Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Control of nitrogen fixation in bacteria that associate with cereals

Nature Microbiology volume 5, pages 314–330 (2020)Cite this article

Subjects

Abstract

Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Transfer of nif clusters across species.
Fig. 2: Transfer of the native K. oxytoca nif cluster across species.
Fig. 3: Transfer of the refactored K. oxytoca nif clusters to R. sp. IRBG74.
Fig. 4: Control of nitrogen fixation in A. caulinodans ORS571.
Fig. 5: Nitrogenase activity of the inducible nif clusters in P. protegens Pf-5.
Fig. 6: Control of nitrogenase activity with sensors that respond to diverse chemicals in the rhizosphere.

Similar content being viewed by others

Data availability

Additional data supporting this study are available from the corresponding author on reasonable request. The RNA-seq and ribosome-profiling data are available in the Sequence Read Archive with the accession code PRJNA579767: K. oxytoca native nif cluster, RNA-seq (SRX7032059, SRX7032060 and SRX7032061) and ribosome-profiling (SRX7034729, SRX7034730, SRX7034731 and SRX7034732); K. oxytoca refactored nif cluster v2.1, RNA-seq (SRX7036110) and ribosome-profiling (SRX7036099); K. oxytoca refactored nif cluster v3.2, RNA-seq (SRX7035703, SRX7035704 and SRX7035705) and ribosome-profiling (SRX7036113, SRX7036114 and SRX7036115).

References

  1. Harlan, J. R. Crops and Man 2nd edn (American Society of Agronomy, 1992).

  2. Simmonds, J. Community Matters: a History of Biological Nitrogen Fixation and Nodulation Research, 1965 to 1995. PhD thesis, Rensselaer Polytechnic Institute (2007).

  3. Erisman, J., Bleeker, A., Galloway, J. & Sutton, M. Reduced nitrogen in ecology and the environment. Environ. Pollut. 150, 140–149 (2007).

    CAS  PubMed  Google Scholar 

  4. Geddes, B. A. et al. Use of plant colonizing bacteria as chassis for transfer of N2-fixation to cereals. Curr. Opin. Biotechnol. 32, 216–222 (2015).

    CAS  PubMed  Google Scholar 

  5. Zaim, S., Bekkar, A. A. & Belabid, L. in Rhizobium Biology and Biotechnology (eds Hansen, A. P. et al.) 25–37 (Springer, 2017).

  6. Nadarajah, K. K. in Rhizobium Biology and Biotechnology (eds Hansen, A. P. et al.) 83–103 (Springer, 2017).

  7. Gutierrez-Zamora, M. & Martınez-Romero, E. Natural endophytic association between Rhizobium etli and maize (Zea mays L.). J. Biotechnol. 91, 117–126 (2001).

    CAS  PubMed  Google Scholar 

  8. McInroy, J. A. & Kloepper, J. W. Survey of indigenous bacterial endophytes from cotton and sweet corn. Plant Soil 173, 337–342 (1995).

    CAS  Google Scholar 

  9. Ramachandran, V. K., East, A. K., Karunakaran, R., Downie, J. A. & Poole, P. S. Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics. Genome Biol. 12, R106 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Frans, J. et al. in Nitrogen Fixation (Ed. Gresshoff, P. M.) 33–44 (Springer, 1990).

  11. Delmotte, N. et al. An integrated proteomics and transcriptomics reference data set provides new insights into the Bradyrhizobium japonicum bacteroid metabolism in soybean root nodules. Proteomics 10, 1391–1400 (2010).

    CAS  PubMed  Google Scholar 

  12. James, E. Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res. 65, 197–209 (2000).

    Google Scholar 

  13. Ars’ene, F., Katupitiya, S., Kennedy, I. R. & Elmerich, C. Use of IacZ fusions to study the expression of nif genes of Azospirillum brasilense. Mol. Plant Microbe Interact. 7, 748–757 (1994).

    Google Scholar 

  14. Boddey, R. M. et al. in Management of Biological Nitrogen Fixation for the Development of More Productive and Sustainable Agricultural Systems (eds Ladha, J. K. & Peoples, M. B.) 195–209 (Springer, 1995).

  15. Bremer, E., Janzen, H. & Gilbertson, C. Evidence against associative N2 fixation as a significant N source in long-term wheat plots. Plant Soil 175, 13–19 (1995).

    CAS  Google Scholar 

  16. Chalk, P. The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes. Plant Soil 132, 29–39 (1991).

    CAS  Google Scholar 

  17. Hurek, T., Reinhold-Hurek, B., Van Montagu, M. & Kellenberger, E. Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J. Bacteriol. 176, 1913–1923 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. ames, E., Olivares, F., Baldani, J. & Döbereiner, J. Herbaspirillum, an endophytic diazotroph colonizing vascular tissue 3 Sorghum bicolor L. Moench. J. Exp. Bot. 48, 785–798 (1997).

    Google Scholar 

  19. Kapulnik, Y., Feldman, M., Okon, Y. & Henis, Y. Contribution of nitrogen fixed by Azospirillum to the N nutrition of spring wheat in Israel. Soil Biology Biochem. 17, 509–515 (1985).

    Google Scholar 

  20. Katupitiya, S. et al. A mutant of Azospirillum brasilense Sp7 impaired in flocculation with a modified colonization pattern and superior nitrogen fixation in association with wheat. Appl. Environ. Microbiol. 61, 1987–1995 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Malik, K. et al. Association of nitrogen-fixing, plant-growth-promoting rhizobacteria (PGPR) with kallar grass and rice. Plant Soil 194, 37–44 (1997).

    CAS  Google Scholar 

  22. Reinhold-Hurek, B. & Hurek, T. Interactions of gramineous plants with Azoarcus spp. and other diazotrophs: identification, localization, and perspectives to study their function. Crit. Rev. Plant Sci. 17, 29–54 (1998).

    Google Scholar 

  23. Stoltzfus, J., So, R., Malarvithi, P., Ladha, J. & De Bruijn, F. Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen. Plant Soil 194, 25–36 (1997).

    CAS  Google Scholar 

  24. Triplett, E. W. Diazotrophic endophytes: progress and prospects for nitrogen fixation in monocots. Plant Soil 186, 29–38 (1996).

    CAS  Google Scholar 

  25. Broek, A. V., Michiels, J., Van Gool, A. & Vanderleyden, J. Spatial-temporal colonization patterns of Azospirillum brasilense on the wheat root surface and expression of the bacterial nifH gene during association. Mol. Plant Microbe Interact. 6, 592–600 (1993).

    Google Scholar 

  26. Yanni, Y. G. et al. in Opportunities for Biological Nitrogen Fixation in Rice and Other Non-Legumes (eds Ladha, J. K. et al.) 99–114 (Springer, 1997).

  27. Okon, Y. Azospirillum as a potential inoculant for agriculture. Trends Biotechnol. 3, 223–228 (1985).

    Google Scholar 

  28. Cocking, E. C. Endophytic colonization of plant roots by nitrogen-fixing bacteria. Plant Soil 252, 169–175 (2003).

    CAS  Google Scholar 

  29. Woodard, H. & Bly, A. Maize growth and yield responses to seed-inoculated N2-fixing bacteria under Dryland production conditions. J. Plant Nutr. 23, 55–65 (2000).

