protists and animals. These communities organize themselves in both time and space, from fast to slow growers, from decomposers to predators, from early colonizers to late arrivers. Despite a growing understanding of community dynamics, the lives of organisms within the soil often remain poorly understood: how do organisms grow, propagate, and disperse within their particle-packed environments? In this essay, I reflect on the microbial life cycles found in soils. Starting from a single gram of soil collected on our campus, I explore the conceptual building blocks underlying life cycle biology, how life cycles emerge within their environment and how they are coupled between species. Microbes express widely diverse life cycle motifs with many unique life stages promoting growth and/or propagation. These life stages have a major impact on community ecology; they determine the fate of microbes within soils and affect how, when, and where microbes interact. A life cycle perspective is thus essential for deepening our understanding of the community dynamics that unfold underfoot.
Bifidobacteria inhabiting the primate gut exhibit host-dependent genetic diversification, particularly in their gene repertoire related to carbohydrate metabolism, suggesting adaptation to host diets. However, these diverse genetic traits remain poorly associated with specific dietary components. Here, through enzymatic and genetic analyses, we demonstrate that several Bifidobacterium species residing in the gut of gummivorous primates, such as marmosets, possess previously uncharacterised pathways for the efficient utilisation of type-II arabinogalactan (AG), the major polysaccharide component of tree gums. The assimilation pathways comprises two key components: a bifunctional β-1,3-galactanase that cleaves the AG backbone via both endo- and exo-mode actions and an ATP-binding cassette transporter that internalises the released arabinogalactan oligosaccharides (AGOs) into cells. Data mining of deposited metagenomic datasets suggested that the endo/exo-β-1,3-galactanase and the AGO transporter contribute to cross-feeding interactions within Bifidobacterium communities in the gut of gummivorous primates. Our study not only highlights molecular strategies employed by certain Bifidobacterium species to adapt to the dietary habits of a host but may also inform probiotic intervention strategies for the health and welfare of these primates in captive settings.
Deoxygenation is driving ecological shifts across vast oceanic volumes. To understand and predict future ocean ecosystem functioning and stability, it is critical to identify the eukaryotic metabolic repertoire that enables survival under anoxia. Foraminiferan protists are one of few eukaryotic lineages that can inhabit anoxic marine sediments, environments relevant today and in Earth's past. Here, we investigate the metabolic strategies employed by a representative of an early-evolving foraminiferan group to persist anoxia. A saccamminid foraminifer inhabiting an anoxic bathyal seafloor in the Santa Barbara Basin (CA, USA) was preserved in situ. (Meta)transcriptomic analyses revealed an aerotolerant mitochondrial metabolism lacking Cytochrome c Oxidase, but expressing alternative oxidase, potentially fueled by internally released oxygen during ROS detoxification, and utilizing a TCA reductive Complex II. This foraminifer uses glutamate oxidation and aspartate-malate shuttle to generate reducing equivalents, driving ATP production via an atypical anaerobic electron-transport chain. Energy metabolism is also tightly linked to phosphate availability, facilitating substrate-level phosphorylation of high-energy intermediates. This foraminifer's competitive advantage is its anaerobic energy metabolism combined with ability to detoxify and reduce oxygen, if present. These unusual capabilities confer a blueprint for understanding how early eukaryotes may have evolved during anoxia, remained resilient during the Neoproterozoic Oxygenation Event, and likely will be ecological winners amid ongoing ocean deoxygenation.
