Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria in a synthetic community

Abstract

Interactive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered a cross-feeding coculture between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excretes essential nitrogen as NH4+ to E. coli, while E. coli excretes essential carbon as fermentation products to R. palustris. Here, we sought to determine whether a reciprocal cross-feeding relationship would evolve spontaneously in cocultures with wild-type R. palustris, which is not known to excrete NH4+. Indeed, we observed the emergence of NH4+ cross-feeding, but driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of fermentation products from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.

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Fig. 1: Synergistic cross-feeding between E. coli and R. palustris is facilitated by NH4+ excretion.
Fig. 2: Coculture doubling times decreased during experimental evolution of WT-based and NifA*-based cocultures.
Fig. 3: Final populations in WT-based cocultures show large increases through serial transfers.
Fig. 4: WT-based and NifA*-based cocultures exhibit distinct metabolic phenotypes.
Fig. 5: Adaptation by E. coli is sufficient to enable growth of WT-based cocultures.
Fig. 6: A missense mutation in E. coli ntrC enables emergent NH4+ cross-feeding by conferring constitutive expression of nitrogen acquisition genes.

References

  1. 1.

    Seth EC, Taga ME. Nutrient cross-feeding in the microbial world. Front Microbiol. 2014;5:350.

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Estrela S, Trisos CH, Brown SP. From metabolism to ecology: cross-feeding interactions shape the balance between polymicrobial conflict and mutualism. Am Nat. 2012;180:566–76.

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Morris BEL, Henneberger R, Huber H, Moissl-Eichinger C. Microbial syntrophy: interaction for the common good. FEMS Microbiol Rev. 2013;37:384–406.

    CAS  PubMed  Google Scholar 

  4. 4.

    Ponomarova O, Patil KR. Metabolic interactions in microbial communities: untangling the Gordian knot. Curr Opin Microbiol. 2015;27:37–44.

    PubMed  Google Scholar 

  5. 5.

    Zelezniak A, Andrejev S, Ponomarova O, Mende DR, Bork P, Patil KR. Metabolic dependencies drive species co-occurrence in diverse microbial communities. Proc Natl Acad Sci USA. 2015;112:6449–54.

    CAS  PubMed  Google Scholar 

  6. 6.

    Embree M, Liu JK, Al-Bassam MM, Zengler K. Networks of energetic and metabolic interactions define dynamics in microbial communities. Proc Natl Acad Sci USA. 2015;112:15450–5.

    CAS  PubMed  Google Scholar 

  7. 7.

    Zengler K, Zaramela LS. The social network of microorganisms—how auxotrophies shape complex communities. Nat Rev Microbiol. 2018;16:383–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Mee MT, Collins JJ, Church GM, Wang HH. Syntrophic exchange in synthetic microbial communities. Proc Natl Acad Sci USA. 2014;111:E2149–56.

    CAS  PubMed  Google Scholar 

  9. 9.

    Bergkessel M, Basta DW, Newman DK. The physiology of growth arrest: uniting molecular and environmental microbiology. Nat Rev Microbiol. 2016;14:549–62.

    CAS  PubMed  Google Scholar 

  10. 10.

    Lennon JT, Jones SE. Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat Rev Microbiol. 2011;9:119–30.

    CAS  PubMed  Google Scholar 

  11. 11.

    Momeni B, Chen C-C, Hillesland KL, Waite A, Shou W. Using artificial systems to explore the ecology and evolution of symbioses. Cell Mol Life Sci. 2011;68:1353–68.

    CAS  PubMed  Google Scholar 

  12. 12.

    Mee MT, Wang HH. Engineering ecosystems and synthetic ecologies. Mol Biosyst. 2012;8:2470–83.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Lindemann SR, Bernstein HC, Song H-S, Fredrickson JK, Fields MW, Shou W, et al. Engineering microbial consortia for controllable outputs. ISME J. 2016;10:2077–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Widder S, Allen RJ, Pfeiffer T, Curtis TP, Wiuf C, Sloan WT, et al. Challenges in microbial ecology: building predictive understanding of community function and dynamics. ISME J. 2016;10:2557–68.

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Hillesland KL, Stahl DA. Rapid evolution of stability and productivity at the origin of a microbial mutualism. Proc Natl Acad Sci USA. 2010;107:2124–9.

    CAS  PubMed  Google Scholar 

  16. 16.

    Harcombe WR, Riehl WJ, Dukovski I, Granger BR, Betts A, Lang AH, et al. Metabolic resource allocation in individual microbes determines ecosystem interactions and spatial dynamics. Cell Rep. 2014;7:1104–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    LaSarre B, McCully AL, Lennon JT, McKinlay JB. Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients. ISME J. 2016;11:337–48.

