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Microbial nitrogen limitation in the mammalian large intestine


Resource limitation is a fundamental factor governing the composition and function of ecological communities. However, the role of resource supply in structuring the intestinal microbiome has not been established and represents a challenge for mammals that rely on microbial symbionts for digestion: too little supply might starve the microbiome while too much might starve the host. We present evidence that microbiota occupy a habitat that is limited in total nitrogen supply within the large intestines of 30 mammal species. Lowering dietary protein levels in mice reduced their faecal concentrations of bacteria. A gradient of stoichiometry along the length of the gut was consistent with the hypothesis that intestinal nitrogen limitation results from host absorption of dietary nutrients. Nitrogen availability is also likely to be shaped by host–microbe interactions: levels of host-secreted nitrogen were altered in germ-free mice and when bacterial loads were reduced via experimental antibiotic treatment. Single-cell spectrometry revealed that members of the phylum Bacteroidetes consumed nitrogen in the large intestine more readily than other commensal taxa did. Our findings support a model where nitrogen limitation arises from preferential host use of dietary nutrients. We speculate that this resource limitation could enable hosts to regulate microbial communities in the large intestine. Commensal microbiota may have adapted to nitrogen-limited settings, suggesting one reason why excess dietary protein has been associated with degraded gut-microbial ecosystems.

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Fig. 1: Mammalian faecal C/N ratio is linked to diet and physiology and controls microbial abundance in vivo.
Fig. 2: Antibiotics change gut nitrogen and host secretions.
Fig. 3: Microbes use nitrogen from host diet and secretions.

Data availability

The 16S rRNA gene nucleotide sequences generated in this study can be downloaded from the European Nucleotide Archive under study accession numbers PRJEB26478 (protein manipulation and NanoSIMS experiments) and PRJEB26446 (antibiotics experiment). NanoSIMS and bulk isotopic data for the dietary and injected 15N study is included in Supplementary Table 4. Other data that support these findings are available from the corresponding author upon request.


  1. 1.

    McFall-Ngai, M. Adaptive immunity: care for the community. Nature 1145, 153 (2007).

    Article  Google Scholar 

  2. 2.

    Sender, R., Fuchs, S. & Milo, R. Are we really vastly outnumbered? Revisiting the ratio of bacterial to host cells in humans. Cell 164, 337–340 (2016).

    CAS  Article  Google Scholar 

  3. 3.

    Lennon, J. T. & Jones, S. E. Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat. Rev. Microbiol. 9, 119–130 (2011).

    CAS  Article  Google Scholar 

  4. 4.

    Hooper, L. V. & Macpherson, A. J. Immune adaptations that maintain homeostasis with the intestinal microbiota. Nat. Rev. Immunol. 10, 159–169, (2010).

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Backhed, F., Ley, R. E., Sonnenburg, J. L., Peterson, D. A. & Gordon, J. I. Host–bacterial mutualism in the human intestine. Science 307, 1915–1920 (2005).

    Article  Google Scholar 

  6. 6.

    Fuller, M. F. & Reeds, P. J. Nitrogen cycling in the gut. Annu. Rev. Nutr. 18, 385–411 (1998).

    CAS  Article  Google Scholar 

  7. 7.

    Walter, J. & Ley, R. The human gut microbiome: ecology and recent evolutionary changes. Annu. Rev. Microbiol. 65, 411–429 (2011).

    CAS  Article  Google Scholar 

  8. 8.

    Carmody, R. N. & Turnbaugh, P. J. Gut microbes make for fattier fish. Cell Host Microbe 12, 259–261 (2012).

    CAS  Article  Google Scholar 

  9. 9.

    Borgstrom, B., Dahlqvist, A., Lundh, G. & Sjovall, J. Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36, 1521–1536 (1957).

    CAS  Article  Google Scholar 

  10. 10.

    Elser, J. J. et al. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol. Lett. 10, 1135–1142 (2007).

    Article  Google Scholar 

  11. 11.

