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The evolution of cooperation within the gut microbiota

Abstract

Cooperative phenotypes are considered central to the functioning of microbial communities in many contexts, including communication via quorum sensing, biofilm formation, antibiotic resistance, and pathogenesis1,2,3,4,5. The human intestine houses a dense and diverse microbial community critical to health1,2,4,5,6,7,8,9, yet we know little about cooperation within this important ecosystem. Here we test experimentally for evolved cooperation within the Bacteroidales, the dominant Gram-negative bacteria of the human intestine. We show that during growth on certain dietary polysaccharides, the model member Bacteroides thetaiotaomicron exhibits only limited cooperation. Although this organism digests these polysaccharides extracellularly, mutants lacking this ability are outcompeted. In contrast, we discovered a dedicated cross-feeding enzyme system in the prominent gut symbiont Bacteroides ovatus, which digests polysaccharide at a cost to itself but at a benefit to another species. Using in vitro systems and gnotobiotic mouse colonization models, we find that extracellular digestion of inulin increases the fitness of B. ovatus owing to reciprocal benefits when it feeds other gut species such as Bacteroides vulgatus. This is a rare example of naturally-evolved cooperation between microbial species. Our study reveals both the complexity and importance of cooperative phenotypes within the mammalian intestinal microbiota.

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Figure 1: Direct and cooperative benefits of polysaccharide digestion by surface glycoside hydrolases (GH).
Figure 2: B. ovatus does not require surface digestion for utilization of inulin.
Figure 3: Cost of inulin digestion by surface glycoside hydrolases.
Figure 4: Interspecies cooperation mediated by surface digestion of inulin is stabilized by reciprocal benefits.

References

  1. Sonnenburg, E. D. et al. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations. Cell 141, 1241–1252 (2010)

    Article  CAS  Google Scholar 

  2. West, S. A., Griffin, A. S., Gardner, A. & Diggle, S. P. Social evolution theory for microorganisms. Nature Rev. Microbiol. 4, 597–607 (2006)

    CAS  Google Scholar 

  3. Rakoff-Nahoum, S., Coyne, M. J. & Comstock, L. E. An ecological network of polysaccharide utilization among human intestinal symbionts. Curr. Biol. 24, 40–49 (2014)

    Article  CAS  Google Scholar 

  4. Drescher, K., Nadell, C. D., Stone, H. A., Wingreen, N. S. & Bassler, B. L. Solutions to the public goods dilemma in bacterial biofilms. Curr. Biol. 24, 50–55 (2014)

    Article  CAS  Google Scholar 

  5. Frank, S. A. A general model of the public goods dilemma. J. Evol. Biol. 23, 1245–1250 (2010)

    Article  Google Scholar 

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

    CAS  Google Scholar 

  7. Subramanian, S. et al. Cultivating healthy growth and nutrition through the gut microbiota. Cell 161, 36–48 (2015)

    Article  CAS  Google Scholar 

  8. Shipman, J. A., Cho, K. H., Siegel, H. A. & Salyers, A. A. Physiological characterization of SusG, an outer membrane protein essential for starch utilization by Bacteroides thetaiotaomicron. J. Bacteriol. 181, 7206–7211 (1999)

    Article  CAS  Google Scholar 

  9. Littman, D. R. & Pamer, E. G. Role of the commensal microbiota in normal and pathogenic host immune responses. Cell Host Microbe 10, 311–323 (2011)

    Article  CAS  Google Scholar 

  10. Waldor, M. K. et al. Where next for microbiome research? PLoS Biol. 13, e1002050 (2015)

    Article  Google Scholar 

  11. Koschwanez, J. H., Foster, K. R. & Murray, A. W. Sucrose utilization in budding yeast as a model for the origin of undifferentiated multicellularity. PLoS Biol. 9, e1001122 (2011)

    Article  CAS  Google Scholar 

  12. Costello, E. K., Stagaman, K., Dethlefsen, L., Bohannan, B. J. M. & Relman, D. A. The application of ecological theory toward an understanding of the human microbiome. Science 336, 1255–1262 (2012)

