Metabolic Reconstruction Research Articles

Disclosure: M. D'silva: None. B. Malachowska: None. Y. Qiu: None. J. Sepulveda: None. R. Chang: None. S. Sidoli: None. J.L. Nadler: None. I.J. Kurland: Scorpion Therapeutics.An important early hallmark of Type I Diabetes Mellitus (T1D) is beta-cell dysfunction, a critical mechanism of which is cytokine mediated. Our exploratory study defined regulatory programs controlling human islet responses to pro-inflammatory cytokines by mapping abundance proteomics , as well as metabolomics and lipidomics from the same islets exposed to inflammation-inducing cytokines TNF-alpha, IL-1β and IFN-γ over the time course at 1,6 and 24 hrs after cytokine exposure. Methods: Pancreatic islets were purchased from Prodo Labs (https://prodolabs.com/). Islet samples were extracted by the Folch method, and targeted mass spectrometric methods employing a Sciex 6500+ QTRAP was used to identified metabolites, neutral and phospholipids and oxylipins/eicosanoids,from the aqueous and chloroform fractions. The Folch protein interface was collected, redissolved and then digested using S-Trap flters (Protif), 5% SDS. Proteome raw files were searched using Proteome Discoverer software. Results: Approximately 3500 proteins were identified, of which approximately 200 were significantly changed with cytokine vs control treatment that were further identified from the RECON genome-scale metabolic reconstruction as potentially important for metabolic network flux determination. ∼100 of these metabolic proteomic changes were seen at 1 hr , and an additional 85 metabolic proteins were significantly changed at 6 hr after cytokine stimulation along 35 proteins that were still changed from 1hr. Overall, cytokine stimulation resulted in an inhibition of metabolic protein expression at these time points, however, the metabolomic analysis indicated that islet metabolism was re-directed to specific pathways rather than simply suppressed. Levels of nucleotides needed for DNA and RNA synthesis could be seen to be increased, as well as pentose and hexosamine pathway intermediates, which depleted glycolytic and TCA cycle intermediates. The inflammatory islet response was reflected by an increase in platelet-activating factor PAF and lyso PAF at 1 and 6 hours, however the brunt of the oxylipin/eicosanoid response was delayed until 24 hours after cytokine stimulation, and secreted (media) oxylipins were composed mainly of cycloxygenase (COX) products of arachidonic (AA) acid, as well as 12- and 15-lipoxygenase (LOX) products of AA and DHA. At 24 hours after cytokine stimulation, the suppression/elevation of the metabolic responses seen at 1 and 6 hrs were largely resolved, along with elevations in HLA proteins and proteosome subunit expression consistent with immune response activation. Conclusion: Interventions for arresting beta cell dysfunction need to consider the pathways which may be activated sequentially and in coordination with immune activation.Presentation: Monday, July 14, 2025

Mitigating perfluorooctanoic acid inhibition in electrochemically-assisted spiral upflow anaerobic membrane reactor for wastewater treatment: EPS interaction-desorption dynamics and metabolic pathway reconstruction.

IntroductionClimate-driven precipitation changes are increasingly threatening alpine ecosystems, yet the adaptive responses of soil microbiomes to rainfall variability remain poorly characterized. This knowledge gaphinders our ability to predict ecosystem resilience under future climate scenarios.MethodsWe combined metagenomic sequencing with detailed physicochemical analyses to examine how natural precipitation events reshape the microbial communities in both rhizosphere and bulk soils associated with Poa alpigena in the alpine sandy ecosystems of Qinghai Lake.ResultsRainfall significantly reduced bacterial alpha diversity, particularly in bulk soils, and triggered a compositional shift from drought-resistant taxa (e.g., Geobacter, Pseudomonas) to moisture-adapted genera (e.g., Azospirillum, Methylobacterium). Actinobacteria remained consistently dominant (31.56-34.62%), while Proteobacteria abundance decreased markedly in the rhizosphere post-rainfall. Metabolic reconstruction revealed a transition from pre-rainfall carbohydrate catabolism to post-rainfall anaerobic energy production and carbon fixation pathways. The rhizosphere microbiome uniquely displayed drought-induced biofilm formation and rainfall-enhanced branched-chain amino acid metabolism. Soil moisture and total carbon were identified as primary drivers of microbial restructuring in bulk soils, whereas root exudates conferred stability to rhizosphere communities against hydrological fluctuations.DiscussionThese results elucidate microbiome-mediated adaptive strategies to precipitation changes in alpine sandy ecosystems, highlighting the critical buffering role of plant-microbe interactions. The study provides a mechanistic basis for predicting and restoring climatevulnerable wetlands under increasingly variable hydrological regimes.