    Gardner, M. L. G. Gastrointestinal absorption of intact proteins. Annu. Rev. Nutr. 8, 329–350 (1988).

    CAS  Article  Google Scholar 

  12. 12.

    Ferraris, R. P. & Carey, H. V. Intestinal transport during fasting and malnutrition. Annu. Rev. Nutr. 20, 195–219 (2000).

    CAS  Article  Google Scholar 

  13. 13.

    Lilburn, T. G. et al. Nitrogen fixation by symbiotic and free-living spirochetes. Science 292, 2495–2498 (2001).

    CAS  Article  Google Scholar 

  14. 14.

    Vecherskii, M. V., Naumova, E. I., Kostina, N. V. & Umarov, M. M. Some specific features of nitrogen fixation in the digestive tract of the European beaver (Castor fiber). Dokl. Biol. Sci. 411, 452–454 (2006).

    CAS  Article  Google Scholar 

  15. 15.

    Wostman, B. S. The germ-free animal in nutritional studies. Annu. Rev. Nutr. 1, 257–279 (1981).

    Article  Google Scholar 

  16. 16.

    Sterner, R. W. & Elser, J. J. Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere (Princeton Univ. Press, Princeton, 2002).

  17. 17.

    Frost, P. C. & Elser, J. J. Growth response of littoral mayflies to the phosphorus content of their food. Ecol. Lett. 5, 232–240 (2002).

    Article  Google Scholar 

  18. 18.

    Elser, J. J. et al. Growth rate–stoichiometry couplings in diverse biota. Ecol. Lett. 6, 936–943 (2003).

    Article  Google Scholar 

  19. 19.

    Fink, P. & Von Elert, E. Physiological responses to stoichiometric constraints: nutrient limitation and compensatory feeding in a freshwater snail. Oikos 115, 484–494 (2006).

    CAS  Article  Google Scholar 

  20. 20.

    Simpson, S. J. & Raubenheimer, D. The Nature of Nutrition: A Unifying Framework from Animal Adaptation to Human Obesity (Princeton Univ. Press, Princeton, 2012).

  21. 21.

    Le Couteur, D. et al. The influence of macronutrients on splanchnic and hepatic lymphocytes in aging mice. J. Gerontol. A Biol. 70, 1499–1507 (2014).

    Article  Google Scholar 

  22. 22.

    Solon-Biet, S. M. et al. Macronutrient balance, reproductive function and lifespan in aging mice. Proc. Natl Acad. Sci. USA 112, 3481–3486 (2015).

    CAS  Article  Google Scholar 

  23. 23.

    Holmes, A. J. et al. Diet-microbiome interactions in health are controlled by intestinal nitrogen source constraints. Cell Metab. 25, 140–151 (2017).

    CAS  Article  Google Scholar 

  24. 24.

    Zimmerman, A. E., Allison, S. D. & Martiny, A. C. Phylogenetic constraints on elemental stoichiometry and resource allocation in heterotrophic marine bacteria. Environ. Microbiol. 16, 1398–1410 (2013).

    Article  Google Scholar 

  25. 25.

    Mouginot, C. et al. Elemental stoichiometry of Fungi and Bacteria strains from grassland leaf litter. Soil Biol. Biochem. 76, 278–285 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Stephen, A. M. & Cummings, J. H. The microbial contribution to human faecal mass. J. Med. Microbiol. 13, 45–56 (1980).

    CAS  Article  Google Scholar 

  27. 27.

    Tjoelker, M. G., Craine, J. M., Wedin, D., Reich, P. B. & Tilman, D. Linking leaf and root trait syndromes among 39 grassland and savannah species. New Phytol. 167, 493–508 (2005).

    CAS  Article  Google Scholar 

  28. 28.

    Kartzinel, T. R. et al. DNA metabarcoding illuminates dietary niche partitioning by African large herbivores. Proc. Natl Acad. Sci. USA 112, 8019–8024 (2015).

    CAS  Article  Google Scholar 

  29. 29.