Although horizontal gene transfer drives bacterial diversification, its contribution to chromosome-scale variation in human gut commensals remains unclear. This study demonstrated that human-associated bifidobacteria undergo extensive chromosomal transfer through natural transformation. Comparative genomics of coexisting Bifidobacterium pseudocatenulatum isolates from a single individual revealed extensive recombination signatures between the lineages. We experimentally reproduced this recombination by co-culturing strains, resulting in the transfer of multiple chromosomal regions and generation of mosaic genomes. Individual recombination tracts reached up to 247 kb per site, with cumulative replacements accounting for up to 28.9% of the recipient chromosome. These transfers occurred with heat-killed donors or purified DNA and were abolished by DNase, thereby identifying natural transformation as the underlying mechanism. Furthermore, we observed that environmental factors strongly influenced transformation frequency, suggesting that gut environmental conditions play a role regulating this process. Using natural transformation, we established a simple markerless genome-editing method that enables efficient gene deletions. Deletions of the Tad pili, ComEA-ComEC, or DprA-ComM-YraN gene clusters abolished transformation, defining the core machinery. The conservation of these genes across the genus Bifidobacterium and experimental demonstration of natural transformation in Bifidobacterium longum and Bifidobacterium breve indicate that natural transformation capacity is widespread within the genus. Our findings establish natural transformation as a key mechanism that promotes genome plasticity and contributes to adaptive evolution in bifidobacteria, thereby expanding our current understanding of horizontal gene transfer in the human gut microbiota.
Diatom blooms influence carbon cycling through organic matter production and its deposition or remineralization - processes mediated by the microbial community. Viruses can influence diatom bloom dynamics and even terminate blooms, yet interactions between diatoms, their viruses, and associated bacteria remain poorly resolved. Here, we examined how infection of the toxigenic diatom Pseudo-nitzschia galaxiae by its ssRNA virus PnGalRNAV reshapes host physiology, microbiome structure, and organic-matter processing in non-axenic batch cultures. Using epi-fluorescence microscopy, 16S rRNA amplicon sequencing, and metatranscriptomics, we linked microbial composition, localisation, and functional activity during viral lysis. Infection rapidly collapsed diatom growth and induced a senescence-like host state, with broad repression of photosynthesis, silicon metabolism, and core biosynthetic pathways, alongside induction of heat-shock and other stress-related genes. Concurrently, phycosphere-associated bacteria declined, detritosphere-associated bacteria increased, and community composition shifted from Marinobacter (Gammaproteobacteria) dominated, towards Flavobacteriaceae (Bacteroidetes) dominated, especially by Polaribacter. In non-infected controls Alphaproteobacteria proved to benefit from the stable healthy phycospheres with a distinct DOM pool. Bacterial metatranscriptomes showed significant upregulation of polysaccharide-degradation-associated genes in infected cultures, indicating active utilisation of lysis-derived diatom glycans. Similar compositional and metabolic profiles in infected cultures and later-stage senescent controls suggest infection accelerated senescence-associated microbial processes. Overall, viral lysis converted a productive diatom culture into a detrital, DOM-rich environment that selects for specialised polysaccharide degraders, redirects carbon through the viral shunt and may accelerate nutrient recycling in coastal systems. Extending this approach to natural microbial communities and diverse diatom-virus systems will help determine whether these mechanisms are broadly conserved.
In zoos, intensive human contact and artificial feeding may create pathways for microplastic (MP) ingestion and gut colonization. We hypothesized that ingested MPs form intestinal plastispheres with elevated pathogenic potential and enhanced environmental persistence. To test this, we surveyed feces from 15 zoo-dwelling species and coupled particle characterization, feces-derived intestinal simulations, metagenomic sequencing, and a subsequent water-exposure experiment. Zoo feces contained more abundant MPs than reported for wild counterparts, with fragments predominating and polyethylene terephthalate (PET)/polystyrene (PS) dominating polymer profiles. MP burdens tracked human-animal interaction patterns, with human-fed species (e.g., Tiger, Elephant) carrying the highest loads (88-212 items/g). MPs supported dense biofilms whose composition diverged from bulk gut communities, exhibiting greater compositional variability and substrate-specific assembly. Metagenomic analyses revealed coordinated enrichment of potentially pathogenic taxa, virulence factor genes (VFGs), and antibiotic resistance genes (ARGs), with ARG profiles dominated by efflux- and inactivation-related mechanisms and tightly associated with mobile genetic elements. Elevated Type II/III/IV/VI secretion systems and effector delivery-related VFGs occurred within extracellular polymeric substance-rich biofilms, suggesting enhanced potential for ARG retention and horizontal gene transfer. During the 35-day aquatic exposure, MP-associated communities persisted longer than non-plastic particle-associated communities and free gut microbiota, suggesting that plastic-specific properties promote microbial persistence. PET/PS plastispheres showed the slowest declines in bacterial activity and favored the persistence of Enterococcus, Enterobacter, and Clostridium. Overall, intestinal MPs in zoo animals may select, enrich, and export high-risk microbiomes, highlighting the need for MP mitigation and evidence-based management of zoos and adjacent ecosystems.
