Single-microbe RNA sequencing uncovers unexplored specialized metabolic functions of keystone species in the human gut

Abstract

The human body is inhabited by trillions of microorganisms that play a crucial role in health and diseases. Our understanding of the species and functional composition of the human gut microbiome is rapidly expanding, but it is still mainly based on taxonomic profiles or gene abundance measurements. As such, little is known about the species–function heterogeneity and dynamic activities in human microecosystem niches. By applying a novel gut-specific single-microbe ribonucleic acid (RNA) sequencing and analytical framework on three healthy donors with distinct enterotypes, we created a comprehensive transcriptional landscape of the human gut microbiome and dissected functional specialization in 38,922 single microbes across 198 species. We investigated the functional redundancy and complementarity involved in short-chain fatty acids related central carbon metabolism and studied the heterogeneity and covariation of single-microbe metabolic capacity. Comparing the human gut microbiome at different times throughout the day, we were able to map diurnal dynamic activities of the gut microbiome and discovered its association with sub-population functional heterogeneous. Remarkably, using single-microbe RNA sequencing, we systematically dissected the metabolic function heterogeneity of Megamonas funiformis, a keystone species in Asian populations. Together with in vitro and in vivo experimental validations, we proved M. funiformis can effectively improve mineral absorption through exogenous phytic acid degradation, which could potentially serve as a probiotic that reduces malnutrition caused by deficiency of mineral elements. Our results indicated that species-function heterogeneity widely exists and plays important roles in the human gut microbiome, and through single-microbe RNA sequencing, we have been able to capture the transcriptional activity variances and identify keystone species with specialized metabolic functions of possible biological and clinical importance.

Keywords

diurnal dynamic / gut microbiome / metabolism / single-microbe RNA sequencing / species functional heterogeneity

References

[1] Rackaityte, Elze, and Susan V. Lynch. 2020. “The Human Microbiome in the 21st Century.” Nature Communications 11: 5256. https://doi.org/10.1038/s41467-020-18983-8

[2] Mann, Elizabeth R., Ying Ka Lam, and Holm H. Uhlig. 2024. “Short-Chain Fatty Acids: Linking Diet, the Microbiome and Immunity.” Nature Reviews Immunology 24: 577-595. https://doi.org/10.1038/s41577-024-01014-8

[3] Vieira-Silva, Sara, Gwen Falony, Youssef Darzi, Gipsi Lima-Mendez, Roberto Garcia Yunta, Shujiro Okuda, Doris Vandeputte, et al. 2016. “Species-Function Relationships Shape Ecological Properties of the Human Gut Microbiome.” Nature Microbiology 1: 16088. https://doi.org/10.1038/nmicrobiol.2016.88

[4] Xu, Meng-Qi, Fei Pan, Li-Hua Peng, and Yun-Sheng Yang. 2024. “Advances in the Isolation, Cultivation, and Identification of Gut Microbes.” Military Medical Research 11: 34. https://doi.org/10.1186/s40779-024-00534-7

[5] Lai, Senying, Yan Yan, Yanni Pu, Shuchun Lin, Jian-Ge Qiu, Bing-Hua Jiang, Marisa Isabell Keller, et al. 2023. “Enterotypes of the Human Gut Mycobiome.” Microbiome 11: 179. https://doi.org/10.1186/s40168-023-01586-y

[6] McCallum, Giselle, and Carolina Tropini. 2024. “The Gut Microbiota and Its Biogeography.” Nature Reviews Microbiology 22: 105-118. https://doi.org/10.1038/s41579-023-00969-0

[7] Keller, Marisa Isabell, Suguru Nishijima, Daniel Podlesny, Chan Yeong Kim, Shahriyar Mahdi Robbani, Christian Schudoma, Anthony Fullam, et al. 2024. “Refined Enterotyping Reveals Dysbiosis in Global Fecal Metagenomes.” bioRxiv 2024: 607711. https://doi.org/10.1101/2024.08.13.607711