    CAS  Google Scholar 

  30. Igiehon, N. O. & Babalola, O. O. Rhizosphere microbiome modulators: contributions of nitrogen fixing bacteria towards sustainable agriculture. Int. J. Environ. Res. Public Health 15, 574 (2018).

    PubMed Central  Google Scholar 

  31. Biswas, J., Ladha, J. & Dazzo, F. Rhizobia inoculation improves nutrient uptake and growth of lowland rice. Soil Sci. Soc. Am. J. 64, 1644–1650 (2000).

    CAS  Google Scholar 

  32. Biswas, J. C., Ladha, J. K., Dazzo, F. B., Yanni, Y. G. & Rolfe, B. G. Rhizobial inoculation influences seedling vigor and yield of rice. Agron. J. 92, 880–886 (2000).

    Google Scholar 

  33. Cummings, S. P. et al. Nodulation of Sesbania species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia. Environ. Microbiol. 11, 2510–2525 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Masson-Boivin, C., Giraud, E., Perret, X. & Batut, J. Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes? Trends Microbiol. 17, 458–466 (2009).

    CAS  PubMed  Google Scholar 

  35. Hoover, T. R., Imperial, J., Ludden, P. W. & Shah, V. K. Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase. Biochemistry 28, 2768–2771 (1989).

    CAS  PubMed  Google Scholar 

  36. Mandon, K. et al. Role of the fixGHI region of Azorhizobium caulinodans in free-living and symbiotic nitrogen fixation. FEMS Microbiol. Lett. 114, 185–189 (1993).

    CAS  PubMed  Google Scholar 

  37. Tsukada, S. et al. Comparative genome-wide transcriptional profiling of Azorhizobium caulinodans ORS571 grown under free-living and symbiotic conditions. Appl. Environ. Microbiol. 75, 5037–5046 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Gopalaswamy, G., Kannaiyan, S., O’Callaghan, K. J., Davey, M. R. & Cocking, E. C. The xylem of rice (Oryza sativa) is colonized by Azorhizobium caulinodans. Proc. Biol. Sci. 267, 103–107 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Webster, G. et al. The flavonoid naringenin stimulates the intercellular colonization of wheat roots by Azorhizobium caulinodans. Plant Cell Environ. 21, 373–383 (1998).

    CAS  Google Scholar 

  40. Webster, G. et al. in Opportunities for Biological Nitrogen Fixation in Rice and Other Non-Legumes (eds Ladha, J. K. et al.) 115–122 (Springer, 1997).

  41. Triplett, E. W., Kaeppler, S. M. & Chelius, M. K. Klebsiella pneumoniae inoculants for enhancing plant growth. US patent 7,393,678 (2008).

  42. Fujii, T. et al. Effect of inoculation with Klebsiella oxytoca and Enterobacter cloacae on dinitrogen fixation by rice–bacteria associations. Plant Soil 103, 221–226 (1987).

    CAS  Google Scholar 

  43. El-Khawas, H. & Adachi, K. Identification and quantification of auxins in culture media of Azospirillum and Klebsiella and their effect on rice roots. Biol. Fertil. Soils 28, 377–381 (1999).

    CAS  Google Scholar 

  44. Palus, J. A., Borneman, J., Ludden, P. W. & Triplett, E. W. A diazotrophic bacterial endophyte isolated from stems of Zea mays L. and Zea luxurians Iltis and Doebley. Plant Soil 186, 135–142 (1996).

    CAS  Google Scholar 

  45. Rediers, H. et al. Development and application of a dapB-based in vivo expression technology system to study colonization of rice by the endophytic nitrogen-fixing bacterium Pseudomonas stutzeri A15. Appl. Environ. Microbiol. 69, 6864–6874 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Haahtela, K. & Korhonen, T. K. In vitro adhesion of N2-fixing enteric bacteria to roots of grasses and cereals. Appl. Environ. Microbiol. 49, 1186–1190 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Desnoues, N. et al. Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiology 149, 2251–2262 (2003).

    CAS  PubMed  Google Scholar 

  48. Paulsen, I. T. et al. Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23, 873 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Walsh, U. F., Morrissey, J. P. & O’Gara, F. Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation. Curr. Opin. Biotechnol. 12, 289–295 (2001).

    CAS  PubMed  Google Scholar 

  50. Dos Santos, P. C., Fang, Z., Mason, S. W., Setubal, J. C. & Dixon, R. Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genomics 13, 162 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Boyd, E. S., Costas, A. M. G., Hamilton, T. L., Mus, F. & Peters, J. W. Evolution of molybdenum nitrogenase during the transition from anaerobic to aerobic metabolism. J. Bacteriol. 197, 1690–1699 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Raymond, J., Siefert, J. L., Staples, C. R. & Blankenship, R. E. The natural history of nitrogen fixation. Molec. Biol. Evol. 21, 541–554 (2004).

    CAS  PubMed  Google Scholar 

  53. Rubio, L. M. & Ludden, P. W. Biosynthesis of the iron-molybdenum cofactor of nitrogenase. Annu. Rev. Microbiol. 62, 93–111 (2008).

    CAS  PubMed  Google Scholar 

  54. Iki, T., Aono, T. & Oyaizu, H. Evidence for functional differentiation of duplicated nifH genes in Azorhizobium caulinodans. FEMS Microbiol. Lett. 274, 173–179 (2007).

    CAS  PubMed  Google Scholar 

  55. Ratet, P., Pawlowski, K., Schell, J. & Bruijn, F. The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status. Mol. Microbiol. 3, 825–838 (1989).

    CAS  PubMed  Google Scholar 

  56. Poudel, S. et al. Electron transfer to nitrogenase in different genomic and metabolic backgrounds. J. Bacteriol. 200, 00757–00717 (2018).

    Google Scholar 

  57. Boyd, E. & Peters, J. W. New insights into the evolutionary history of biological nitrogen fixation. Front. Microbiol. 4, 201 (2013).

    PubMed  PubMed Central  Google Scholar 

  58. Shah, V. K., Stacey, G. & Brill, W. J. Electron transport to nitrogenase. Purification and characterization of pyruvate: flavodoxin oxidoreductase. The nifJ gene product. J. Biol. Chem. 258, 12064–12068 (1983).

    CAS  PubMed  Google Scholar 

  59. Schmehl, M. et al. Identification of a new class of nitrogen fixation genes in Rhodobacter capsalatus: a putative membrane complex involved in electron transport to nitrogenase. Mol. Gen. Genet. 241, 602–615 (1993).

    CAS  PubMed  Google Scholar 

  60. Edgren, T. & Nordlund, S. The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase. J. Bacteriol. 186, 2052–2060 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Pascuan, C., Fox, A. R., Soto, G. & Ayub, N. D. Exploring the ancestral mechanisms of regulation of horizontally acquired nitrogenases. J. Mol. Evol. 81, 84–89 (2015).

    CAS  PubMed  Google Scholar 

  62. Yan, Y. et al. Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proc. Natl Acad. Sci. USA 105, 7564–7569 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Kechris, K. J., Lin, J. C., Bickel, P. J. & Glazer, A. N. Quantitative exploration of the occurrence of lateral gene transfer by using nitrogen fixation genes as a case study. Proc. Natl Acad. Sci. USA 103, 9584–9589 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Thöny, B., Anthamatten, D. & Hennecke, H. Dual control of the Bradyrhizobium japonicum symbiotic nitrogen fixation regulatory operon fixR nifA: analysis of cis-and trans-acting elements. J. Bacteriol. 171, 4162–4169 (1989).