    Article  ADS  CAS  Google Scholar 

  13. Estrela, S., Whiteley, M. & Brown, S. P. The demographic determinants of human microbiome health. Trends Microbiol. 23, 134–141 (2015)

    Article  CAS  Google Scholar 

  14. Mitri, S. & Foster, K. R. The genotypic view of social interactions in microbial communities. Annu. Rev. Genet. 47, 247–273 (2013)

    Article  CAS  Google Scholar 

  15. Oliveira, N. M., Niehus, R. & Foster, K. R. Evolutionary limits to cooperation in microbial communities. Proc. Natl Acad. Sci. USA 111, 17941–17946 (2014)

    Article  ADS  CAS  Google Scholar 

  16. Faith, J. J. et al. The long-term stability of the human gut microbiota. Science 341, 1237439 (2013)

    Article  Google Scholar 

  17. Zitomersky, N. L., Coyne, M. J. & Comstock, L. E. Longitudinal analysis of the prevalence, maintenance, and IgA response to species of the order Bacteroidales in the human gut. Infect. Immun. 79, 2012–2020 (2011)

    Article  CAS  Google Scholar 

  18. Flint, H. J., Bayer, E. A., Rincon, M. T., Lamed, R. & White, B. A. Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis. Nature Rev. Microbiol. 6, 121–131 (2008)

    CAS  Google Scholar 

  19. Elhenawy, W., Debelyy, M. O. & Feldman, M. F. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles. mBio 5, e00909–14 (2014)

    Article  Google Scholar 

  20. Cuskin, F. et al. Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism. Nature 517, 165–169 (2015)

    Article  ADS  CAS  Google Scholar 

  21. Sachs, J. L., Mueller, U. G., Wilcox, T. P. & Bull, J. J. The evolution of cooperation. Q. Rev. Biol. 79, 135–160 (2004)

    Article  Google Scholar 

  22. Foster, K. R. & Wenseleers, T. A general model for the evolution of mutualisms. J. Evol. Biol. 19, 1283–1293 (2006)

    Article  CAS  Google Scholar 

  23. Gore, J., Youk, H. & van Oudenaarden, A. Snowdrift game dynamics and facultative cheating in yeast. Nature 459, 253–256 (2009)

    Article  ADS  CAS  Google Scholar 

  24. Martens, E. C. et al. Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts. PLoS Biol. 9, e1001221 (2011)

    Article  CAS  Google Scholar 

  25. Orel, R. & Kamhi Trop, T. Intestinal microbiota, probiotics and prebiotics in inflammatory bowel disease. World J. Gastroenterol. 20, 11505–11524 (2014)

    Article  Google Scholar 

  26. Cameron, E. A. et al. Multifunctional nutrient-binding proteins adapt human symbiotic bacteria for glycan competition in the gut by separately promoting enhanced sensing and catalysis. mBio 5, e01441–14 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Momeni, B., Waite, A. J. & Shou, W. Spatial self-organization favors heterotypic cooperation over cheating. Elife 2, e00960 (2013)

    Article  Google Scholar 

  28. Ng, K. M. et al. Microbiota-liberated host sugars facilitiate post-antobiotic expansion of enteric pathogens. Nature 502, 96–99 (2013)

    Article  ADS  CAS  Google Scholar 

  29. Degnan, P. H., Barry, N. A., Mok, K. C., Taga, M. E. & Goodman, A. L. Human gut microbes use multiple transporters to distinguish vitamin B12 analogs and compete in the gut. Cell Host Microbe 15, 47–57 (2014)

    Article  CAS  Google Scholar 

  30. Fischbach, M. A. & Sonnenburg, J. L. Eating for two: how metabolism establishes interspecies interactions in the gut. Cell Host Microbe 10, 336–347 (2011)

    Article  CAS  Google Scholar 

  31. Stevens, A. M., Shoemaker, N. B. & Salyers, A. A. The region of a Bacteroides conjugal chromosomal tetracycline resistance element which is responsible for production of plasmidlike forms from unlinked chromosomal DNA might also be involved in transfer of the element. J. Bacteriol. 172, 4271–4279 (1990)