[8] Costea, Paul I., Falk Hildebrand, Manimozhiyan Arumugam, Fredrik Bäckhed, Martin J. Blaser, Frederic D. Bushman, Willem M. de Vos, et al. 2018. “Enterotypes in the Landscape of Gut Microbial Community Composition.” Nature Microbiology 3: 8-16. https://doi.org/10.1038/s41564-017-0072-8

[9] Risely, Alice, Kerstin Wilhelm, Tim Clutton-Brock, Marta B. Manser, and Simone Sommer. 2021. “Diurnal Oscillations in Gut Bacterial Load and Composition Eclipse Seasonal and Lifetime Dynamics in Wild Meerkats.” Nature Communications 12: 6017. https://doi.org/10.1038/s41467-021-26298-5

[10] Shen, Yifei, Qinghong Qian, Liguo Ding, Wenxin Qu, Tianyu Zhang, Mengdi Song, Yingjuan Huang, et al. 2024. “High-Throughput Single-Microbe RNA Sequencing Reveals Adaptive State Heterogeneity and Host-Phage Activity Associations in Human Gut Microbiome.” Protein & Cell 16: pwae027. https://doi.org/10.1093/procel/pwae027

[11] Shen, Yifei, Fei Yu, Dan Zhang, Qianda Zou, Mengxiao Xie, Xiao Chen, Lingjun Yuan, et al 2022. “Dynamic Alterations in the Respiratory Tract Microbiota of Patients With COVID-19 and Its Association With Microbiota in the Gut.” Advanced Science 9: 2200956. https://doi.org/10.1002/advs.202200956

[12] Tett, Adrian, Edoardo Pasolli, Giulia Masetti, Danilo Ercolini, and Nicola Segata. 2021. “Prevotella Diversity, Niches and Interactions With the Human Host.” Nature Reviews Microbiology 19: 585-599. https://doi.org/10.1038/s41579-021-00559-y

[13] Segui-Perez, Celia, Rivka de Jongh, Robin L. W. Jonkergouw, Paula Pelayo, Emily P. Balskus, Aldert Zomer, and Karin Strijbis. 2024. “Prevotella Timonensis Degrades the Vaginal Epithelial Glycocalyx Through High Fucosidase and Sialidase Activities.” mBio 15: e00691-00624. https://doi.org/10.1128/mbio.00691-24

[14] Liou, Jong-Shian, Chien-Hsun Huang, Nao Ikeyama, Ai-Yun Lee, I-Ching Chen, Jochen Blom, Chih-Chieh Chen, et al. 2020. “Prevotella Hominis Sp. Nov., Isolated From Human Faeces.” International Journal of Systematic and Evolutionary Microbiology 70: 4767-4773. https://doi.org/10.1099/ijsem.0.004342

[15] Yoshida, Naofumi, Takuo Emoto, Tomoya Yamashita, Hikaru Watanabe, Tomohiro Hayashi, Tokiko Tabata, Namiko Hoshi, et al. 2018. “Bacteroides Vulgatus and Bacteroides Dorei Reduce Gut Microbial Lipopolysaccharide Production and Inhibit Atherosclerosis.” Circulation 138: 2486-2498. https://doi.org/10.1161/CIRCULATIONAHA.118.033714

[16] Chen, Bo, Lulu Sun, Guangyi Zeng, Zhe Shen, Kai Wang, Limin Yin, Feng Xu, et al. 2022. “Gut Bacteria Alleviate Smoking-Related NASH by Degrading Gut Nicotine.” Nature 610: 562-568. https://doi.org/10.1038/s41586-022-05299-4

[17] Zha, Xu, Xicheng Liu, Mengping Wei, Huanwei Huang, Jiaqi Cao, Shuo Liu, Xiaomei Bian, et al. 2025. “Microbiota-Derived Lysophosphatidylcholine Alleviates Alzheimer's Disease Pathology via Suppressing Ferroptosis.” Cell Metabolism 37: 169-186.e9. https://doi.org/10.1016/j.cmet.2024.10.006

[18] Liu, Xiaochun, Yang Luo, Xingyu Chen, Mingyang Wu, Xiaoqiang Xu, Jingru Tian, Yingxia Gao, et al. 2024. “Fecal Microbiota Transplantation Against Moderate-to-Severe Atopic Dermatitis: A Randomized, Double-Blind Controlled Explorer Trial.” Allergy. (In Press). https://doi.org/10.1111/all.16372

[19] Yachida, Shinichi, Sayaka Mizutani, Hirotsugu Shiroma, Satoshi Shiba, Takeshi Nakajima, Taku Sakamoto, Hikaru Watanabe, et al. 2019. “Metagenomic and Metabolomic Analyses Reveal Distinct Stage-Specific Phenotypes of the Gut Microbiota in Colorectal Cancer.” Nature Medicine 25: 968-976. https://doi.org/10.1038/s41591-019-0458-7

[20] Lee, Giljae, Hyun Ju You, Jasmohan S. Bajaj, Sae Kyung Joo, Junsun Yu, Seoyeon Park, Hyena Kang, et al. 2020. “Distinct Signatures of Gut Microbiome and Metabolites Associated With Significant Fibrosis in Non-Obese NAFLD.” Nature Communications 11: 4982. https://doi.org/10.1038/s41467-020-18754-5

[21] Almeida, Alexandre, Stephen Nayfach, Miguel Boland, Francesco Strozzi, Martin Beracochea, Zhou Jason Shi, Katherine S. Pollard, et al. 2021. “A Unified Catalog of 204,938 Reference Genomes From the Human Gut Microbiome.” Nature Biotechnology 39: 105-114. https://doi.org/10.1038/s41587-020-0603-3

[22] Becht, Etienne, Leland McInnes, John Healy, Charles-Antoine Dutertre, Immanuel W. H. Kwok, Lai Guan Ng, Florent Ginhoux, and Evan W. Newell. 2019. “Dimensionality Reduction for Visualizing Single-Cell Data Using UMAP.” Nature Biotechnology 37: 38-44. https://doi.org/10.1038/nbt.4314

[23] Rey, Federico E., Mark D. Gonzalez, Jiye Cheng, Meng Wu, Philip P. Ahern, and Jeffrey I. Gordon. 2013. “Metabolic Niche of a Prominent Sulfate-Reducing Human Gut Bacterium.” Proceedings of the National Academy of Sciences 110: 13582-13587. https://doi.org/10.1073/pnas.1312524110

[24] Qin, Youwen, Aki S. Havulinna, Yang Liu, Pekka Jousilahti, Scott C. Ritchie, Alex Tokolyi, Jon G. Sanders, et al. 2022. “Combined Effects of Host Genetics and Diet on Human Gut Microbiota and Incident Disease in a Single Population Cohort.” Nature Genetics 54: 134-142. https://doi.org/10.1038/s41588-021-00991-z

[25] Clasen, Sara J., Michael E. W. Bell, Andrea Borbón, Du-Hwa Lee, Zachariah M. Henseler, Jacobo de la Cuesta-Zuluaga, Katarzyna Parys, et al. “Silent Recognition of Flagellins From Human Gut Commensal Bacteria by Toll-Like Receptor 5.” Science Immunology 8: eabq7001. https://doi.org/10.1126/sciimmunol.abq7001

[26] Lu, Zheng, and James A. Imlay. 2021. “When Anaerobes Encounter Oxygen: Mechanisms of Oxygen Toxicity, Tolerance and Defence.” Nature Reviews Microbiology 19: 774-785. https://doi.org/10.1038/s41579-021-00583-y

[27] Culp, Elizabeth J., and Andrew L. Goodman. 2023. “Cross-Feeding in the Gut Microbiome: Ecology and Mechanisms.” Cell Host & Microbe 31: 485-499. https://doi.org/10.1016/j.chom.2023.03.016

[28] Vishnu Prasoodanan, P. K., Ashok K. Sharma, Shruti Mahajan, Darshan B. Dhakan, Abhijit Maji, Joy Scaria, and Vineet K. Sharma. 2021. “Western and Non-Western Gut Microbiomes Reveal New Roles of Prevotella in Carbohydrate Metabolism and Mouth-Gut Axis.” npj Biofilms and Microbiomes 7: 77. https://doi.org/10.1038/s41522-021-00248-x

[29] Zhao, Tingting, Han Yue, Junfeng Peng, Yingmin Nie, Longzhen Wu, Tingting Li, Wei Niu, et al 2023. “Degradation of Xylan by Human Gut Bacteroides Xylanisolvens XB1A.” Carbohydrate Polymers 315: 121005. https://doi.org/10.1016/j.carbpol.2023.121005

[30] Stincone, Anna, Alessandro Prigione, Thorsten Cramer, Mirjam M. C. Wamelink, Kate Campbell, Eric Cheung, Viridiana Olin-Sandoval, et al. 2015. “The Return of Metabolism: Biochemistry and Physiology of the Pentose Phosphate Pathway.” Biological Reviews 90: 927-963. https://doi.org/10.1111/brv.12140

[31] Koendjbiharie, Jeroen G., Richard van Kranenburg, and Servé W. M. Kengen. 2021. “The PEP-Pyruvate-Oxaloacetate Node: Variation at the Heart of Metabolism.” FEMS Microbiology Reviews 45: fuaa061. https://doi.org/10.1093/femsre/fuaa061

[32] Zhang, Silai, Satoshi Wakai, Naoya Sasakura, Hiroko Tsutsumi, Yoji Hata, Chiaki Ogino, and Akihiko Kondo. 2020. “Pyruvate Metabolism Redirection for Biological Production of Commodity Chemicals in Aerobic Fungus Aspergillus Oryzae.” Metabolic Engineering 61: 225-237. https://doi.org/10.1016/j.ymben.2020.06.010

[33] Hackmann, Timothy J. 2022. “Redefining the Coenzyme A Transferase Superfamily With a Large Set of Manually Annotated Proteins.” Protein Science 31: 864-881. https://doi.org/10.1002/pro.4277

[34] Ikeyama, Nao, Takumi Murakami, Atsushi Toyoda, Hiroshi Mori, Takao Iino, Moriya Ohkuma, and Mitsuo Sakamoto. 2020. “Microbial Interaction Between the Succinate-Utilizing Bacterium Phascolarctobacterium Faecium and the Gut Commensal Bacteroides Thetaiotaomicron.” Microbiologyopen 9: e1111. https://doi.org/10.1002/mbo3.1111

[35] Wang, Xingyu, Zhongle Cai, Qiaoling Wang, Chao Wu, Yingkai Sun, Zhifeng Wang, Xiaoqiang Xu, et al 2024. “Bacteroides methylmalonyl-CoA Mutase Produces Propionate That Promotes Intestinal Goblet Cell Differentiation and Homeostasis.” Cell Host & Microbe 32: 63-78.e67. https://doi.org/10.1016/j.chom.2023.11.005

[36] Qiu, Xiaojie, Qi Mao, Ying Tang, Li Wang, Raghav Chawla, Hannah A. Pliner, and Cole Trapnell. 2017. “Reversed Graph Embedding Resolves Complex Single-Cell Trajectories.” Nature Methods 14: 979-982. https://doi.org/10.1038/nmeth.4402

[37] Ma, Luyan Z., Di Wang, Yiwei Liu, Zhenyu Zhang, and Daniel J. Wozniak. 2022. “Regulation of Biofilm Exopolysaccharide Biosynthesis and Degradation in Pseudomonas Aeruginosa.” Annual Review of Microbiology 76: 413-433. https://doi.org/10.1146/annurev-micro-041320-111355

[38] Zhou, Diao, Ying Zhao, Jing Li, Vinothkannan Ravichandran, Leli Wang, Qiuyun Huang, Cang Chen, Hengjia Ni, and Jia Yin. 2021. “Effects of Phytic Acid-Degrading Bacteria on Mineral Element Content in Mice.” Frontiers in Microbiology 12: 753195. https://doi.org/10.3389/fmicb.2021.753195