    PubMed  PubMed Central  Google Scholar 

  65. Li, X.-X., Liu, Q., Liu, X.-M., Shi, H.-W. & Chen, S.-F. Using synthetic biology to increase nitrogenase activity. Microb. Cell Fact. 15, 43 (2016).

    PubMed  PubMed Central  Google Scholar 

  66. Han, Y. et al. Interspecies transfer and regulation of Pseudomonas stutzeri A1501 nitrogen fixation Island in Escherichia coli. J. Microbiol. Biotechnol. 25, 1339–1348 (2015).

    CAS  PubMed  Google Scholar 

  67. Dixon, R. A. & Postgate, J. R. Genetic transfer of nitrogen fixation from Klebsiella pneumoniae to Escherichia coli. Nature 237, 102 (1972).

    CAS  PubMed  Google Scholar 

  68. Cannon, F., Dixon, R., Postgate, J. & Primrose, S. Chromosomal integration of Klebsiella nitrogen fixation genes in Escherichia coli. Microbiology 80, 227–239 (1974).

    CAS  Google Scholar 

  69. Cannon, F., Dixon, R., Postgate, J. & Primrose, S. Plasmids formed in nitrogen-fixing Escherichia coli–Klebsiella pneumoniae hybrids. Microbiology 80, 241–251 (1974).

    CAS  Google Scholar 

  70. Wang, L. et al. A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli. PLoS Genet. 9, e1003865 (2013).

    PubMed  PubMed Central  Google Scholar 

  71. Temme, K., Zhao, D. & Voigt, C. A. Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc. Natl Acad. Sci. USA 109, 7085–7090 (2012).

    PubMed  PubMed Central  Google Scholar 

  72. Smanski, M. J. et al. Functional optimization of gene clusters by combinatorial design and assembly. Nat. Biotechnol. 32, 1241 (2014).

    CAS  PubMed  Google Scholar 

  73. Zhang, L., Liu, X., Li, X. & Chen, S. Expression of the N2 fixation gene operon of Paenibacillus sp. WLY78 under the control of the T7 promoter in Escherichia coli BL21. Biotechnol. Lett. 37, 1999–2004 (2015).

    CAS  PubMed  Google Scholar 

  74. Postgate, J. R. & Kent, H. M. Qualitative evidence for expression of Klebsiella pneumoniae nif in Pseudomonas putida. Microbiology 133, 2563–2566 (1987).

    CAS  Google Scholar 

  75. Setten, L. et al. Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions. PLoS ONE 8, e63666 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Dixon, R. & Kahn, D. Genetic regulation of biological nitrogen fixation. Nat. Rev. Microbiol. 2, 621–631 (2004).

    CAS  PubMed  Google Scholar 

  77. Fischer, H.-M. Environmental regulation of rhizobial symbiotic nitrogen fixation genes. Trends Microbiol. 4, 317–320 (1996).

    CAS  PubMed  Google Scholar 

  78. Tsoy, O. V., Ravcheev, D. A., Čuklina, J. & Gelfand, M. S. Nitrogen fixation and molecular oxygen: comparative genomic reconstruction of transcription regulation in Alphaproteobacteria. Front. Microbiol. 7, 1343 (2016).

    PubMed  PubMed Central  Google Scholar 

  79. Hill, S., Kennedy, C., Kavanagh, E., Goldberg, R. B. & Hanau, R. Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. peumoniae. Nature 290, 424–426 (1981).

    CAS  PubMed  Google Scholar 

  80. Mandon, K. et al. Poly-β-hydroxybutyrate turnover in Azorhizobium caulinodans is required for growth and affects nifA expression. J. Bacteriol. 180, 5070–5076 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Little, R., Reyes‐Ramirez, F., Zhang, Y., van Heeswijk, W. C. & Dixon, R. Signal transduction to the Azotobacter vinelandii NIFL–NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein. EMBO J. 19, 6041–6050 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Poole, P., Ramachandran, V. & Terpolilli, J. Rhizobia: from saprophytes to endosymbionts. Nat. Rev. Microbiol. 16, 291–303 (2018).

    CAS  PubMed  Google Scholar 

  83. Kong, Q., Wu, Q., Ma, Z. & Shen, S. Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae. J. Bacteriol. 166, 353–356 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Brooks, S. J., Collins, J. J. & Brill, W. J. Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature. J. Bacteriol. 157, 460–464 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Peoples, M., Ladha, J. & Herridge, D. Biological nitrogen fixation: an efficient source of nitrogen for sustainable agricultural production? Plant Soil 174, 3–28 (1995).

    CAS  Google Scholar 

  86. Barakat, M., Cheviron, B. & Angulo-Jaramillo, R. Influence of the irrigation technique and strategies on the nitrogen cycle and budget: a review. Agric. Water Manag. 178, 225–238 (2016).

    Google Scholar 

  87. Burger, M. & Jackson, L. E. Microbial immobilization of ammonium and nitrate in relation to ammonification and nitrification rates in organic and conventional cropping systems. Soil Biol. Biochem. 35, 29–36 (2003).

    CAS  Google Scholar 

  88. MacNeil, D. & Brill, W. Mutations in nif genes that cause Klebsiella pneumoniae to be derepressed for nitrogenase synthesis in the presence of ammonium. J. Bacteriol. 144, 744–751 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Bali, A., Blanco, G., Hill, S. & Kennedy, C. Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl. Environ. Microbiol. 58, 1711–1718 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Brewin, B., Woodley, P. & Drummond, M. The basis of ammonium release in nifL mutants of Azotobacter vinelandii. J. Bacteriol. 181, 7356–7362 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Arsène, F., Kaminski, P. A. & Elmerich, C. Control of Azospirillum brasilense NifA activity by PII: effect of replacing Tyr residues of the NifA N-terminal domain on NifA activity. FEMS Microbiol. Lett. 179, 339–343 (1999).

    PubMed  Google Scholar 

  92. Souza, E., Pedrosa, F., Drummond, M., Rigo, L. & Yates, M. Control of Herbaspirillum seropedicae NifA activity by ammonium ions and oxygen. J. Bacteriol. 181, 681–684 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Paschen, A., Drepper, T., Masepohl, B. & Klipp, W. Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium. FEMS Microbiol. Lett. 200, 207–213 (2001).

    CAS  PubMed  Google Scholar 

  94. Rey, F. E., Heiniger, E. K. & Harwood, C. S. Redirection of metabolism for biological hydrogen production. Appl. Environ. Microbiol. 73, 1665–1671 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Smanski, M. J. et al. Synthetic biology to access and expand nature’s chemical diversity. Nat. Rev. Microbiol. 14, 135–149 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Scupham, A. J. et al. Inoculation with Sinorhizobium meliloti RMBPC-2 increases alfalfa yield compared with inoculation with a nonengineered wild-type strain. Appl. Environ. Microbiol. 62, 4260–4262 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Fox, A. R. et al. Major cereal crops benefit from biological nitrogen fixation when inoculated with the nitrogen‐fixing bacterium Pseudomonas protegens Pf-5 X940. Environ. Microbiol. 18, 3522–3534 (2016).