Chloroplast-stealing (a.k.a., "kleptoplastidic") plankton transiently obtain and integrate prey metabolic machinery into their own cells. To do so, they must address a series of challenges, including physical localization of acquired machinery, metabolic integration of organelles and their products, and maintenance of machinery. Here, we study this transient integration in the marine ciliate Mesodinium chamaeleon by pulse feeding the ciliate with cryptophyte algal prey (Storeatula major) and then tracking changes in ciliate cellular ultrastructure, physiology (growth and photosynthesis), and gene expression over time. We demonstrate a predictable series of changes, from the reconfiguration of metabolic activity fueled by recent ingestion, to a period of rapid growth, to a reduction in physiological performance as prey organelle number and functionality become limiting. Because M. chamaeleon also steals transcriptionally active prey nuclei, gene expression is a complex milieu of host and cryptophyte expression, with much of prey metabolism apparently intact in the new host, albeit at low levels. Collectively, our results highlight the holistic orchestration of stolen organelles to produce rapid-yet transient-growth in a new host.
Coral resilience under climate change depends on the stability of coral-Symbiodiniaceae symbioses. Vertically transmitting corals inherit symbionts directly from parental colonies, yet the extent to which symbiont cellular traits are conserved across host life stages remains unclear. Here, we examined cell-surface glycan profiles of Symbiodiniaceae in parental colonies and eggs of the coral Montipora capitata. Glycan signatures were structured by symbiont species and differed between coral life stages, with mannose/glucose- and galactose-containing glycoproteins as primary drivers of variation. Despite life-stage differences, parent-offspring comparisons revealed significant conservation of glycan profiles, indicating intergenerational transmission of symbiont cellular traits that differed between Cladocopium C31 and Durusdinium glynnii and were driven by distinct glycan classes. These results suggest that vertical transmission preserves key recognition-relevant glycans while allowing flexibility in other symbionts' surface traits, consistent with a role in maintaining symbiosis stability.
Dryland ecosystems rely on infrequent rainfall pulses to activate soil microbial communities, yet the fraction and identity of microbes resuscitating after hydration remain unclear. We applied bioorthogonal non-canonical amino acid tagging coupled with fluorescence-activated cell sorting (BONCAT-FACS) and 16S rRNA gene sequencing to identify translationally active bacteria in early (L-BSC) and late (D-BSC) successional cyanobacteria-dominated biocrusts subjected to 3 mm simulated rainfall under light and dark conditions. Our results reveal that only a small subset of the microbial community resumes activity within six hours, with higher active cell abundances in mature crusts. Microbial activity patterns were largely independent of light exposure and showed partial decoupling from total community composition, indicating that presence does not predict short-term function. These findings suggest that biocrust maturity shapes microbial activation dynamics and that functional responses to precipitation pulses are governed by a conserved pool of fast responders, informing predictions of dryland soil microbiome resilience under changing precipitation regimes.