    Article  CAS  Google Scholar 

  32. Smith, C. J., Rogers, M. B. & McKee, M. L. Heterologous gene expression in Bacteroides fragilis. Plasmid 27, 141–154 (1992)

    Article  CAS  Google Scholar 

  33. Coyne, M. J., Fletcher, C. M., Reinap, B. & Comstock, L. E. UDP-glucuronic acid decarboxylases of Bacteroides fragilis and their prevalence in bacteria. J. Bacteriol. 193, 5252–5259 (2011)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. B. Ogbunugafor, J. Ordovas-Montanes, U. von Andrian and M. Waldor for suggestions. M. Delaney for SCFA analysis, V. Yeliseyev for assistance with gnotobiotics. Inulin and FOS were provided by Beneo-Orafti. Mice were provided by the HDDC, NIH Grant P30 DK34845. S.R.-N. is supported by the PIDS-St Jude Research Hospital Fellowship Program in Basic Research, a K12 Child Health Research Center grant through Boston Children’s Hospital and a Pilot Feasibility Award funded by HDDC P30 DK034854. K.R.F. is supported by European Research Council Grant 242670. This work was supported by Public Health Service grant R01AI081843 (to L.E.C.) from the NIH/NIAID.

Author information

Authors and Affiliations

Authors

Contributions

S.R.-N. performed mutant construction bacterial cultures, gnotobiotic experiments, protein purification and TLC, L.E.C. assisted with mutant construction. S.R.-N. and L.E.C. analysed the data. S.R.-N., K.R.F. and L.E.C. designed the study and wrote the paper.

Corresponding author

Correspondence to Seth Rakoff-Nahoum.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Limited cooperation during polysaccharide utilization by B. thetaiotaomicron.

ad, Independent experiments for Fig. 1b–e. a, b, Upper panels n = 2 biological replicates, lower panels n = 1; c, d, n = 2 biological replicates. e, TLC analysis of conditioned media from B. thetaiotaomicron grown in amylopectin (left panel) or levan (right panel) minimal media. EL, early log; ML, late log; LL, late log; Stat, stationary phase; Glu, glucose; Fru, fructose; Suc, sucrose. See Supplementary Information Fig. 1 for uncropped scanned images. In all panels, error bars represent standard error; P values derived from two-tailed Student’s t-test.

Source data

Extended Data Figure 2 Bo 04502 and 04503 mutants grow equivalently to wild type on limiting concentrations of inulin and do not require surface digestion for utilization of inulin.

a, Growth of Bo WT, Δ04502, Δ04503 and Δ04502/3 mutants in varying concentrations of inulin as indicated. Biological replicates of each condition are plotted as individual lines (n = 2 cell culture biological replicates). Upper and lower panels are independent experiments. b, Independent experiments for Fig. 2d. EL, early log; ML, mid log; LL, late log; St, stationary phase. See Supplementary Information Fig. 1 for uncropped scanned images. c, Complementation of Bo Δ04502 and Δ04502/3 mutants with the respective genes in trans. TLC analysis of conditioned media from Bo Δ04502/3 (left panel) complemented in trans with BACOVA_04502, BACOVA_04503, BACOVA_04502/3 or vector alone (pFD340) and Bo Δ04502 (right panel) with BACOVA_04502 or vector alone grown in defined inulin media. See Supplementary Information Fig. 1 for uncropped scanned images. S, stationary phase.

Source data

Extended Data Figure 3 SusC and SusD homologues BACOVA_04505 and BACOVA_04505 are required for inulin utilization.

a, Independent experiments for Fig. 2e. Left panel n = 2 biological replicates, right panel each line represents n = 1 sample per condition. b, Complementation of Bo Δ04504, Bo Δ04505 and Δ04504/5 mutants with the genes in trans. Growth of Bo Δ04504, Bo Δ04505, Bo Δ04504/5 with BACOVA_04504, BACOVA_04505, BACOVA_04504/5 or vector alone (pFD340) in trans in defined inulin media. Each line represents n = 1 sample per condition. In all panels, error bars represent standard error; P values derived from two-tailed Student’s t-test.