Candidatus Acidulodesulfobacterales, a formerly proposed bacterial order within the Deltaproteobacteria lineage, represents an ecologically significant group in sulfur-rich environments. Their diversity and functional potential in artificial acid mine drainage (AMD) ecosystems have been well studied; however, their distribution and ecological role in marine hydrothermal sulfides remain poorly understood. Here we integrated publicly available metagenome-assembled genomes (MAGs) with a newly reconstructed MAG from hydrothermal sulfides to perform comprehensive phylogenetic, metabolic, and host-virus interaction analyses. Phylogenomic and 16S rRNA gene analyses indicated that this lineage represents a distinct phylum-level clade, leading us to propose the designation Ca. Acidulodesulfobacteriota. Metabolic reconstructions indicated a versatile lifestyle, encompassing pathways for carbon fixation, nitrogen fixation, sulfur metabolism, iron oxidation, and hydrogen oxidation. Notably, the concatenated DsrAB protein phylogeny and the mixed enzyme types involved in Dsr-dependent dissimilatory sulfur metabolism suggest that Ca. Acidulodesulfobacteriota may represent a transitional lineage in the evolutionary shift from reductive to oxidative Dsr metabolism. Viral auxiliary metabolic genes (AMGs) associated with this phylum were predicted to modulate host metabolic pathways, including folate biosynthesis and sulfur metabolism, highlighting intricate host-virus interactions. These findings advance our understanding of the evolution, metabolic potential, and ecological roles of Ca. Acidulodesulfobacteriota in biogeochemical cycling.

The WOR-3 phylum is widely distributed in various environments, including hot springs, marine ecosystems, and hydrothermal vents, yet its ecological roles and metabolic capabilities remain poorly understood. In this study, we analyzed 181 medium- to high-quality metagenome-assembled genomes, including 59 newly reconstructed from environmental samples and 122 retrieved from public databases. Phylogenetic analyses resolved the WOR-3 lineage into four subgroups (subgroup 1-4). Metabolic reconstruction revealed significant divergence of the carbon, sulfur, nitrogen, and hydrogen metabolism pathways among the different subgroups. Subgroup 1 was characterized by fermentative metabolism involving formate and ethanol and uniquely exhibited potential for carbon fixation via the Calvin cycle, as indicated by the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) gene. Notably, WOR-3 RuBisCO is phylogenetically affiliated with archaeal form III, although the carbon fixation pathway follows the canonical bacterial Calvin cycle-a feature of potential evolutionary significance. Subgroup 3 exhibits metabolic versatility, including genes for dissimilatory sulfate reduction, sulfur oxidation, partial denitrification, and fatty acid degradation. In addition, all subgroups harbored key components of hydrogen metabolism, including widespread NiFe hydrogenases, supporting H2-dependent electron transfer and energy conservation. Within the WOR-3 lineage, the coexistence of two respiratory enzyme systems-the Rnf complex and the oxidative phosphorylation respiratory chain-indicates distinct anaerobic and aerobic metabolic lifestyles, respectively. Collectively, this study expands the genomic framework for the WOR-3 phylum and provides novel insights into the metabolic versatility and ecological functions of this previously uncharacterized lineage in biogeochemical cycles of carbon, nitrogen, and sulfur.IMPORTANCEThe WOR-3 phylum represents a widespread but poorly understood bacterial lineage inhabiting diverse various environments. By integrating 181 metagenome-assembled genomes, including 59 newly reconstructed, this study provides the most comprehensive genomic framework to date for WOR-3. Phylogenomic and metabolic reconstruction revealed four distinct subgroups with divergent capacities for carbon, sulfur, and nitrogen metabolism. Notably, subgroup 1 encodes a complete Calvin-Benson-Bassham cycle featuring an archaeal-type form III ribulose-1,5-bisphosphate carboxylase/oxygenase, suggesting an unusual evolutionary trajectory for carbon fixation in this lineage. Subgroup 3 exhibits versatile metabolic potential, including dissimilatory sulfur metabolism, partial denitrification, and fatty acid degradation, highlighting its possible roles in multiple biogeochemical processes. These findings not only expand the taxonomic and functional landscape of the WOR-3 phylum but also offer key insights into its ecological roles in global element cycling.