Microorganisms assemble into spatially structured communities in which neighboring cells are separated by highly heterogeneous cell-cell distances. These spatial distances are not merely geometric features but key ecological variables that determine whether cells can exchange metabolites, compete through toxins, sense one another, or transfer genes, thereby profoundly influencing community assembly, productivity, and the evolution of microorganisms residing in communities. In this review, we synthesize recent progress on how cell-cell distance in microbial communities is determined, as well as how it modulates microbial interactions that lead to diverse ecological and evolutionary consequences. Specifically, we propose several testable conceptual frameworks that help quantify complicated distance-interaction relationships. We discuss emerging imaging, automated analysis, and spatial engineering approaches that now make it possible to quantify and manipulate cell-cell distance with increasing precision. Viewing microbial communities through the lens of cell-cell distance provides a unifying framework for linking microscale spatial organization to microbiome function, offering new opportunities to predict and engineer microbial communities.
Sulfate-reducing bacteria (SRB) are widespread in marine and terrestrial environments, where they often form syntrophic associations with bacteria, archaea, and eukaryotes. Among the most intimate of these are multipartite symbioses in gutless marine oligochaete worms, which host SRB and sulfur-oxidizing endosymbionts that engage in a syntrophic exchange of sulfur compounds. Despite decades of research on free-living SRB, the metabolic traits that enable SRB to persist in symbiosis, and how these differ across hosts and environments, remain poorly understood. We show that a globally distributed clade of symbiotic SRB, which we named Candidatus Desulfoconcordia, has a conserved core metabolism that diverges from free-living relatives. Using comparative genomics and metaproteomics, we reveal that these endosymbionts retain key traits of SRB such as sulfate reduction, complete oxidation of acetate to CO2, amino acid degradation for nitrogen acquisition, and transport of essential nutrients. However, they exhibit a more oxygen-tolerant metabolism and lack typical nutrient-scavenging mechanisms of free-living SRB. One trait, the glyoxylate bypass, was consistently expressed in situ and may serve both in reactive oxygen species defence and in biomass generation. The expression of oxygen-tolerant pathways, coupled with the loss of nutrient-scavenging functions, indicate specialization to a host-associated, redox-fluctuating environment distinct from that of free-living SRB. The symbiont genomes are also larger than those of free-living relatives, contrasting with genome reduction in many endosymbionts and reinforcing the importance of metabolic versatility. Our findings provide a framework for understanding how metabolic flexibility enables SRB to persist in long-term multipartite symbioses across diverse marine ecosystems.
Mountain gradients act as natural experiments, and elevational mitochondrial DNA clines in insects are often interpreted as signatures of local metabolic adaptation. However, mitochondrial DNA is maternally co-inherited with heritable endosymbionts that can promote cytoplasmic hitchhiking and, when abundant, dominate marker-gene microbiome profiles, complicating inference about environmental forcing. Here, we evaluate a symbiont-aware cytoplasmic-axis framework in the tea green leafhopper Empoasca onukii using a densely replicated elevational survey across tea agroecosystems. Across 790 adults from 79 sites spanning 11 to 2750 meters above sea level, we quantified Wolbachia infection prevalence, within-host burden, and strain composition, and related these measures to mitochondrial haplotypes, a conservative nuclear reference marker, and whole-insect bacterial community profiles. Wolbachia prevalence, burden, and strain composition varied along elevation, with pronounced strain turnover. Mitochondrial diversity declined and haplotypes homogenized at high elevations, whereas the nuclear reference marker showed weak spatial structure, yielding mitochondrial-nuclear discordance consistent with cytoplasmic hitchhiking and sweep-like mtDNA homogenization. Bacterial community separation was strongest when Wolbachia features were retained but persisted after Wolbachia removal and renormalization, indicating both compositional dominance by Wolbachia and residual restructuring among non-Wolbachia taxa. In a balanced subset, mitochondrial coding variation and host energetic readouts provided observational functional context for the Wolbachia-centered cytoplasmic-axis pattern. Together, these results place Wolbachia at the center of a testable cytoplasmic framework linking elevational mitochondrial turnover with microbiome restructuring, and they highlight the broader importance of dominant heritable symbionts.