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    McCully AL, LaSarre B, McKinlay JB. Recipient-biased competition for an intracellularly generated cross-fed nutrient is required for coexistence of microbial mutualists. mBio. 2017;8:e01620–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    McCully AL, LaSarre B, McKinlay JB. Growth-independent cross-feeding modifies boundaries for coexistence in a bacterial mutualism. Environ Microbiol. 2017;19:3538–50.

    CAS  PubMed  Google Scholar 

  20. 20.

    McCully AL, Behringer MG, Gliessman JR, Pilipenko EV, Mazny JL, Lynch M, et al. An Escherichia coli nitrogen starvation response is important for mutualistic coexistence with Rhodopseudomonas palustris. Appl Environ Microbiol. 2018;84:e00404–18.

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Kim M, Zhang Z, Okano H, Yan D, Groisman A, Hwa T. Need‐based activation of ammonium uptake in Escherichia coli. Mol Syst Biol. 2012;8:616.

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Walter A, Gutknecht J. Permeability of small nonelectrolytes through lipid bilayer membranes. J Membr Biol. 1986;90:207–17.

    CAS  PubMed  Google Scholar 

  23. 23.

    Dixon R, Kahn D. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol. 2004;2:621–31.

    CAS  PubMed  Google Scholar 

  24. 24.

    McKinlay JB, Harwood CS. Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. Proc Natl Acad Sci USA. 2010;107:11669–75.

    CAS  PubMed  Google Scholar 

  25. 25.

    Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, et al. The complete genome sequence of Escherichia coli K-12. Science. 1997;277:1453–62.

    CAS  PubMed  Google Scholar 

  26. 26.

    Kim M-K, Harwood CS. Regulation of benzoate-CoA ligase in Rhodopseudomonas palustris. FEMS Microbiol Lett. 1991;83:199–203.

    CAS  Google Scholar 

  27. 27.

    Quandt J, Hynes MF. Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene. 1993;127:15–21.

    CAS  PubMed  Google Scholar 

  28. 28.

    Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000;97:6640–5.

    CAS  PubMed  Google Scholar 

  29. 29.

    Rey FE, Oda Y, Harwood CS. Regulation of uptake hydrogenase and effects of hydrogen utilization on gene expression in Rhodopseudomonas palustris. J Bacteriol. 2006;188:6143–52.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    McKinlay JB, Zeikus JG, Vieille C. Insights into Actinobacillus succinogenes fermentative metabolism in a chemically defined growth medium. Appl Environ Microbiol. 2005;71:6651–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Huang JJ, Heiniger EK, McKinlay JB, Harwood CS. Production of hydrogen gas from light and the inorganic electron donor thiosulfate by Rhodopseudomonas palustris. Appl Environ Microbiol. 2010;76:7717–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Zhou K, Zhou L, Lim QE, Zou R, Stephanopoulos G, Too H-P. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol Biol. 2011;12:18.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Deatherage DE, Barrick JE. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. In: Sun L, Shou W, editors. Engineering and analyzing multicellular systems: methods and protocols. New York, NY: Springer New York; 2014. p. 165–88.

  35. 35.

    Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly. 2012;6:80–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Harcombe WR, Chacón JM, Adamowicz EM, Chubiz LM, Marx CJ. Evolution of bidirectional costly mutualism from byproduct consumption. Proc Natl Acad Sci USA. 2018;115:12000–4.

    CAS  PubMed  Google Scholar 

  38. 38.

    Pande S, Kaftan F, Lang S, Svatoš A, Germerodt S, Kost C. Privatization of cooperative benefits stabilizes mutualistic cross-feeding interactions in spatially structured environments. ISME J. 2015;10:1413–23.

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Harcombe W. Novel cooperation experimentally evolved between species. Evolution. 2010;64:2166–72.

    PubMed  Google Scholar 

  40. 40.

    Pechter KB, Yin L, Oda Y, Gallagher L, Yang J, Manoil C, et al. Molecular basis of bacterial longevity. mBio. 2017;8:e01726–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Lipson DA. The complex relationship between microbial growth rate and yield and its implications for ecosystem processes. Front Microbiol. 2015;6:615.

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Wortel MT, Noor E, Ferris M, Bruggeman FJ, Liebermeister W. Metabolic enzyme cost explains variable trade-offs between microbial growth rate and yield. PLoS Comp Biol. 2018;14:e1006010.

    Google Scholar 

  43. 43.

    Cheng C, O’Brien EJ, McCloskey D, Utrilla J, Olson C, LaCroix RA, et al. Laboratory evolution reveals a two-dimensional rate-yield tradeoff in microbial metabolism. PLoS Comp Biol. 2019;15:e1007066.

    CAS  Google Scholar 

  44. 44.

    McDowall JS, Murphy BJ, Haumann M, Palmer T, Armstrong FA, Sargent F. Bacterial formate hydrogenlyase complex. Proc Natl Acad Sci USA. 2014;111:E3948–56.

    CAS  PubMed  Google Scholar 

  45. 45.

    Sangani AA, McCully AL, LaSarre B, McKinlay JB. Fermentative Escherichia coli makes a substantial contribution to H2 production in coculture with phototrophic Rhodopseudomonas palustris. FEMS Microbiol Lett. 2019;366:fnz162.

    CAS  PubMed  Google Scholar 

  46. 46.

    Zimmer DP, Soupene E, Lee HL, Wendisch VF, Khodursky AB, Peter BJ, et al. Nitrogen regulatory protein C-controlled genes of Escherichia coli: scavenging as a defense against nitrogen limitation. Proc Natl Acad Sci USA. 2000;97:14674–9.

    CAS  PubMed  Google Scholar 

  47. 47.

    Brown DR, Barton G, Pan Z, Buck M, Wigneshweraraj S. Nitrogen stress response and stringent response are coupled in Escherichia coli. Nat Commun. 2014;5:4115.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Switzer A, Brown DR, Wigneshweraraj S. New insights into the adaptive transcriptional response to nitrogen starvation in Escherichia coli. Biochem Soc Trans. 2018;46:1721–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Jensen KF. The Escherichia coli K-12 “wild types” W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels. J Bacteriol. 1993;175:3401–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    LaCroix RA, Sandberg TE, O’Brien EJ, Utrilla J, Ebrahim A, Guzman GI, et al. Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K-12 MG1655 on glucose minimal medium. Appl Environ Microbiol. 2015;81:17–30.

    PubMed  Google Scholar 

  51. 51.

    Ko M, Park C. H-NS-Dependent regulation of flagellar synthesis is mediated by a LysR family protein. J Bacteriol. 2000;182:4670–2.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Krin E, Danchin A, Soutourina O. Decrypting the H-NS-dependent regulatory cascade of acid stress resistance in Escherichia coli. BMC Microbiol. 2010;10:273.

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Weglenski P, Ninfa AJ, Ueno-Nishio S, Magasanik B. Mutations in the glnG gene of Escherichia coli that result in increased activity of nitrogen regulator I. J Bacteriol. 1989;171:4479–85.

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Dixon R, Eydmann T, Henderson N, Austin S. Substitutions at a single amino acid residue in the nitrogen-regulated activator protein NTRC differentially influence its activity in response to phosphorylation. Mol Microbiol. 1991;5:1657–67.

    CAS  PubMed  Google Scholar 

  55. 55.

    Hart SFM, Pineda JMB, Chen C-C, Green R, Shou W. Disentangling strictly self-serving mutations from win-win mutations in a mutualistic microbial community. eLife. 2019;8:e44812.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Waite AJ, Shou W. Adaptation to a new environment allows cooperators to purge cheaters stochastically. Proc Natl Acad Sci USA. 2012;109:19079–86.

    CAS  PubMed  Google Scholar 

  57. 57.

    Estrela S, Morris JJ, Kerr B. Private benefits and metabolic conflicts shape the emergence of microbial interdependencies. Environ Microbiol. 2016;18:1415–27.

    PubMed  Google Scholar 

  58. 58.

    Warsi OM, Andersson DI, Dykhuizen DE. Different adaptive strategies in E. coli populations evolving under macronutrient limitation and metal ion limitation. BMC Evol Biol. 2018;18:72.

    PubMed  PubMed Central  Google Scholar 

  59. 59.

    Rosenzweig RF, Sharp RR, Treves DS, Adams J. Microbial evolution in a simple unstructured environment: genetic differentiation in Escherichia coli. Genetics. 1994;137:903–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Treves DS, Manning S, Adams J. Repeated evolution of an acetate-crossfeeding polymorphism in long-term populations of Escherichia coli. Mol Biol Evol. 1998;15:789–97.

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported in part by the US Army Research Office grants W911NF-14–1–0411 and W911NF-17–1–0159, a National Science Foundation CAREER award MCB-1749489, the US Department of Energy, Office of Science, Office of Biological and Environmental Research, under award DE-SC0008131, and the Joint Genome Institute Community Science Program, CSP 502893. The work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02–05CH11231. We thank A.L. Posto, J.R. Gliessman, and M.C. Onyeziri for coculture passaging and initial characterizations, J.T. Lennon and B.K. Lehmkuhl for equipment and assistance with qRT-PCR, and J. Ford and A.M. Buechlein at the IU Center for Genomics and Bioinformatics.

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Correspondence to James B. McKinlay.

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Fritts, R.K., Bird, J.T., Behringer, M.G. et al. Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria in a synthetic community. ISME J 14, 2816–2828 (2020). https://doi.org/10.1038/s41396-020-00737-5

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