    CAS  PubMed  Google Scholar 

  98. Suthar, H., Hingurao, K., Vaghashiya, J. & Parmar, J. Fermentation: a process for biofertilizer production. Microorg. Green Revol. 6, 229–252 (2017).

    Google Scholar 

  99. Lucy, M., Reed, E. & Glick, B. R. Applications of free living plant growth-promoting rhizobacteria. Antonie Van Leeuwenhoek 86, 1–25 (2004).

    CAS  PubMed  Google Scholar 

  100. Mahmood, A., Turgay, O. C., Farooq, M. & Hayat, R. Seed biopriming with plant growth promoting rhizobacteria: a review. FEMS Microbiol. Ecol. 92, fiw112 (2016).

    PubMed  Google Scholar 

  101. Velusamy, P., Immanuel, J. E., Gnanamanickam, S. S. & Thomashow, L. Biological control of rice bacterial blight by plant-associated bacteria producing 2, 4-diacetylphloroglucinol. Can. J. Microbiol. 52, 56–65 (2006).

    CAS  PubMed  Google Scholar 

  102. Mishra, J. Development and Evaluation of Pseudomonas Based Bioformulation for Disease Control and Growth Enhancement of Zea Mays L. PhD thesis, Babasaheb Bhimrao Ambedkar University (2016).

  103. Weller, D. M. et al. Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots. Plant Biol. 9, 4–20 (2007).

    CAS  PubMed  Google Scholar 

  104. Kaiser, C. et al. Exploring the transfer of recent plant photosynthates to soil microbes: mycorrhizal pathway vs direct root exudation. New Phytol. 205, 1537–1551 (2015).

    CAS  PubMed  Google Scholar 

  105. Mus, F. et al. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Appl. Environ. Microbiol. 82, 3698–3710 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Perrine-Walker, F. M., Prayitno, J., Rolfe, B. G., Weinman, J. J. & Hocart, C. H. Infection process and the interaction of rice roots with rhizobia. J. Exp. Bot. 58, 3343–3350 (2007).

    CAS  PubMed  Google Scholar 

  107. Gough, C. et al. Specific flavonoids promote intercellular root colonization of Arabidopsis thaliana by Azorhizobium caulinodans ORS571. Mol. Plant Microbe Interact. 10, 560–570 (1997).

    CAS  PubMed  Google Scholar 

  108. O’Callaghan, K. J. et al. Effects of glucosinolates and flavonoids on colonization of the roots of Brassica napus by Azorhizobium caulinodans ORS571. Appl. Environ. Microbiol. 66, 2185–2191 (2000).

    PubMed  PubMed Central  Google Scholar 

  109. Mongiardini, E. J. et al. The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation. FEMS Microbiol. Ecol. 65, 279–288 (2008).

    CAS  PubMed  Google Scholar 

  110. Matilla, M. A., Espinosa-Urgel, M., Rodríguez-Herva, J. J., Ramos, J. L. & Ramos-González, M. I. Genomic analysis reveals the major driving forces of bacterial life in the rhizosphere. Genome Biol. 8, R179 (2007).

    PubMed  PubMed Central  Google Scholar 

  111. Casas, M. I., Duarte, S., Doseff, A. I. & Grotewold, E. Flavone-rich maize: an opportunity to improve the nutritional value of an important commodity crop. Front. Plant Sci. 5, 440 (2014).

    PubMed  PubMed Central  Google Scholar 

  112. Ogo, Y., Ozawa, K., Ishimaru, T., Murayama, T. & Takaiwa, F. Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits. Plant Biotechnol. J. 11, 734–746 (2013).

    CAS  PubMed  Google Scholar 

  113. Bacilio-Jiménez, M. et al. Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria. Plant Soil 249, 271–277 (2003).

    Google Scholar 

  114. Pini, F. et al. Lux bacterial biosensors for in vivo spatiotemporal mapping of root secretion. Plant Physiol. 174, 1289–1306 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Ryan, P. R., Dessaux, Y., Thomashow, L. S. & Weller, D. M. Rhizosphere engineering and management for sustainable agriculture. Plant Soil 321, 363–383 (2009).

    CAS  Google Scholar 

  116. Dessaux, Y., Grandclément, C. & Faure, D. Engineering the rhizosphere. Trends Plant Sci. 21, 266–278 (2016).

    CAS  PubMed  Google Scholar 

  117. Geddes, B. A. et al. Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria. Nat. Comm. 10, 3430 (2019).

    Google Scholar 

  118. Arnold, W., Rump, A., Klipp, W., Priefer, U. B. & Pühler, A. Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J. Mol. Biol. 203, 715–738 (1988).

    CAS  PubMed  Google Scholar 

  119. de Salamone, I. G., Döbereiner, J., Urquiaga, S. & Boddey, R. M. Biological nitrogen fixation in Azospirillum strain-maize genotype associations as evaluated by the 15N isotope dilution technique. Biol. Fert. Soils 23, 249–256 (1996).

    Google Scholar 

  120. Pedrosa, F. & Elmerich, C. in Associative and Endophytic Nitrogen-fixing Bacteria and Cyanobacterial Associations (eds Elmerich, C. & Newton, W. E.) 41–71 (Springer, 2007).

  121. Welsh, E. A. et al. The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle. Proc. Natl Acad. Sci. USA 105, 15094–15099 (2008).

    PubMed  PubMed Central  Google Scholar 

  122. Hamilton, T. L. et al. Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii. J. Bacteriol. 193, 4477–4486 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Oda, Y. et al. Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris. J. Bacteriol. 187, 7784–7794 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Haselkorn, R. & Kapatral, V. in Genomes and Genomics of Nitrogen-fixing Organisms (eds Palacios, R. & Newton, W. E.) 71–82 (Springer, 2005).

  125. Jeong, H.-S. & Jouanneau, Y. Enhanced nitrogenase activity in strains of Rhodobacter capsulatus that overexpress the rnf genes. J. Bacteriol. 182, 1208–1214 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. Curatti, L., Brown, C. S., Ludden, P. W. & Rubio, L. M. Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii. Proc. Natl Acad. Sci. USA 102, 6291–6296 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Kaminski, P. A. et al. Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene. Mol. Gen. Genet. 214, 496–502 (1988).

    CAS  PubMed  Google Scholar 

  128. Wientjens, R. The Involvement of the fixABCX Genes and the Respiratory Chain in the Electron Transport to Nitrogenase in Azotobacter vinelandii. PhD thesis, Agricultural University (1993).

  129. McDermott, J. E. et al. A model of cyclic transcriptomic behavior in the cyanobacterium Cyanothece sp. ATCC 51142. Mol. Biosyst. 7, 2407–2418 (2011).

    CAS  PubMed  Google Scholar 

  130. Sevilla, M., Burris, R. H., Gunapala, N. & Kennedy, C. Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and nif mutant strains. Mol. Plant Microbe Interact. 14, 358–366 (2001).

    CAS  PubMed  Google Scholar 

  131. Eskin, N., Vessey, K. & Tian, L. Research progress and perspectives of nitrogen fixing bacterium, Gluconacetobacter diazotrophicus, in monocot plants. Int. J. Agron. https://doi.org/10.1155/2014/208383 (2014).