    Kleynhans, E. J., Jolles, A. E., Bos, M. & Olff, H. Resource partitioning along multiple niche dimensions in differently sized African savanna grazers. Oikos 120, 591–600 (2011).

    Article  Google Scholar 

  30. 30.

    Müller, D. W. H. et al. Assessing the Jarman–Bell Principle: scaling of intake, digestibility, retention time andgut fill with body mass in mammalian herbivores. Comp. Biochem. Phys. A 164, 129–140 (2013).

    Article  Google Scholar 

  31. 31.

    Hirakawa, H. Coprophagy in leporids and other mammalian herbivores. Mamm. Rev. 31, 61–80 (2001).

    Article  Google Scholar 

  32. 32.

    Kashyap, P. C. et al. Complex interactions among diet, gastrointestinal transit, and gut microbiota in humanized mice. Gastroenterology 144, 967–977 (2013).

    Article  Google Scholar 

  33. 33.

    Reikvam, D. H. et al. Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression. PLoS ONE 6, e17996 (2011).

    CAS  Article  Google Scholar 

  34. 34.

    Wlodarska, M. et al. Antibiotic treatment alters the colonic mucus layer and predisposes the host to exacerbated Citrobacter rodentium-induced colitis. Infect. Immun. 79, 1536–1545 (2011).

    CAS  Article  Google Scholar 

  35. 35.

    Johansson, M. E. et al. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. USA 105, 15064–15069 (2008).

    CAS  Article  Google Scholar 

  36. 36.

    Johansson, M. E. et al. Normalization of host intestinal mucus layers requires long-term microbial colonization. Cell Host. Microbe 18, 582–592 (2015).

    CAS  Article  Google Scholar 

  37. 37.

    Li, H. et al. The outer mucus layer hosts a distinct intestinal microbial niche. Nat. Commun. 6, 3292 (2015).

    Article  Google Scholar 

  38. 38.

    Berry, D. et al. Host-compound foraging by intestinal microbiota revealed by single-cell stable isotope probing. Proc. Natl Acad. Sci USA 110, 4720–4725 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Tailford, L. E., Crost, E. H., Kavanaugh, D. & Juge, N. Mucin glycan foraging in the human gut microbiome. Front. Genet. 6, 81 (2015).

    Article  Google Scholar 

  40. 40.

    Ley, R. E., Lozupone, C. A., Hamady, M., Knight, R. & Gordon, J. I. Worlds within worlds: evolution of the vertebrate gut microbiota. Nat. Rev. Microbiol. 6, 776–788 (2008).

    CAS  Article  Google Scholar 

  41. 41.

    den Besten, G. et al. The role of short-chain fatty acids in the interplay between diet, gut microbiotaand host energy metabolism. J. Lipid Res. 54, 2325–2340 (2013).

    Article  Google Scholar 

  42. 42.

    Koropatkin, N. M., Cameron, E. A. & Martens, E. C. How glycan metabolism shapes the human gut microbiota. Nat. Rev. Microbiol. 10, 323–335 (2012).

    CAS  Article  Google Scholar 

  43. 43.

    Carey, H. V., Walters, W. A. & Knight, R. Seasonal restructuring of the ground squirrel gut microbiota over the annual hibernation cycle. Am. J. Physiol. Regul. Integ. Comp. Physiol. 304, R33–R42 (2013).

    CAS  Article  Google Scholar 

  44. 44.

    Costello, E. K., Gordon, J. I., Secor, S. M. & Knight, R. Postprandial remodeling of the gut microbiota in Burmese pythons. ISME J. 4, 1375–1385 (2010).

    CAS  Article  Google Scholar 

  45. 45.

    McFall-Ngai, M. et al. Animals in a bacterial world, a new imperative for the life sciences. Proc. Natl Acad. Sci. USA 110, 3229–3236 (2013).

    CAS  Article  Google Scholar 

  46. 46.

    Cho, I. et al. Antibiotics in early life alter the murine colonic microbiome and adiposity. Nature 488, 621–626 (2012).