Source data

Extended Data Figure 4 Costs of extracellular inulin digestion by B. ovatus.

a, b, Independent experiments for Fig. 3a, c. n = 3 biological replicates at day 1, 2; n = 2, biological replicates at day 4 (a). In upper and upped middle panels biological replicates of each condition are plotted as individual lines (n = 2 cell culture biological replicates); in lower middle and lower panels each line represents n = 1 sample per condition (b). c, TLC analysis of conditioned media from Bo WT and Bo Δ04502/3 cultured in 0.5% inulin with trace (0.06%) amounts of fructose. St, stationary phase. See Supplementary Information Fig. 1 for uncropped scanned images. In all panels, error bars represent standard error; P values derived from two-tailed Student’s t-test.

Source data

Extended Data Figure 5 Preferential utilization of undigested inulin by B. ovatus and costs of inulin digestion by 04502/3.

a, Time to mid-log (estimated at 50% maximal OD (OD50) by linear regression analysis) of Fig. 3d. b, Additional independent experiments for Fig. 3d. Each line represents n = 1 sample per condition. c, Competition of Bo WT and Bo Δ04502/3 co-cultured in 0.5% inulin with trace (0.06%) amounts of fructose. n = 2 cell culture biological replicates; d, e, Growth (d) and TLC analysis of conditioned media (e) of Bo WT and Bo Δ04502/3 cultured in 0.1% inulin with trace (0.06%) amounts of fructose. See Supplementary Information Fig. 1 for uncropped scanned images. For d, each panel is an independent experiment. Each condition is plotted as individual lines in each panel (n = 2 cell culture biological replicates). In all panels, error bars represent standard error; P values derived from two-tailed Student’s t-test.

Source data

Extended Data Figure 6 A complex mouse microbiota differentially affects B. ovatus WT and Δ04502/3 pre-colonized gnotobiotic mice and analysis of cross feeding of the predominant murine Bacteroidales of the murine gut microbiota.

a, Germ-free mice were monocolonized with Bo WT or Δ04502/3 and maintained on a diet supplemented with inulin as the sole polysaccharide and housed under gnotobiotic conditions for 2 weeks. Bacteria were enumerated from faeces before gavage with intestinal microbiota of conventionally raised mice (n = 5 mice, cell culture biological replicates). b, Growth of two dominant mouse microbiota Bacteroidales strains (Parabacteroides goldsteinii and a strain with 96% 16S rRNA gene identity to B. thetaiotaomicron) with inulin breakdown products derived from the conditioned media of B. ovatus grown in inulin (all inulin had been digested) or undigested inulin minimal media. Each data point is a different isolate of the indicated species from the caeca of the conventionally raised mice used for gavage (n = 11 isolates for upper panel, n = 6 for lower panel). In all panels, error bars represent standard error.

Source data

Extended Data Figure 7 The B. ovatus, but not B. fragilis benefits from B. vulgatus in co-culture in inulin.

a, b, Independent experiments for Fig. 4b, d. The left panel corresponds to starting culture with ~107 CFU Bo corresponding to starting culture of ~106 CFU Bo in Fig. 4d. The two right panels are a duplicate pair of experiments of starting CFU of 106 and 107. In each panel n = 2 biological replicates. c, Enumeration of B. fragilis in monoculture or co-culture with Bv on defined inulin plates, n = 3 cell culture biological replicates. Letters in parentheses refer to values correlating to colour of line used for statistical analysis. In a, for example, p (g,r) refers to comparison of values of green (g) and red (r) values at the time-point indicated. In b, colour of P value indicates comparison of monoculture and co-culture for the given condition at the time-point indicated. For all panels, error bars represent standard error; P values derived from two-tailed Student’s t-test.