    Google Scholar 

  132. Lee, S., Reth, A., Meletzus, D., Sevilla, M. & Kennedy, C. Characterization of a major cluster of nif, fix, and associated genes in a sugarcane endophyte, Acetobacter diazotrophicus. J. Bacteriol. 182, 7088–7091 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Shanks, R. M. et al. Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria. Appl. Environ. Microbiol. 72, 5027–5036 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. Hakoyama, T. et al. Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation. Nature 462, 514–517 (2009).

    CAS  PubMed  Google Scholar 

  135. Li, G.-W., Oh, E. & Weissman, J. S. The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria. Nature 484, 538–541 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. Lalanne, J.-B. et al. Evolutionary convergence of pathway-specific enzyme expression stoichiometry. Cell 173, 749–761 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Li, G.-W., Burkhardt, D., Gross, C. & Weissman, J. S. Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157, 624–635 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Poza-Carrión, C., Jiménez-Vicente, E., Navarro-Rodríguez, M., Echavarri-Erasun, C. & Rubio, L. M. Kinetics of nif gene expression in a nitrogen-fixing bacterium. J. Bacteriol. 196, 595–603 (2014).

    PubMed  PubMed Central  Google Scholar 

  139. Tezcan, F. A., Kaiser, J. T., Howard, J. B. & Rees, D. C. Structural evidence for asymmetrical nucleotide interactions in nitrogenase. J. Am. Chem. Soc. 137, 146–149 (2014).

    PubMed  PubMed Central  Google Scholar 

  140. Klugkist, J. & Haaker, H. Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii. J. Bacteriol. 157, 148–151 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Song, M. et al. Control of type III protein secretion using a minimal genetic system. Nat. Comm. 8, 14737 (2017).

    CAS  Google Scholar 

  142. Guo, C.-J. et al. Discovery of reactive microbiota-derived metabolites that inhibit host proteases. Cell 168, 517–526 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. Ren, H., Hu, P. & Zhao, H. A plug-and‐play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae. Biotechnol. Bioeng. 114, 1847–1854 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. MacLellan, S. R., MacLean, A. M. & Finan, T. M. Promoter prediction in the rhizobia. Microbiology 152, 1751–1763 (2006).

    CAS  PubMed  Google Scholar 

  145. Ramírez-Romero, M. A., Masulis, I., Cevallos, M. A., González, V. & Davila, G. The Rhizobium etli σ70 (SigA) factor recognizes a lax consensus promoter. Nucleic Acids Res. 34, 1470–1480 (2006).

    PubMed  PubMed Central  Google Scholar 

  146. Becker, A. et al. Riboregulation in plant-associated α-proteobacteria. RNA Biol. 11, 550–562 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. Robledo, M., Frage, B., Wright, P. R. & Becker, A. A stress-induced small RNA modulates alpha-rhizobial cell cycle progression. PLoS Genet. 11, e1005153 (2015).

    PubMed  PubMed Central  Google Scholar 

  148. Giacomini, A., Ollero, F. J., Squartini, A. & Nuti, M. P. Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in Gram-negative bacteria. Gene 144, 17–24 (1994).

    CAS  PubMed  Google Scholar 

  149. Khan, S. R., Gaines, J., Roop, R. M. & Farrand, S. K. Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl. Environ. Microbiol. 74, 5053–5062 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. Tett, A. J. et al. Regulatable vectors for environmental gene expression in Alphaproteobacteria. Appl. Environ. Microbiol. 78, 7137–7140 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. Mostafavi, M. et al. Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. BMC Microbiol. 14, 295 (2014).

    PubMed  PubMed Central  Google Scholar 

  152. Anderson, J. et al. BglBricks: a flexible standard for biological part assembly. J. Biol. Eng. 4, 1 (2010).

    PubMed  PubMed Central  Google Scholar 

  153. Farasat, I. et al. Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria. Mol. Sys. Biol. 10, 731 (2014).

    Google Scholar 

  154. Cambray, G. et al. Measurement and modeling of intrinsic transcription terminators. Nucleic Acids Res. 41, 5139–5148 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  155. Chen, Y.-J. et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat. Methods 10, 659–664 (2013).

    CAS  PubMed  Google Scholar 

  156. Temme, K., Hill, R., Segall-Shapiro, T. H., Moser, F. & Voigt, C. A. Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Res. 40, 8773–8781 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  157. Price, M. N., Arkin, A. P. & Alm, E. The life-cycle of operons. PLoS Genet. 2, e96 (2006).

    PubMed  PubMed Central  Google Scholar 

  158. Touchon, M. & Rocha, E. P. Coevolution of the organization and structure of prokaryotic genomes. Cold Spring Harb. Persp. Biol. 8, a018168 (2016).

    Google Scholar 

  159. Price, M. N., Huang, K. H., Arkin, A. P. & Alm, E. Operon formation is driven by co-regulation and not by horizontal gene transfer. Genome Res. 15, 809–819 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  160. Kaminski, P. A. & Elmerich, C. The control of Azorhizobium caulinodans nifA expression by oxygen, ammonia and by the HF‐I‐like protein, NrfA. Mol. Microbiol. 28, 603–613 (1998).

    CAS  PubMed  Google Scholar 

  161. Pawlowski, K., Klosse, U. & De Bruijn, F. J. Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism. Mol. Gen. Genet. 231, 124–138 (1991).

    CAS  PubMed  Google Scholar 

  162. Kaminski, P. & Elmerich, C. Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans. Mol. Microbiol. 5, 665–673 (1991).

    CAS  PubMed  Google Scholar 

  163. Kaminski, P., Mandon, K., Arigoni, F., Desnoues, N. & Elmerich, C. Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK‐like gene, a positive regulator of nifA. Mol. Microbiol. 5, 1983–1991 (1991).

  164. Michel-Reydellet, N. & Kaminski, P. A. J. Azorhizobium caulinodans PII and GlnK proteins control nitrogen fixation and ammonia assimilation. J. Bacteriol. 181, 2655–2658 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  165. Drepper, T. et al. Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus. Microbiology 149, 2203–2212 (2003).

    CAS  PubMed  Google Scholar 

  166. Martínez-García, E. & de Lorenzo, V. Molecular tools and emerging strategies for deep genetic/genomic refactoring of Pseudomonas. Curr. Opin. Biotechnol. 47, 120–132 (2017).

    PubMed  Google Scholar 

  167. Calero, P., Jensen, S. I. & Nielsen, A. T. Broad-host-range ProUSER vectors enable fast characterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440. ACS Synth. Biol. 5, 741–753 (2016).

    CAS  PubMed  Google Scholar 

  168. Choi, K.-H. & Schweizer, H. P. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat. Protoc. 1, 153–161 (2006).

    CAS  PubMed  Google Scholar 

  169. Poole, R. K. & Hill, S. Respiratory protection of nitrogenase activity in Azotobacter vinelandii—roles of the terminal oxidases. Biosci. Rep. 17, 303–317 (1997).

    CAS  PubMed  Google Scholar 

  170. Sabra, W., Zeng, A.-P., Lünsdorf, H. & Deckwer, W.-D. Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase. Appl. Environ. Microbiol. 66, 4037–4044 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  171. Schlesier, J., Rohde, M., Gerhardt, S. & Einsle, O. A conformational switch triggers nitrogenase protection from oxygen damage by Shethna protein II (FeSII). J. Am. Chem. Soc. 138, 239–247 (2015).