    CAS  Article  Google Scholar 

  47. 47.

    Foster, K. R., Schluter, J., Coyte, K. Z. & Rakoff-Nahoum, S. The evolution of the host microbiome as an ecosystem on a leash. Nature 548, 43–51 (2017).

    CAS  Article  Google Scholar 

  48. 48.

    Kaspari, M. & Powers, J. S. Biogeochemistry and geographical ecology: embracing all twenty-five elements required to build organisms. Am. Nat. 188, S62–S73 (2016).

    Article  Google Scholar 

  49. 49.

    David, L. A. et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559–563 (2014).

    CAS  Article  Google Scholar 

  50. 50.

    Scott, K. P., Gratz, S. W., Sheridan, P. O., Flint, H. J. & Duncan, S. H. The influence of diet on the gut microbiota. Pharmacol. Res. 69, 52–60 (2013).

    CAS  Article  Google Scholar 

  51. 51.

    Smith, V. H., Tilman, G. D. & Nekola, J. C. Eutrophication: impacts of excess nutrient inputs on freshwater, marine and terrestrial ecosystems. Environ. Pollut. 100, 179–196 (1999).

    CAS  Article  Google Scholar 

  52. 52.

    Stevens, C. E. & Hume, I. D. Comparative Physiology of the Vertebrate Digestive System 2nd edn (Cambridge Univ. Press, Cambridge, 1995).

  53. 53.

    Pérez, W., Lima, M. & Clauss, M. Gross anatomicy of the intestine in the giraffe (Giraffa camelopardalis). Anat. Histol. Embryol. 38, 432–435 (2009).

    Article  Google Scholar 

  54. 54.

    Kararli, T. T. Comparison of the gastrointestinal anatomicy, physiology and biochemistry of humans and commonly used laboratory animals. Biopharm. Drug Dispos. 16, 351–380 (1995).

    CAS  Article  Google Scholar 

  55. 55.

    Aust, G. et al. Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth. MBoC 24, 2256–2268 (2013).

    CAS  Article  Google Scholar 

  56. 56.

    Ley, R. E. et al. Evolution of mammals and their gut microbes. Science 320, 1647–1651 (2008).

    CAS  Article  Google Scholar 

  57. 57.

    Bergström, A. et al. Introducing GUt Low-Density Array (GULDA) – a validated approach for qPCR-based intestinal microbial community analysis. FEMS Microbiol. Lett. 337, 38–47 (2012).

    Article  Google Scholar 

  58. 58.

    Taberlet, P. et al. Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding. Nucleic Acids Res. 3, e14 (2007).

    Article  Google Scholar 

  59. 59.

    De Barba, M. et al. DNA metabarcoding multiplexing and validation of data accuracy for diet assessment: application to omnivorous diet. Mol. Ecol. Resour. 14, 306–323 (2014).

    Article  Google Scholar 

  60. 60.

    Cerling, T. E., Harris, J. M. & Passey, B. H. Diets of East African Bovidae based on stable isotope analysis. J. Mammal. 84, 456–470 (2003).

    Article  Google Scholar 

  61. 61.

    Frank, J. A. et al. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl. Environ. Microb. 74, 2461–2470 (2008).

    CAS  Article  Google Scholar 

  62. 62.

    Rettedal, E. A., Gumpert, H. & Sommer, M. O. Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria. Nat. Commun. 5, 4714 (2014).

    CAS  Article  Google Scholar 

  63. 63.

    Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl Acad. Sci. USA 108, 4516–4522 (2011).

    CAS  Article  Google Scholar 

  64. 64.

    Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 6, 1621–1624 (2012).

    CAS  Article  Google Scholar 

  65. 65.

    Maurice, C. F., Haiser, H. J. & Turnbaugh, P. J. Xenobiotics shape the physiology and gene expression of the active human gut microbiome. Cell 152, 39–50 (2013).

    CAS  Article  Google Scholar 

  66. 66.

    Bates, D., Maechler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015).

    Article  Google Scholar 

  67. 67.

    Park, S. W. et al. The protein disulphide isomerase AGR2 is essential for production of intestinal mucus. Proc. Natl Acad. Sci. USA 106, 6950–6955 (2009).

    CAS  Article  Google Scholar 

  68. 68.

    McClatchy, D. B., Dong, M.-Q., Wu, C. C., Venable, J. D. & Yates, J. R. III 15N metabolic labelling of mammalian tissue with slow protein turnover. J.Proteome Res. 6, 2005–2010 (2007).

    CAS  Article  Google Scholar 

  69. 69.

    McClatchy, D. B., Liao, L., Park, S. K., Venable, J. D. & Yates, J. R. III Quantification of the synaptosomal proteome of the rat cerebellum during post-natal development. Genome Res. 17, 1378–1388 (2007).

    CAS  Article  Google Scholar 

  70. 70.

    Franks, A. H. et al. Variations of bacterial populations in human faeces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 64, 3336–3345 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71.

    Manz, W., Amann, R., Ludwig, W., Vancanneyt, M. & Schleifer, K. H. Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum Cytophaga–Flavobacter–Bacteroides in the natural environment. Microbiology 142, 1097–1106 (1996).

  72. 72.

    Daims, H., Stoecker, K. & Wagner, M. in Molecular Microbial Ecology (eds Osborn, M. & Smith, C) Ch. 9 (Taylor & Francis, London, 2004).

  73. 73.

    Slodzian, G., Hillion, F., Stadermann, F. J. & Zinner, E. QSA influences on isotopic ratio measurements. Appl. Surf. Sci. 231, 874–877 (2004).

    Article  Google Scholar 

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W. Cook carried out C/N ratio measurements in the Duke Environmental Isotope Laboratory. Samples were provided by S. Mills and D. Lafferty (snowshoe hare); E. Ehmke (lemurs); L. McGraw, A. Vogel and C. Clement (prairie vole); D. Koeberl, V. Sakach and L. Morgan (dog); C. Drea (meerkat). Statistical advice was provided by K. Choudhury and S. Mukherjee. The manuscript was improved thanks to comments from J. Heffernan, J. Rawls and P. Turnbaugh. This work was funded by an NSF Doctoral Dissertation Improvement grant to A.T.R., J.P.W. and L.A.D. (grant no. DEB-1501495) and grants from the Hartwell Foundation, Alfred P. Sloan Foundation and Searle Scholars Programme to L.A.D. A.T.R. was supported by the NSF Graduate Research Fellowship Programme under grant no. DGE 1106401. F.C.P. was supported by a European Research Council Marie Curie Individual Fellowship (grant no. 658718). D.B. was supported in part by Austrian Science Fund (grant nos. P26127-B20 and P27831-B28) and European Research Council (Starting Grant: FunKeyGut 741623). M.W. was supported by the European Research Council via the Advanced Grant project ‘NITRICARE 294343’. The contents of this paper are the responsibility of the authors and do not necessarily represent the views of the funding institutions.

Author information




A.T.R., F.P., A.S., D.B. and M.W. carried out FISH / NanoSIMS work. A.T.R., X.Z. and R.P. performed diet manipulation experiments. L.P.H., S.J. and H.K.D. processed samples. T.M.O., S.C.A., T.R.K. and R.M.P. contributed data. A.T.R. performed all other experiments. A.M.D., R.R.D. and J.P.W. were involved in study design. A.T.R. and L.A.D. designed the study, analysed data and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Lawrence A. David.

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Supplementary Information

Supplementary Figures 1–7, Supplementary Tables 1–3, Supplementary Tables 5–9.

Reporting Summary

Supplementary Table 4

Isotope data for single-cell and whole-gut contents for data presented in Fig. 3, Supplementary Fig. 7.

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Reese, A.T., Pereira, F.C., Schintlmeister, A. et al. Microbial nitrogen limitation in the mammalian large intestine. Nat Microbiol 3, 1441–1450 (2018).

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