Bacteria showcase remarkable metabolic diversity and traits, even among strains of the same species. In recent years, a large number of bacterial genomes have been sequenced, leading to the elucidation and documentation of genomic differences and commonalities across and within species. Genome-scale metabolic reconstructions, which are often defined and curated using data from phenotype microarrays, elucidate the differences in metabolic traits resulting from genomic diversity. These microarrays measure cellular respiration on a variety of carbon, nitrogen, phosphorus, and sulfur sources and various stressors and inhibitors over a period of time to determine the metabolic activity of a given strain. Despite their popularity in measuring bacterial metabolic activity and traits, no public databases that allow researchers to warehouse, access, and analyze this information currently exist. Additionally, there are no publicly available tools that allow researchers to view the variance of these metabolic traits across bacterial strains. To address this need, we present Phenotype Microarray Knowledgebase (PMkbase [version 1.0], https://pmkbase.com/), an interactive database that acts as a repository of phenotype microarray (PM) data with integrated sequence information. Binarized activity calls, along with associated kinetic parameters, are made for all metabolic substrates and inhibitors. Users can upload their own data for analysis and visualization and to perform quality checks on their experiments. PMkbase will address an unmet need to track and view bacterial metabolic traits and provide researchers with valuable information to develop metabolic models, enrich pangenomic analyses, and design new experiments.IMPORTANCEBacterial species can be differentiated by their metabolic profiles or the type of nutrients they consume. Interestingly, strains within the same species also display differences in nutrient consumption. Phenotype microarrays are a high-throughput, widely used technology to measure which substrates can be metabolized by various microbial strains and the extent to which inhibitors can affect it. Despite their widespread use, public databases to parse and access this data type at scale do not exist. PMkbase, which contains 9,024 data points for nitrogen substrate utilization, 41,664 data points for carbon substrate utilization, 8,448 data points for phosphorus/sulfur substrate utilization, and 27,264 data points on various antibiotics across three species (Escherichia coli, Pseudomonas putida, and Staphylococcus aureus), has been developed to allow researchers to freely access PM data, along with enriching the data with sequence information.

The bacterial genus Endozoicomonas is a predominant member of the coral microbiome, widely recognised for its ubiquity and ability to form high-density aggregates within coral tissues. Hence, investigating its metabolic interplay with coral hosts offers critical insights into its ecological roles and contributions to coral health and resilience. Using long- and short-read whole-genome sequencing of 11 Endozoicomonas strains from Acropora loripes, genome sizes were found to range between 5.8 and 7.1 Mbp. Phylogenomic analysis identified two distinct clades within the family Endozoicomonadaceae. Metabolic reconstruction uncovered clade-specific pathways, including the degradation of holobiont-derived carbon and lipids (e.g., galactose, starch, triacylglycerol, D-glucuronate), the latter of which suggests involvement of Endozoicomonas in host 'sex-type' steroid hormone metabolism. A clade-specific type 6 Secretion System (T6SS) and predicted effector molecules were identified, potentially facilitating coral-bacterium symbiosis. Additionally, genomic analyses revealed diverse phosphorus acquisition strategies, implicating Endozoicomonas in holobiont phosphorus cycling and stress responses. This study reveals clade-specific genomic signatures of Endozoicomonas supporting its mutualistic lifestyle within corals. Findings suggests possible roles in nutrient cycling, reproductive health, and stress resilience, offering novel insights into coral holobiont functioning.