    PubMed  Google Scholar 

  172. Ledbetter, R. N. et al. The electron bifurcating FixABCX protein complex from Azotobacter vinelandii: generation of low-potential reducing equivalents for nitrogenase catalysis. Biochemistry 56, 4177–4190 (2017).

    CAS  PubMed  Google Scholar 

  173. Li, B. et al. Root exudates drive interspecific facilitation by enhancing nodulation and N2 fixation. Proc. Natl Acad. Sci. USA 113, 6496–6501 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  174. Wang, Y., Zhang, J., Sun, Y., Feng, J. & Zhang, X. Evaluating the potential value of vatural product cuminic acid against plant pathogenic fungi in cucumber. Molecules 22, 1914 (2017).

    PubMed Central  Google Scholar 

  175. Zhou, X. & Wu, F. Vanillic acid changed cucumber (Cucumis sativus L.) seedling rhizosphere total bacterial, Pseudomonas and Bacillus spp. communities. Sci. Rep. 8, 4929 (2018).

    PubMed  PubMed Central  Google Scholar 

  176. Sun, Y., Wang, Y., Han, L., Zhang, X. & Feng, J. Antifungal activity and action mode of cuminic acid from the seeds of Cuminum cyminum L. against Fusarium oxysporum f. sp. Niveum (FON) causing fusarium wilt on watermelon. Molecules 22, 2053 (2017).

    PubMed Central  Google Scholar 

  177. Wang, Y., Sun, Y., Zhang, Y., Zhang, X. & Feng, J. Antifungal activity and biochemical response of cuminic acid against Phytophthora capsici Leonian. Molecules 21, 756 (2016).

    PubMed Central  Google Scholar 

  178. Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S. & Vivanco, J. M. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol. 57, 233–266 (2006).

    CAS  PubMed  Google Scholar 

  179. Rasmann, S. & Turlings, T. C. Root signals that mediate mutualistic interactions in the rhizosphere. Curr. Opin. Plant Biol. 32, 62–68 (2016).

    CAS  PubMed  Google Scholar 

  180. Soto, M. J., Sanjuan, J. & Olivares, J. Rhizobia and plant-pathogenic bacteria: common infection weapons. Microbiology 152, 3167–3174 (2006).

    CAS  PubMed  Google Scholar 

  181. Poonguzhali, S., Madhaiyan, M. & Sa, T. Quorum-sensing signals produced by plant-growth promoting Burkholderia strains under in vitro and in planta conditions. Res. Microbiol. 158, 287–294 (2007).

    CAS  PubMed  Google Scholar 

  182. Sanchez-Contreras, M., Bauer, W. D., Gao, M., Robinson, J. B. & Allan Downie, J. Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes. Philos. Trans. R. Soc. B 362, 1149–1163 (2007).

    CAS  Google Scholar 

  183. Chagas, F. O., de Cassia Pessotti, R., Caraballo-Rodríguez, A. M. & Pupo, M. T. Chemical signaling involved in plant–microbe interactions. Chem. Soc. Rev. 47, 1652–1704 (2018).

    CAS  PubMed  Google Scholar 

  184. Schikora, A., Schenk, S. T. & Hartmann, A. Beneficial effects of bacteria–plant communication based on quorum sensing molecules of the N-acyl homoserine lactone group. Plant Mol. Biol. 90, 605–612 (2016).

    CAS  PubMed  Google Scholar 

  185. Westervelt, P., Bloom, M. L., Mabbott, G. A. & Fekete, F. A. The isolation and identification of 3, 4-dihydroxybenzoic acid formed by nitrogen-fixing Azomonas macrocytogenes. FEMS Microbiol. Lett. 30, 331–335 (1985).

    CAS  Google Scholar 

  186. Collinson, S. K., Doran, J. L. & Page, W. J. Production of 3, 4-dihydroxybenzoic acid by Azomonas macrocytogenes and Azotobacter paspali. Can. J. Microbiol. 33, 169–175 (1987).

    CAS  Google Scholar 

  187. Bhattacharya, A., Sood, P. & Citovsky, V. The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection. Mol. Plant Path. 11, 705–719 (2010).

    CAS  Google Scholar 

  188. Mathesius, U. et al. Extensive and specific responses of a eukaryote to bacterial quorum-sensing signals. Proc. Natl Acad. Sci. USA 100, 1444–1449 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  189. Hernández-Reyes, C., Schenk, S. T., Neumann, C., Kogel, K. H. & Schikora, A. N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens. Microbial Biotechnol. 7, 580–588 (2014).

    Google Scholar 

  190. Pérez-Montaño, F. et al. Rice and bean AHL-mimic quorum-sensing signals specifically interfere with the capacity to form biofilms by plant-associated bacteria. Res. Microbiol. 164, 749–760 (2013).

    PubMed  Google Scholar 

  191. Bressan, M. et al. Exogenous glucosinolate produced by Arabidopsis thaliana has an impact on microbes in the rhizosphere and plant roots. ISME J. 3, 1243–1257 (2009).

    CAS  PubMed  Google Scholar 

  192. Badri, D. V. et al. An ABC transporter mutation alters root exudation of phytochemicals that provoke an overhaul of natural soil microbiota. Plant Physiol. 151, 2006–2017 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  193. Abdel-Ghany, S. E., Day, I., Heuberger, A. L., Broeckling, C. D. & Reddy, A. S. Production of phloroglucinol, a platform chemical, in Arabidopsis using a bacterial gene. Sci. Rep. 6, 38483 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  194. Oger, P., Petit, A. & Dessaux, Y. Genetically engineered plants producing opines alter their biological environment. Nat. Biotechnol. 15, 369 (1997).

    CAS  PubMed  Google Scholar 

  195. Mondy, S. et al. An increasing opine carbon bias in artificial exudation systems and genetically modified plant rhizospheres leads to an increasing reshaping of bacterial populations. Mol. Ecol. 23, 4846–4861 (2014).

    PubMed  Google Scholar 

  196. Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J. & Voigt, C. A. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat. Chem. Biol. 15, 196–204 (2018).

    PubMed  Google Scholar 

  197. Poole, P. S., Schofiel, N. A., Reid, C. J., Drew, E. M. & Walshaw, D. L. Identification of chromosomal genes located downstream of dctD that affect the requirement for calcium and the lipopolysaccharide layer of Rhizobium leguminosarum. Microbiology 140, 2797–2809 (1994).

    CAS  PubMed  Google Scholar 

  198. Wilson, P. W. & Knight, S. G. Experiments in Bacterial Physiology (Burgess, 1952).

  199. Udvardi, M. & Poole, P. S. Transport and metabolism in legume-rhizobia symbioses. Ann. Rev. Plant Biol. 64, 781–805 (2013).

    CAS  Google Scholar 

  200. Ferri, L., Gori, A., Biondi, E. G., Mengoni, A. & Bazzicalupo, M. Plasmid electroporation of Sinorhizobium strains: the role of the restriction gene hsdR in type strain Rm1021. Plasmid 63, 128–135 (2010).

    CAS  PubMed  Google Scholar 

  201. Selbitschka, W. et al. Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae. Mol. Gen. Genet. 229, 86–95 (1991).

    CAS  PubMed  Google Scholar 

  202. Prell, J., Boesten, B., Poole, P. & Priefer, U. B. The Rhizobium leguminosarum bv. viciae VF39 γ-aminobutyrate (GABA) aminotransferase gene (gabT) is induced by GABA and highly expressed in bacteroids. Microbiology 148, 615–623 (2002).