Source data

Extended Data Figure 8 Secreted factors from Bo and isogenic mutants, but not B. fragilis (Bf), support Bv survival.

a, Growth of Bv in conditioned media from Bo WT, Δ04502, Δ04502/3 or Bf grown in defined media with inulin as the sole carbohydrate and in inulin media. End time-point corresponded to peak growth of Bv in conditioned media derived from Bo WT. B. fragilis, which utilizes inulin1,3 but similar to Bo ∆04502/3 does not liberate inulin breakdown products3, does not support the survival of Bv during co-culture. Left and right panels are independent experiments. Left panel; start, n = 4 biological replicates; end, n = 2 biological replicates. Right panel; t0, n = 4 cell culture biological replicates; t13 and 24, n = 2 biological replicates. b, Growth of Bv in dialysed (2 kDa MW membrane) or undialysed conditioned media from Bo Δ04502/3 grown in inulin, n = 2 cell culture biological replicates. c, Gas chromatographic analysis of acetate, propionate and succinate in conditioned media during growth of Bo WT and Δ04502/3 in defined media with inulin as the sole carbohydrate. Other volatile and non-volatile substances (as listed in Methods) were undetectable, n = 2 cell culture biological replicates, except Δ04502/3 stationary phase, n = 1. d, Growth of Bv in defined medium with inulin as the sole carbohydrate with or without addition with 15 mM of acetate, propionate or succinate, t = 0, n = 4 biological replicates; t = 13 and 24 h, n = 2 biological replicates. CM, conditioned media; UD, undetected. For all panels, error bars represent standard error; P values derived from two-tailed Student’s t-test.

Source data

Extended Data Figure 9 Spatial aspects of mutualism between Bo and Bv via cross feeding and interspecies cooperation in vivo via 04502/3.

a, Independent experiments for Fig. 4c, n = 4 biological replicates. b, Independent experiments for Fig. 4e, n = 3 cell culture biological replicates. c, Scatter plot of experiment in Fig. 4f, n = 3 cell culture biological replicates. d, Ratios of wild type and Δ04502/3 in faeces 21 days after co-colonization of three germ-free mice (n = 3 mice biological replicates) on a diet of inulin as the sole dietary polysaccharide (pre-Bv) and then four days after introduction of Bv (post-Bv). Each panel shows the ratio pre- and post- Bv of an individual mouse. P values are Fisher exact test comparing the frequency of Bo WT and Δ04502/3 pre- and post- colonization with Bv for each individual mouse. At day 21, all mice were colonized with a higher ratio of the mutant (ranging to 86% in the mouse depicted in the lowest panel), with each mouse showing a statistically significant increase in the proportion of the wild type after introduction of Bv. e, Growth of B. ovatus in 0.5% inulin (upper panel) or fructose (lower panel) in minimal media to which filter sterilized conditioned media from early log, A600nm matched growth of Bv or Bo in 0.125% fructose minimal media or fresh 0.125% fructose minimal media control was added at 1:1 ratio. In e, numbers refer to P values (*P < 0.05, **P < 0.01, ***P < 0.001) of comparison of values of green (g), red (r) or black (b) values by unpaired, two-tailed student t-test at the time-point indicated, n = 2 cell culture biological replicates. For all panels, error bars represent standard error; for all panels except d, P values derived from two-tailed Student’s t-test.

Source data

Extended Data Figure 10 Schematic of forms of cooperativity via polysaccharide digestion among Bacteroidales.

a, Limited cooperation. Privatization of extracellularly digested public goods by the individual performing the digestion leads to greater individual (thick arrow) than shared benefits (thin arrow) as seen in Bt during growth on levan and amylopectin. b, Cooperation between species is seen between Bo and Bv during growth on inulin. Surface digestion of inulin by Bo creates breakdown products that it does not need to grow on inulin. Rather, inulin breakdown represents a dedicated cross-feeding system that provides benefits to Bv, with reciprocal fitness benefits to Bo.

Supplementary information

Supplementary Information

Supplementary Figure 1 shows the uncropped TLC scans for Figures 2b, 2d (left), 2d (right) and Extended Data Figures 1e, 2b, 2c, 4c, 5e and Supplementary Table 1 which shows the primers used in this study. (PDF 136 kb)

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Rakoff-Nahoum, S., Foster, K. & Comstock, L. The evolution of cooperation within the gut microbiota. Nature 533, 255–259 (2016). https://doi.org/10.1038/nature17626

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