Avian pathogenic Escherichia coli (APEC) are a genetically diverse pathotype primarily associated with extra-intestinal infections in birds. APEC lineages are predicted to have unique metabolic capabilities contributing to virulence and survival in the host environment. Here, we present a genome-scale metabolic model for the APEC pathotype based on 114 APEC genome sequences and lineage-specific models for the phylogroups B2, C and G based on a representative isolate for each phylogroup. A total of 1,848 metabolic reactions were predicted in the 114 APEC isolates before gap filling and manual correction. Of these, 89% represented core reactions, whilst the 11% accessory reactions were mostly associated with carbon and nitrogen metabolism. Predictions of auxotrophy were confirmed by inactivation of the conditionally essential lysA and the non-essential potE genes. The APEC metabolic model outperformed the E. coli K-12 iJO1366 model in the Biolog Phenotypic Array platform. Sub-models specific to phylogroups B2, C and G predicted differences in the metabolism of 3-hydroxyphenylacetate (3-HPAA), a phenolic acid derived from the flavonoid quercetin, which is commonly added to poultry feed. Two 3-HPAA-associated reactions/genes distinguished APEC phylogroup C from APEC phylogroups B2 and G, and 3-HPAA supported the growth of APEC phylogroup C in minimal media, but not phylogroups B2 and G. In conclusion, we have constructed genome-scale metabolic models for the three major APEC phylogroups B2, C and G and have identified a metabolic pathway distinguishing phylogroup C APEC. This demonstrates the importance of lineage- and pathotype-specific metabolic models when investigating genetically diverse microbial pathogens.

Multidimensional insights into efficiency-stability trade-off in anaerobic digestion from organic overloading stress: Kinetic behavior, energy harvesting, and genome-centric metagenomics microbial self-adaptation.

Genomic analysis of Pseudoalteromonas sp. SD03 reveals its potential in chitin hydrolysis.

Microorganisms have driven Earth's sulfur cycle since the emergence of life1-6, yet the sulfur-cycling capacities of microorganisms and their integration with other element cycles remain incompletely understood. One such uncharacterized metabolism is the coupling of sulfide oxidation with iron(III) oxide reduction, a ubiquitous environmental process hitherto considered to be strictly abiotic7,8. Here we present a comprehensive genomic analysis of sulfur metabolism across prokaryotes, and reveal bacteria that are capable of oxidizing sulfide using extracellular solid phase iron(III). Based on a phylogenetic framework of over hundred genes involved in dissimilatory transformation of sulfur compounds, we recorded sulfur-cycling capacity in most bacterial and archaeal phyla. Metabolic reconstructions predicted co-occurrence of sulfur compound oxidation and iron(III) oxide respiration in diverse members of 37 prokaryotic phyla. Physiological and transcriptomic evidence demonstrated that a cultivated representative, Desulfurivibrio alkaliphilus, grows autotrophically by oxidizing dissolved sulfide or iron monosulfide (FeS) to sulfate with ferrihydrite as an extracellular iron(III) electron acceptor. The biological process outpaced the abiotic process at environmentally relevant sulfide concentrations. These findings expand the known diversity of sulfur-cycling microorganisms and unveil a biological mechanism that links sulfur and iron cycling in anoxic environments, thus highlighting the fundamental role of microorganisms in global element cycles.

Understanding the molecular mechanisms behind plant response to stress can enhance breeding strategies and help us design crop varieties with improved stress tolerance, yield, and quality. To investigate resource redistribution from growth- to defense-related processes in an essential tuber crop, potato, here we generate a large-scale compartmentalized genome-scale metabolic model (GEM), potato-GEM. Apart from a large-scale reconstruction of primary metabolism, the model includes the full known potato secondary metabolism, spanning over 566 reactions that facilitate the biosynthesis of 182 distinct potato secondary metabolites. Constraint-based modeling identifies that the activation of the largest amount of secondary (defense) pathways occurs at a decrease of the relative growth rate of potato leaf, due to the costs incurred by defense. We then obtain transcriptomics data from experiments exposing potato leaves to two biotic stress scenarios, a herbivore and a viral pathogen, and apply them as constraints to produce condition-specific models. We show that these models recapitulate experimentally observed decreases in relative growth rates under treatment as well as changes in metabolite levels between treatments, enabling us to pinpoint the metabolic rewiring underlying growth-defense trade-offs. Potato-GEM thus presents a useful resource to study and broaden our understanding of potato and general plant defense responses under stress conditions.

Abstract Magnetotactic bacteria (MTB) are distinguished by their ability to navigate along Earth's magnetic field and form diverse intracellular minerals, including nanocrystals of magnetite or greigite (i.e., magnetosomes). Although MTB are widespread in oxic‐anoxic transition zones of aquatic systems worldwide, their presence in deep‐sea environments has been less explored largely due to challenges in sampling and analytical methods. Here, we investigated deep‐sea sediments from two active cold seeps in the South China Sea using metagenomic, magnetic, and microscopic techniques and extended our study to a global‐scale metagenomic analysis of cold seep ecosystems. Our results reveal a wide distribution and high phylogenetic diversity of MTB in cold seeps worldwide with the phylum Desulfobacterota being particularly prevalent. Genome‐scale metabolic reconstructions suggest that MTB contribute to iron and sulfur cycling potentially coupled with anaerobic methane oxidation in these deep‐sea habitats. These findings not only broaden our understanding of MTB diversity and distribution in the deep sea but also underscore their important roles in biogeochemical processes within cold seep ecosystems.

Microbial-targeted drug delivery systems represent an emerging and rapidly developing therapeutic strategy, garnering widespread attention for their potential. By utilizing engineered microorganismssuch as probiotics, Escherichia coli, and bacteriophagesas drug carriers, these systems enable precise, targeted drug delivery and controlled release, particularly in the treatment of tumors, autoimmune diseases, and inflammatory conditions. Microorganisms offer distinct advantages by navigating complex physiological and pathological environments through their inherent chemotaxis, metabolic adaptability, and responsiveness to specific lesions, thus significantly enhancing drug efficacy while minimizing side effects on healthy tissues . This review explores the latest advancements in microbial-targeted drug delivery systems, highlighting key technologies such as microbial engineering, metabolic pathway reconstruction, cell surface modification, and microbial targeting optimization. Additionally, we address the challenges these systems face in clinical application, including biosafety concerns, drug delivery efficiency, and regulatory and ethical issues. With ongoing innovations in engineering microbiology, microbial-targeted drug delivery systems hold promise for achieving more precise and effective treatments in the future.

Chemolithoautotrophic members from the Campylobacteria class are dominant key players in sulfidic habitats, where they make up a stable portion of sulfide-oxidizing bacterial communities. Nevertheless, few isolates have so far been cultivated and studied in situ, and most are derived from chemosynthetic ecosystems, limiting our understanding of their physiological and metabolic features as well as ecological roles in the global marine environments. In this study, seven potentially new species were successfully isolated from mangrove sediments and further diverged into three potentially new genera within the class Campylobacteria. These isolates were obligate chemolithoautotrophs that could grow through hydrogen oxidation as well as sulfur oxidation, reduction, and disproportionation. Metabolic reconstructions revealed that these isolates contained diverse sulfide:quinone oxidoreductase and flavocytochrome c sulfide dehydrogenase for sulfide oxidation, distinct Sox gene cluster for sulfur oxidation, as well as group I, II, and IV hydrogenases for hydrogen consumption and production. Notably, these strains lacked the complete denitrification pathway, instead having all genes for nitrogen fixation, which might facilitate their survival in the nitrogen-limited mangrove sediments. Moreover, they also demonstrated the ability to adapt to low O2 conditions, such as a more efficient 2-oxoglutarate:ferredoxin oxidoreductase complex for CO2 fixation and diverse terminal oxidases including Cco, Cox, and Cyd. Metatranscriptomic analysis further confirmed their activity and different adaptation mechanisms in in situ mangrove sediments. Assessing their occurrences indicated that these lineages were globally distributed in hypoxic and anoxic environments and dominant members of marine and mangrove sediments. Overall, these results indicate that these new Campylobacteria members are metabolically versatile and play an underappreciated role in the biogeochemical cycling of carbon-rich mangrove sediments.IMPORTANCEChemolithoautotrophic Campylobacteria spp. are generally associated with sulfide-rich environments, where they play a key role in the cycling of carbon, nitrogen, and sulfur. Yet, only a limited number of cultured isolates are currently available. In this study, we isolated seven potentially new species belonging to three new genera from mangrove sediments, which significantly expanded our understanding of the species diversity within the class Campylobacteria. These isolates demonstrated diverse and unique metabolic potentials for CO2 fixation, sulfur oxidation, hydrogen oxidation, nitrogen metabolism, and oxygen respiration, making them well adapted to the sulfur-rich, nitrogen-limited, and low-oxygen habitats they inhabit. The frequent detection of these novel species in marine and mangrove sediments, as revealed by 16S rRNA gene sequences in public databases, indicates a potential preference for oxygen-limited environments. Overall, this study promotes our understanding of the in situ function and ecological role of Campylobacteria, especially in previously overlooked carbon-rich sediment ecosystems.

The colonic epithelium plays a key role in the host-microbiome interactions, allowing uptake of various nutrients and driving important metabolic processes. To unravel detailed metabolic activities in the human colonic epithelium, our present study focuses on the generation of the first cell-type-specific genome-scale metabolic model (GEM) of human colonic epithelial cells, named iColonEpithelium. GEMs are powerful tools for exploring reactions and metabolites at the systems level and predicting the flux distributions at steady state. Our cell-type-specific iColonEpithelium metabolic reconstruction captures genes specifically expressed in the human colonic epithelial cells. iColonEpithelium is also capable of performing metabolic tasks specific to the colonic epithelium. A unique transport reaction compartment has been included to allow for the simulation of metabolic interactions with the gut microbiome. We used iColonEpithelium to identify metabolic signatures associated with inflammatory bowel disease. We used single-cell RNA sequencing data from Crohn’s Diseases (CD) and ulcerative colitis (UC) samples to build disease-specific iColonEpithelium metabolic networks in order to predict metabolic signatures of colonocytes in both healthy and disease states. We identified reactions in nucleotide interconversion, fatty acid synthesis and tryptophan metabolism were differentially regulated in CD and UC conditions, relative to healthy control, which were in accordance with experimental results. The iColonEpithelium metabolic network can be used to identify mechanisms at the cellular level, and we show an initial proof-of-concept for how our tool can be leveraged to explore the metabolic interactions between host and gut microbiota.

Quantifying supply and demand in the pea aphid-Buchnera symbiosis reveals the metabolic Achilles' heels of this interaction.