    CAS  PubMed  Google Scholar 

  203. Silva-Rocha, R. et al. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41, D666–D675 (2012).

    PubMed  PubMed Central  Google Scholar 

  204. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  205. Yan, Y. et al. Global transcriptional analysis of nitrogen fixation and ammonium repression in root-associated Pseudomonas stutzeri A1501. BMC Genomics 11, 11 (2010).

    PubMed  PubMed Central  Google Scholar 

  206. Jacob, G., Schaefer, J., Garbow, J. & Stejskal, E. Solid-state NMR studies of Klebsiella pneumoniae grown under nitrogen-fixing conditions. J. Biol. Chem. 262, 254–259 (1987).

  207. Blomberg, P., Wagner, E. & Nordström, K. Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III. EMBO J. 9, 2331–2340 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  208. Gorochowski, T. E. et al. Genetic circuit characterization and debugging using RNA‐seq. Mol. Syst. Biol. 13, 952 (2017).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Science Foundation (grant no. NSF-1331098), the Abdul Latif Jameel Water and Food Security Lab (J-WAFS) at the Massachusetts Institute of Technology, the US National Science Foundation Synthetic Biology Engineering Research Center (grant no. SynBERC EEC0540879) and the Office of Naval Research Multidisciplinary University Research Initiative (MURI grant no. N00014-13-1-0074). We thank G. O’Toole of Dartmouth College for the yeast shuttle vectors.

Author information

Authors and Affiliations

  1. Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Min-Hyung Ryu, Jing Zhang, Tyler Toth & Christopher A. Voigt

  2. Departments of Bacteriology and Agronomy, University of Wisconsin–Madison, Madison, WI, USA

    Devanshi Khokhani & Jean-Michel Ané

  3. Department of Plant Sciences, University of Oxford, Oxford, UK

    Barney A. Geddes & Philip S. Poole

  4. Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

    Florence Mus, Amaya Garcia-Costas & John W. Peters

  5. Institute of Biological Chemistry, Washington State University, Pullman, WA, USA

    Florence Mus & John W. Peters

  6. Department of Biology, Colorado State University–Pueblo, Pueblo, CO, USA

    Amaya Garcia-Costas

Authors
  1. Min-Hyung Ryu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tyler Toth
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Devanshi Khokhani
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Barney A. Geddes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Florence Mus
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Amaya Garcia-Costas
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. John W. Peters
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Philip S. Poole
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jean-Michel Ané
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Christopher A. Voigt
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.-H.R. and C.A.V. conceived the study and designed the experiments. J.Z. and M.-H.R. performed the RNA-seq and ribosome-profiling experiments and analysed the data. T.T. and M.-H.R. performed the opine experiments and analysed the data. D.K., J.-M.A., F.M. and J.W.P. performed the 15N-incorporation experiments and analysed the data. B.A.G. and P.S.P. performed the diazotrophic growth experiments and analysed the data. M.-H.R. performed all other experiments and analysed the data. M.-H.R. and C.A.V. wrote the manuscript with input from all of the authors.

Corresponding author

Correspondence to Christopher A. Voigt.

Ethics declarations

Competing interests

M.-H.R. and C.A.V. have filed a patent application (US provisional application no. 62/820,765) on this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Nitrogenase activity in wild-type R. sp. IRBG74 and a Δnif mutant strain, in which the native nif clusters are deleted.

(a) The nif clusters in R. sp. IRBG74. The deleted regions generated by the suicide plasmids pMR45–46 are marked (Methods). (b) Transfer of native nif constructs into R. sp. IRBG74. Nitrogenase activity was detected only from the transfer of the R. sphaeroides nif cluster into R. sp. IRBG74 MR17 (ΔhsdR, recA) but not into R. sp. IRBG74 MR19 (ΔhsdR, recA Δnif). Expression of A. caulinodans nifV on the plasmid pMR49 in R. sp. IRBG74 MR17 was induced by 0.5 mM IPTG. The co-transfer of the complete A. caulinodans nif cluster on the two plasmids pMR19 and pMR20 did not yield activity in R. sp. IRBG74 MR17. Error bars represent standard deviation from three independent experiments on different days. Asterisk indicate ethylene production below the detection limit. Rsp, R. sphaeroides; Aca, A. caulinodans. (c) Plasmid maps used in (b).

Extended Data Fig. 2 Ribosome profiling data for the K. oxytoca nif cluster.

Ribosome profiling data for the K. oxytoca nif cluster in its native host (top) and when transferred into different strains are shown.

Extended Data Fig. 3 The effect of NifA overexpression on the nifH promoter activity in R. sp. IRBG74.

(a) The design of the reporter constructs used to measure nifH promoter activity shown. The nifH promoter activity was analysed in R. sp. IRBG74 using flow cytometry. Overexpression of R. sp. IRBG74 NifA increased the activity of the R. sp. IRBG74 nifH promoter but failed to complement or enhance the activities of the other nifH promoters including K. oxytoca, P. stutzeri and A. caulinodans. Error bars represent standard deviation from three independent experiments on different days. WT, wild-type; Rsp, R. sp. IRBG74; Kox, K. oxytoca M5al; Pst, P. stutzeri A1501; Aca, A. caulinodans ORS571 (b) Plasmid maps used to assess the effect of nifA overexpression in R. sp. IRBG74.

Extended Data Fig. 4 Nitrogenase activity when different inducible nif clusters are transferred to E. coli MG1655.

(a) The same universal controller system based on K. oxytoca nifA was optimized and used for all three clusters (Supplementary Figure 15, 17b). The controller plasmid pMR104 and genetic parts are provided in Supplementary Table 3 and 4. (b) The nif clusters from K. oxytoca, P. stutzeri, and A. vinelandii are shown. The deleted regions corresponding the NifLA regulators are marked, and their corresponding genomic locations are provided in Supplementary Table 3. The dotted lines indicate that multiple regions from the genome were cloned and combined to form the nif cluster. The clusters were carried on the plasmids pMR23–25 (Supplementary Table 3). (c) The induction of the nifH promoters from each species by the controller are shown (+, 50 μM IPTG). (d) The nitrogenase activities of the native cluster (intact nifLA) are compared to the inducible clusters in the presence and absence of 50 μM IPTG. The dashed lines indicate the activity of the native clusters in the wild-type context (top to bottom, K. oxytoca M5al, P. stutzeri A1501 and A. vinelandii DJ). (e) Regulation of nitrogenase activity by ammonium. Ammonium tolerance of nitrogenase from the native (black bar) and inducible (grey bar) systems was tested in the presence of 17.1 mM ammonium acetate and 50 μM IPTG (inducible). Asterisks indicate ethylene production below the detection limit. (f) Regulation of nitrogenase activity by oxygen. The native nif cluster is compared to the inducible version including the controller plasmid and 50 μM IPTG. Nitrogenase activities were measured after 3 h of incubation at constant oxygen concentrations (0 to 3%) in the headspace (Methods). Error bars represent standard deviation from three independent experiments on different days.

Extended Data Fig. 5 Transfer of the refactored nif cluster v3.2 in P. protegens Pf-5.