[39] De Vos, Willem M., Minh Nguyen Trung, Mark Davids, Guizhen Liu, Melany Rios-Morales, Henning Jessen, Dorothea Fiedler, Max Nieuwdorp, and Thi Phuong Nam Bui. 2024. “Phytate Metabolism Is Mediated by Microbial Cross-Feeding in the Gut Microbiota.” Nature Microbiology 9: 1812-1827. https://doi.org/10.1038/s41564-024-01698-7

[40] Wu, Shu-en, Seika Hashimoto-Hill, Vivienne Woo, Emily M. Eshleman, Jordan Whitt, Laura Engleman, Rebekah Karns, et al. 2020. “Microbiota-Derived Metabolite Promotes HDAC3 Activity in the Gut.” Nature 586: 108-112. https://doi.org/10.1038/s41586-020-2604-2

[41] Lopez, Hubert W., Charles Coudray, Marie-Anne Levrat-Verny, Christine Feillet-Coudray, Christian Demigné, and Christian Rémésy. 2000. “Fructooligosaccharides Enhance Mineral Apparent Absorption and Counteract the Deleterious Effects of Phytic Acid on Mineral Homeostasis in Rats.” The Journal of Nutritional Biochemistry 11: 500-508. https://doi.org/10.1016/s0955-2863(00)00109-1

[42] Yatsunenko, Tanya, Federico E. Rey, Mark J. Manary, Indi Trehan, Maria Gloria Dominguez-Bello, Monica Contreras, Magda Magris, et al. 2012. “Human Gut Microbiome Viewed Across Age and Geography.” Nature 486: 222-227. https://doi.org/10.1038/nature11053

[43] Fassarella, Marina, Ellen E. Blaak, John Penders, Arjen Nauta, Hauke Smidt, and Erwin G. Zoetendal. 2021. “Gut Microbiome Stability and Resilience: Elucidating the Response to Perturbations in Order to Modulate.” Gut 70: 595. https://doi.org/10.1136/gutjnl-2020-321747

[44] Li, Leyuan, Tong Wang, Zhibin Ning, Xu Zhang, James Butcher, Joeselle M. Serrana, Caitlin M. A. Simopoulos, et al. 2023. “Revealing Proteome-Level Functional Redundancy in the Human Gut Microbiome Using Ultra-Deep Metaproteomics.” Nature Communications 14: 3428. https://doi.org/10.1038/s41467-023-39149-2

[45] Wu, Gary D., Jun Chen, Christian Hoffmann, Kyle Bittinger, Ying-Yu Chen, Sue A. Keilbaugh, Meenakshi Bewtra, et al. 2011. “Linking Long-Term Dietary Patterns With Gut Microbial Enterotypes.” Science 334: 105-108. https://doi.org/10.1126/science.1208344

[46] Wolter, Mathis, Erica T. Grant, Marie Boudaud, Alex Steimle, Gabriel V. Pereira, Eric C. Martens, and Mahesh S. Desai. 2021. “Leveraging Diet to Engineer the Gut Microbiome.” Nature Reviews Gastroenterology & Hepatology 18: 885-902. https://doi.org/10.1038/s41575-021-00512-7

[47] Schlemmer, Ulrich, Wenche Frølich, Rafel M. Prieto, and Felix Grases. 2009. “Phytate in Foods and Significance for Humans: Food Sources, Intake, Processing, Bioavailability, Protective Role and Analysis.” Molecular Nutrition & Food Research 53: S330-S375. https://doi.org/10.1002/mnfr.200900099

[48] Kumar, Awadhesh, Milan Kumar Lal, Soumya Kumar Sahoo, Goutam Kumar Dash, Upasana Sahoo, Biswaranjan Behera, Lopamudra Nayak, and Torit Baran Bagchi. 2023. “The Diversity of Phytic Acid Content and Grain Processing Play Decisive Role on Minerals Bioavailability in Rice.” Journal of Food Composition and Analysis 115: 105032. https://doi.org/10.1016/j.jfca.2022.105032

[49] Berdy, Brittany, Amy L. Spoering, Losee L. Ling, and Slava S. Epstein. 2017. “In Situ Cultivation of Previously Uncultivable Microorganisms Using the Ichip.” Nature Protocols 12: 2232-2242. https://doi.org/10.1038/nprot.2017.074

[50] Dobin, Alexander, Carrie A. Davis, Felix Schlesinger, Jorg Drenkow, Chris Zaleski, Sonali Jha, Philippe Batut, Mark Chaisson, and Thomas R. Gingeras. 2013. “STAR: Ultrafast Universal RNA-seq Aligner.” Bioinformatics 29: 15-21. https://doi.org/10.1093/bioinformatics/bts635

[51] Liao, Yang, Gordon K. Smyth, and Wei Shi. 2014. “Featurecounts: an Efficient General Purpose Program for Assigning Sequence Reads to Genomic Features.” Bioinformatics 30: 923-930. https://doi.org/10.1093/bioinformatics/btt656

[52] Smith, Tom, Andreas Heger, and Ian Sudbery. 2017. “UMI-Tools: Modeling Sequencing Errors in Unique Molecular Identifiers to Improve Quantification Accuracy.” Genome Research 27: 491-499. https://doi.org/10.1101/gr.209601.116

[53] Hao, Yuhan, Stephanie Hao, Erica Andersen-Nissen, William M. Mauck,, Shiwei Zheng, Andrew Butler, Maddie J. Lee, et al. 2021. “Integrated Analysis of Multimodal Single-Cell Data.” Cell 184: 3573-3587.e29. https://doi.org/10.1016/j.cell.2021.04.048

[54] Barbie, David A., Pablo Tamayo, Jesse S. Boehm, So Young Kim, Susan E. Moody, Ian F. Dunn, Anna C. Schinzel, et al. 2009. “Systematic RNA Interference Reveals That Oncogenic KRAS-Driven Cancers Require TBK1.” Nature 462: 108-112. https://doi.org/10.1038/nature08460

[55] Aibar, Sara, Carmen Bravo González-Blas, Thomas Moerman, Vân Anh Huynh-Thu, Hana Imrichova, Gert Hulselmans, Florian Rambow, et al. 2017. “SCENIC: Single-Cell Regulatory Network Inference and Clustering.” Nature Methods 14: 1083-1086. https://doi.org/10.1038/nmeth.4463

[56] DeTomaso, David, Matthew G. Jones, Meena Subramaniam, Tal Ashuach, Chun J. Ye, and Nir Yosef. 2019. “Functional Interpretation of Single Cell Similarity Maps.” Nature Communications 10: 4376. https://doi.org/10.1038/s41467-019-12235-0

[57] Hänzelmann, Sonja, Robert Castelo, and Justin Guinney. 2013. “GSVA: Gene Set Variation Analysis for Microarray and RNA-Seq Data.” BMC Bioinformatics 14: 7. https://doi.org/10.1186/1471-2105-14-7

[58] Trapnell, Cole, Davide Cacchiarelli, Jonna Grimsby, Prapti Pokharel, Shuqiang Li, Michael Morse, Niall J. Lennon, et al. 2014. “The Dynamics and Regulators of Cell Fate Decisions Are Revealed by Pseudotemporal Ordering of Single Cells.” Nature Biotechnology 32: 381-386. https://doi.org/10.1038/nbt.2859

[59] Sakon, H., F. Nagai, M. Morotomi, and R. Tanaka. 2008. “Sutterella Parvirubra Sp. Nov. and Megamonas Funiformis Sp. Nov., Isolated From Human Faeces.” International Journal of Systematic and Evolutionary Microbiology 58: 970-975. https://doi.org/10.1099/ijs.0.65456-0

[60] Ting, H. U. O., M. A. O. Bao-hong, L. I. Ya-li, and R. E. N. Wei. 2022. “Determination of 30 Trace Elements in Blood by Microwave Digestion-Inductively Coupled Plasma-Mass Spectrometry.” Analysis and Testing Technology and Instruments 28: 198-204. https://doi.org/10.16495/j.1006-3757.2022.02.014

RIGHTS & PERMISSIONS

2025 The Author(s). iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.