SUMMARYMetabolite identification remains a significant challenge in mass spectrometry (MS)‐based metabolomics research. To address this issue, we combined a triple‐labeled precursor‐based isotope tracing approach (TLEMMA) with high‐resolution liquid chromatography‐MS for metabolite identification and metabolic network construction. As a demonstration, we fed duckweed (Spirodela polyrhiza) with four forms of phenylalanine (Phe) including unlabeled Phe, Phe‐5H2, Phe‐8H2, and Phe‐13C915N1. The distinctive isotopic pattern obtained from MS spectra greatly facilitated data processing, enabling comprehensive extraction of all Phe‐derived metabolites. Importantly, the labeling pattern allowed efficient metabolite identification by significantly reducing the number of structural and positional isomers. Using this approach, 47 phenylalanine‐derived metabolites were putatively identified. To further evaluate the efficiency of metabolite identification in relation to the number of differently labeled precursors used, we compared the number of filtered candidates based solely on the labeling patterns obtained from unlabeled, single, dual, and triple isotope‐labeled precursor tracing experiments. On average, TLEMMA eliminates the number of false candidates by 99.1% compared with unlabeled samples, 95% compared with single isotope‐labeled samples, and 66.7% compared with dual isotope‐labeled samples. This significant reduction in the number of false positives, along with the ability to identify previously unreported metabolites, demonstrates the power of TLEMMA in advancing the field of metabolomics and metabolic network reconstruction.

Scaling metabolic model reconstruction up to the pan-genome level: A systematic review and prospective applications to photosynthetic organisms.

Salt-tolerant and halophilic microorganisms are critical drivers of ecosystem stability and biogeochemical cycling in athalassohaline environments. Lake Barkol, a high-altitude inland saline lake, provides a valuable natural setting for investigating microbial community dynamics and adaptation mechanisms under extreme salinity. In this study, we employed high-throughput metagenomic sequencing to characterize the taxonomic composition, metabolic potential, and ecological functions of microbial communities in both water and sediment samples from Lake Barkol. We reconstructed 309 metagenome-assembled genomes (MAGs), comprising 279 bacterial and 30 archaeal genomes. Notably, approximately 97% of the MAGs could not be classified at the species level, indicating substantial taxonomic novelty in this ecosystem. Dominant bacterial phyla included Pseudomonadota, Bacteroidota, Desulfobacterota, Planctomycetota, and Verrucomicrobiota, while archaeal communities were primarily composed of Halobacteriota, Thermoplasmatota, and Nanoarchaeota. Metabolic reconstruction revealed the presence of diverse carbon fixation pathways, including the Calvin-Benson-Bassham (CBB) cycle, the Arnon-Buchanan reductive tricarboxylic acid (rTCA) cycle, and the Wood-Ljungdahl pathway. Autotrophic sulfur-oxidizing bacteria, alongside members of Cyanobacteria and Desulfobacterota, were implicated in primary production and carbon assimilation. Nitrogen metabolism was predominantly mediated by Gammaproteobacteria, with evidence for both nitrogen fixation and denitrification processes. Sulfur cycling was largely driven by Desulfobacterota and Pseudomonadota, contributing to sulfate reduction and sulfur oxidation pathways. Microbial communities exhibited distinct osmoadaptation strategies. The “salt-in” strategy was characterized by ion transport systems such as Trk/Ktr potassium uptake and Na+/H+ antiporters, enabling active intracellular ion homeostasis. In contrast, the “salt-out” strategy involved the biosynthesis and uptake of compatible solutes including ectoine, trehalose, and glycine betaine. These strategies were differentially enriched between water and sediment habitats, suggesting spatially distinct adaptive responses to local salinity gradients and nutrient regimes. Additionally, genes encoding microbial rhodopsins were widely distributed, suggesting that rhodopsin-based phototrophy may contribute to supplemental energy acquisition under osmotic stress conditions. The integration of functional and taxonomic data highlights the metabolic versatility and ecological roles of microbial taxa in sustaining biogeochemical processes under hypersaline conditions. Overall, this study reveals extensive taxonomic novelty and functional plasticity among microbial communities in Lake Barkol and underscores the influence of salinity in structuring microbial assemblages and metabolic pathways in athalassohaline ecosystems.