High-throughput sequencing has revolutionized microbiome research, yet distinguishing viable from nonviable microbes in complex communities remains challenging. Microbial viability underlines microbiome function, which is mostly driven by living, active members. Transcriptional activity of genes has been suggested as a viability marker but not systematically explored in complex communities. Here, we find that both community-wide transcription and cpn60 gene activity can effectively quantify community composition and identify viable members. For targeted cpn60 amplicons, protein-coding RNA activity better captured the differential structure of the active community than 16S rRNA amplicons. To enable scalable application, we constructed an expanded database integrating diverse cpn60 protein and nucleotide sequences, substantially extending the existing cpnDB database. In Human Microbiome Project II data, cpn60 protein-based taxonomy strongly agreed with shotgun metagenomic profiles. Cpn60 transcript abundance correlated positively with bacterial growth rates estimated by the bPTR method, indicating its potential as a marker of microbial transcriptional activity and viability. It also distinguished resident gut microbes from potentially transient (e.g., oral-typical) members. Together, these results demonstrate that integrating DNA and RNA sequences at the single cpn60 locus enables accurate profiling of microbiome composition and activity, offering a cost-effective framework extendable to other rationally selected genes across diverse ecosystems.
Obesity is closely associated with gut microbial dysbiosis and chronic low-grade inflammation, yet how microbial signals orchestrate systemic immune-metabolic dysfunction remains unclear. In adiposity-prone Ningxiang pigs (NF), we observed an expansion of Gram-negative bacteria, elevated circulating lipopolysaccharide (LPS), and reduced abundance of Clostridium butyricum and its metabolite butyrate. Fecal microbiota transplantation (FMT) from NF donors induced adiposity in lean Duroc-Landrace-Yorkshire (CF) recipients, accompanied by a significant decrease in C. butyricum colonization. In high-fat diet (HFD)-fed mice, supplementation with C. butyricum and butyrate effectively restored gut microbial homeostasis and ameliorated obesity. Integrated multi-organ transcriptomic analyses across pigs and long-tailed macaques revealed that early growth response 1 (EGR1) is a central regulator of obesity-associated inflammation, with consistent enrichment of PI3K-Akt signaling. Single-cell RNA sequencing further demonstrated that EGR1-high macrophages adopt a pro-inflammatory M1 phenotype, whereas EGR1-low macrophages exhibit an anti-inflammatory M2 profile. Leveraging EGR1 macrophage-specific differentially expressed genes, both in silico knockout of EGR1 and pharmacological inhibition (EGR1i; IT25) confirmed the functional involvement of PI3K-Akt signaling downstream of EGR1. Integrating metagenomic and single-cell data, we propose a conserved LPS-EGR1 axis that governs macrophage polarization in obesity. To experimentally validate this model, we established an LPS-potentiated HFD-induced obesity model in mice and an LPS-induced M1 polarization model in RAW264.7 macrophages. Treatment with C. butyricum, butyrate, arginine, or EGR1i significantly attenuated weight gain, downregulated EGR1 expression, and effectively suppressed M1 macrophage polarization, thereby restoring the M1/M2 balance. Notably, this process is accompanied by significant alterations in genes related to the PI3K-Akt signaling pathway, suggesting that this pathway may act in concert with EGR1 within this regulatory network. In summary, these cross-species experiments elucidate a fundamental mechanism whereby gut-derived LPS regulates macrophage polarization via EGR1, thereby linking microbial dysbiosis to metabolic inflammation in mammals. CB and BA break the LPS-EGR1-M1 loop to combat obesity. CB: Clostridium butyricum. BA: Butyric acid. LPS: Lipopolysaccharide. GNB: Gram-negative bacteria. EGR1: Early Growth Response 1.
Human anelloviruses (ANVs) represent one of the most diverse, prevalent, and persistent viral communities within the human virome and constitute dominant members of the commensal viral ecosystem. Despite their ubiquity and emerging associations with immune modulation and hematologic malignancy, the genomic diversity and community organization of the human ANV virome (anellome) remain incompletely resolved. This limitation is driven in part by fragmented reference datasets, inconsistent taxonomic annotation, and geographic underrepresentation, particularly for Asian populations. A geographically expanded reference and standardized analytical framework are therefore needed to profile the human anellome at species-level and lineage-level resolution. We integrated metagenomic sequencing data from 6348 Chinese samples with globally available ANV genomes to construct an expanded phylogenomic resource for the human anellome. This integration improved Asian representation and expanded represented human ANV diversity to 771 species-level taxa, comprising 91 ICTV-recognized human-associated species and 680 provisional new species (PNS), corresponding to an approximately eightfold expansion in represented species-level diversity. The resulting framework enabled high-resolution profiling of personal anellomes. The richest individual profile contained 305 species-level taxa and 2071 lineages, indicating extensive intra-host ANV diversity under the expanded reference framework. Across analyzed cohorts and public metadata, we identified recurrent core anellome components characterized by high detection frequency, abundance, geographic representation, cross-cohort reproducibility, and longitudinal persistence. Several core lineages showed significant inverse associations with CD4 T-cell counts in HIV-infected individuals. Paired peripheral blood mononuclear cell (PBMC) and plasma analyses further identified compartment-associated differences in diversity, composition, and lineage enrichment. Within this framework, a TTMV::RARA-associated Betatorquevirus clade showed higher ORF2 conservation, RUNX1 motif enrichment, and PBMC-biased distribution compared with background clades. These findings support the human anellome as a structured commensal viral community that can be resolved at species-level and lineage-level resolution. The HANVdb Resource provides a reusable framework for investigating ANV diversity, anellome ecology, immune-associated variation, compartmental organization, and lineage-level hypotheses relevant to viral integration-associated disease.
Iron, the most abundant redox-active metal in the Earth’s crust, is coupled to numerous biogeochemical cycles. However, the mechanisms of iron oxidation and the organisms involved remain incompletely understood. Banded iron formations (BIFs) are a major reservoir of iron ore in the Precambrian sedimentary record, yet the biological contribution to their genesis remains a subject of unresolved debate. While large-scale BIF deposition largely ceased after the Proterozoic, microbial activity in modern Holocene sediments under fluctuating redox conditions provides a unique opportunity to examine mechanisms reminiscent of ancient iron cycling. Here, we report the stratigraphic sequence of microbiome profiles recorded in laminated facies with iron-containing crystalline illite in the embayment sediments beneath the Larsen C Ice Shelf (LCIS) in Antarctica during the Holocene. LCIS sediments record microbial community shifts tightly coupled to environmental changes throughout the Holocene. Metagenomic analyses revealed three dominant microbial phases corresponding to geological facies boundaries. The open marine setting (phase A) showed higher taxonomic richness, whereas the sub-ice shelf sediments (phases B and C) were largely anoxic and characterized by diverse chemolithoautotrophic metabolisms. Keystone taxa including uncultured members of Thermodesulfovibrionia, as well as unique microbial communities and metabolisms, were evident in the aphotic, anoxic seawater; metagenomic analyses further revealed chemolithotrophy. The Thermodesulfovibrionia bacterium, visualized using fluorescence in situ hybridization and designated as “Candidatus Mariimomonas ferrooxydans”, formed a novel clade in the phylum Nitrospirota. Metagenome-assembled genome analysis identified a putative outer-membrane Fe(II) oxidase, Cyc2, whose Fe(II)-oxidation activity was experimentally confirmed. Our findings document the interaction between microbiome and environment, illustrating how LCIS sediments preserve a dynamic record of microbial community responses to environmental transitions. These results provide critical insights into microbial iron mineralization, echoing the biogeochemistry of the geologic past, particularly synglacial iron formation during the Neoproterozoic Snowball Earth.
Alterations in the oral microbiota are increasingly recognized as an integral component of the tumor microenvironment, forming a clinically relevant oncobiome that may modulate tumorigenesis, therapeutic response, and patient prognosis in head and neck squamous cell carcinoma (HNSCC). In this prospective study, we characterized the salivary microbiome of 124 Spanish HNSCC patients (86 laryngeal and 38 pharyngeal carcinomas) and 66 healthy controls from northern Spain using 16S rRNA gene sequencing. Microbial community structure, differential abundance, and associations with tumor site and treatment received were analyzed using ANCOM-BC, while prognostic associations were assessed through univariable and multivariable Cox regression models and survival analyses. Distinct microbial community profiles were observed between patients and controls, revealing pronounced site-specific dysbiosis between pharyngeal and laryngeal tumors. Microbiome composition also varied according to treatment modality. Chemoradiotherapy induced a marked remodeling of the oncobiome, characterized by a post-treatment enrichment of Lactobacillus. More importantly, a specific 10-bacteria salivary signature was significantly associated with poorer survival outcomes. including disease-specific survival (DSS), overall survival (OS), and disease-free survival (DFS). This 10-bacterial signature remained independently associated with poor prognosis in multivariable analysis for DSS (HR = 6.215; p < 0.001), OS (HR = 5.417; p < 0.001), and DFS (HR = 6.842; p < 0.001), after adjustment for established clinicopathological variables. Furthermore, we identified tumor site–specific prognostic taxa within the oral microbiome of HNSCC patients. The presence of putative Prevotella, Porphyromonas and Fusobacterium species specifically and significantly correlated with improved DSS, OS and DFS in pharyngeal carcinomas, whereas putative Stomatobaculum longum and Dialister were associated with favorable survival outcomes in laryngeal carcinomas. Our findings reveal distinct oral microbial patterns linked to HNSCC prognosis and recurrence risk, demonstrating that the salivary microbiome may represent a source of non-invasive, dynamic biomarkers for risk stratification and personalized management. The identification of tumor site–specific bacterial signatures associated with both favorable and unfavorable survival outcomes supports the concept of an anatomically contextualized oncobiome, although validation in independent cohorts and higher-resolution microbiome studies will be required before clinical implementation.