(a) Controllers whose output is T7 RNAP integrated on the genome of P. protegens Pf-5 are described. Substituted genetic parts for the controller optimization compared to the controller module pKT249 in E. coli MG1655 are highlighted in red. The response functions for the controllers with the reporter plasmid pMR81 was measured in the P. protegens Pf- 5 controller strain MR7. The controller driving the expression of GFP by the T7 promoter led to 96-fold induction by IPTG. (b) The genetic parts used to build the refactored v3.2 nif gene cluster are shown (provided in Supplementary Table 4). (c) The activity of the refactored nif cluster v3.2. Nitrogenase expression was induced by 1 mM IPTG. (d) The function of the transcriptional parts of the cluster v3.2 was analysed by RNA- seq (Supplementary Figure 18). The performance of the promoters (left) and terminators (right) was calculated (Methods). (e) The translation efficiency of the nif genes v3.2 as calculated using ribosome profiling and RNA-seq. Lines connect points that occur in the same operon. (f) The ribosome density (RD) is compared for the refactored v3.2 nif genes in P. protegens Pf-5 versus that measured for the nif genes from the native K. oxytoca cluster in K. oxytoca (→Klebsiella: R2 = 0.68). Error bars represent standard deviation from three independent experiments on different days.

Extended Data Fig. 6 Control of nitrogenase fixation in A. caulinodans ORS571 under changing environmental conditions.

(a) The effect of the absence or presence of 10 mM ammonium chloride is shown. The WT NifA from A. caulinodans ORS571 is compared to different combinations of amino acid substitutions. NifA/RpoN expression is induced by 1 mM IPTG (+) for A. caulinodans ΔnifA containing the controller plasmid pMR124–127 (+). An asterisk indicates ethylene production below the detection limit. (b) The nitrogenase activity is shown as a function of the oxygen concentration in the headspace (Methods). The native nif cluster (wild-type A. caulinodans ORS571, black) is compared to the inducible version (grey) including the controller plasmid and 1 mM IPTG. Error bars represent standard deviation from three independent experiments on different days.

Extended Data Fig. 7 Ammonium repression of the transferred nif clusters in E. coli MG1655 and P. protegens Pf-5.

Nitrogenase sensitivity to ammonium was measured by acetylene reduction assay in the absence (-) or presence (+) of 17.1 mM ammonium acetate. The sensitivity of the native and inducible nif clusters in E. coli MG1655 (a) and P. protegens Pf- 5 (b). Note that the data are from Fig. 4 and Supplementary Figure 8. (c) The specific nitrogenase activities of the native A. vinelandii nif cluster are compared to the inducible A. vinelandii cluster in the presence (+) and absence (-) of 17.1 mM ammonium acetate in P. protegens Pf-5. The nif clusters from the inducible version were induced by 50 μM and 0.5 mM IPTG in E. coli MG1655 and P. protegens Pf-5, respectively. Asterisks indicate ethylene production below the detection limit. Error bars represent standard deviation from three independent experiments on different days.

Extended Data Fig. 8 The effect of oxygen on the activity of the nifH promoters.

Expression from the nifH promoters was analysed in E. coli MG1655 containing the controller plasmid pMR104, P. protegens Pf-5 MR10 (for K. oxytoca) and MR9 (for P. stutzeri and A. vinelandii) at varying initial oxygen levels in the headspace. The three nifH promoters were induced with 0.05 mM IPTG and 0.5 mM IPTG in E. coli MG1655 and P. protegens Pf-5, respectively, and incubated at varying initial oxygen concentrations. Error bars represent standard deviation from three independent experiments on different days.

Extended Data Fig. 9 The effect of the rnf and fix complex on nitrogenase activity.

The modified nif clusters of A. vinelandii on the plasmids pMR25–28 were analysed in the controller strain P. protegens Pf-5 MR9. The deleted regions from the clusters were provided in Supplementary Table 3. Nitrogenase was induced with 0.5 mM IPTG. Dots in the DNA line indicate where multiple regions were cloned from genomic DNA and combined to form one large plasmid-borne nif cluster. An asterisk indicates ethylene production below the detection limit. Error bars represent standard deviation from three independent experiments on different days.

Extended Data Fig. 10 Regulation of nitrogenase activity in the E. coli MG1655 ‘Marionette’ strain.

(a) Controller plasmids used to drive expression of T7 promoters. (b) Inducibility of the T7 promoter by the controller plasmids encoding T7 RNAP under the regulation of the 12 sensors was tested with a reporter plasmid pMR123 (right). (c) Inducible control of nitrogenase activity in response to 12 inducers was tested with each of 12 controller plasmid and the plasmid pMR138 (right) carrying the refactored nif cluster v2.1 on pBBR1 origin. The choline-Cl inducible system was omitted for activity assay as the system was not inducible. For the DAPG-, DHBA-, and vanillic acid-inducible system, the refactored cluster v2.1 was carried on a lower copy number plasmid pMR31 (right) as there was no colony formation from the transformation of the plasmid pMR138. The inducer concentrations are: 400 μM arabinose, 1 mM choline-Cl, 500 nM 3OC14HSL, 50 μM cuminic acid, 25 nM 3OC6HSL, 25 μM DAPG, 500 μM DHBA, 1 mM IPTG, 100 nM aTc, 250 μM naringenin, 50 μM vanillic acid, and 250 μM salicylic acid. Plasmid and genetic parts are provided in Supplementary Table 3 and 4. Error bars represent standard deviation from three independent experiments on different days.

Supplementary information

Supplementary Information

Supplementary Figs. 1–22 and Supplementary Tables 1–6.

Reporting Summary

Supplementary Data 1

Plasmid pMR3_Native nif cluster of Klebsiella oxytoca M5al.

Supplementary Data 2

Plasmid pMR5_Native nif cluster of Pseudomonas stutzeri A1501.

Supplementary Data 3

Plasmid pMR7_Native nif cluster of Azotobacter vinelandii DJ.

Supplementary Data 4

Plasmid pMR9_Native nif cluster of Cyanothece ATCC51142.

Supplementary Data 5

Plasmid pMR11_Native nif cluster of Paenibacillus polymyxa WLY78.

Supplementary Data 6

Plasmid pMR13_Native nif cluster of Azospirillium brasilense Sp7.

Supplementary Data 7

Plasmid pMR15_Native nif cluster of Rhodobacter sphaeroides 2.4.1.

Supplementary Data 8

Plasmid pMR17_Native nif cluster of Rhodopseudomonas palustris CGA009.

Supplementary Data 9

Plasmid pMR19_Native nif cluster of Azorhizobium caulinodans ORS571 (Part 1 of 2).

Supplementary Data 10

Plasmid pMR20_Native nif cluster of Azorhizobium caulinodans ORS571 (Part 2 of 2).

Supplementary Data 11

Plasmid pMR21_Native nif cluster of Gluconacetobacter diazotrophicus PA1 5.

Supplementary Data 12

Plasmid pMR29_Refactored nif cluster v2.1.

Supplementary Data 13

Plasmid pMR38_Refactored nif cluster v3.2.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ryu, MH., Zhang, J., Toth, T. et al. Control of nitrogen fixation in bacteria that associate with cereals. Nat Microbiol 5, 314–330 (2020). https://doi.org/10.1038/s41564-019-0631-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-019-0631-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology