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Evolutionary origin and ecological implication of a unique nif island in free-living Bradyrhizobium lineages


The alphaproteobacterial genus Bradyrhizobium has been best known as N2-fixing members that nodulate legumes, supported by the nif and nod gene clusters. Recent environmental surveys show that Bradyrhizobium represents one of the most abundant free-living bacterial lineages in the world’s soils. However, our understanding of Bradyrhizobium comes largely from symbiotic members, biasing the current knowledge of their ecology and evolution. Here, we report the genomes of 88 Bradyrhizobium strains derived from diverse soil samples, including both nif-carrying and non-nif-carrying free-living (nod free) members. Phylogenomic analyses of these and 252 publicly available Bradyrhizobium genomes indicate that nif-carrying free-living members independently evolved from symbiotic ancestors (carrying both nif and nod) multiple times. Intriguingly, the nif phylogeny shows that the vast majority of nif-carrying free-living members comprise an independent cluster, indicating that horizontal gene transfer promotes nif expansion among the free-living Bradyrhizobium. Comparative genomics analysis identifies that the nif genes found in free-living Bradyrhizobium are located on a unique genomic island of ~50 kb equipped with genes potentially involved in coping with oxygen tension. We further analyze amplicon sequencing data to show that Bradyrhizobium members presumably carrying this nif island are widespread in a variety of environments. Given the dominance of Bradyrhizobium in world’s soils, our findings have implications for global nitrogen cycles and agricultural research.

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Fig. 1: The maximum-likelihood phylogenomic tree of Bradyrhizobium and inferred lifestyle evolutionary history.
Fig. 2: Nif gene phylogeny of Bradyrhizobium.
Fig. 3: The comparison of the gene arrangement of the nif gene cluster located in the nif island and in the symbiosis island.
Fig. 4: Normalized abundance of free-living and symbiotic nifH of Bradyrhizobium in amplicon sequencing samples collected from different types of environments.

Data availability

The genomic sequences and raw reads of the 88 sequenced Bradyrhizobium and five Afipia strains are available at NCBI BioProject (accession: PRJNA698083).

Code availability

The Python [72] and Ruby [73] codes used for phylogenomic and comparative genomics analyses are deposited in the online repository


  1. 1.

    Ormeno-Orrillo E, Martinez-Romero E. A genomotaxonomy view of the Bradyrhizobium genus. Front Microbiol. 2019;10:1334.

    PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Avontuur JR, Palmer M, Beukes CW, Chan WY, Coetzee MPA, Blom J, et al. Genome-informed Bradyrhizobium taxonomy: where to from here? Syst Appl Microbiol. 2019;42:427–39.

    PubMed  Article  Google Scholar 

  3. 3.

    Andrews M, Andrews ME. Specificity in legume-rhizobia symbioses. Int J Mol Sci. 2017;18:705.

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  4. 4.

    Parker MA. The spread of Bradyrhizobium lineages across host legume clades: from Abarema to Zygia. Micro Ecol. 2015;69:630–40.

    Article  Google Scholar 

  5. 5.

    Garrido-Oter R, Nakano RT, Dombrowski N, Ma KW, McHardy AC, Schulze-Lefert P, et al. Modular traits of the Rhizobiales root microbiota and their evolutionary relationship with symbiotic rhizobia. Cell Host Microbe. 2018;24:155–67.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Delgado-Baquerizo M, Oliverio AM, Brewer TE, Benavent-Gonzalez A, Eldridge DJ, Bardgett RD, et al. A global atlas of the dominant bacteria found in soil. Science. 2018;359:320–5.

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Hollowell AC, Regus JU, Gano KA, Bantay R, Centeno D, Pham J, et al. Epidemic spread of symbiotic and non-symbiotic Bradyrhizobium genotypes across California. Micro Ecol. 2016;71:700–10.

    CAS  Article  Google Scholar 

  8. 8.

    VanInsberghe D, Maas KR, Cardenas E, Strachan CR, Hallam SJ, Mohn WW. Non-symbiotic Bradyrhizobium ecotypes dominate North American forest soils. ISME J. 2015;9:2435–41.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Wang S, Meade A, Lam H, Luo H. Evolutionary timeline and genomic plasticity underlying the lifestyle diversity in Rhizobiales. Msystems. 2020;5:e00438–00420.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Peoples MB, Craswell ET. Biological nitrogen fixation: Investments, expectations and actual contributions to agriculture. Plant Soil. 1992;141:13–39.

    CAS  Article  Google Scholar 

  11. 11.

    Cleveland CC, Townsend AR, Schimel DS, Fisher H, Howarth RW, Hedin LO, et al. Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Glob Biogeochem Cy. 1999;13:623–45.

    CAS  Article  Google Scholar 

  12. 12.

    Herridge DF, Peoples MB, Boddey RM. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil. 2008;311:1–18.

    CAS  Article  Google Scholar 

  13. 13.

    Vitousek PM, Menge DN, Reed SC, Cleveland CC. Biological nitrogen fixation: rates, patterns and ecological controls in terrestrial ecosystems. Philos Trans R Soc Lond B Biol Sci. 2013;368:20130119.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  14. 14.

    Reed SC, Cleveland CC, Townsend AR. Relationships among phosphorus, molybdenum and free-living nitrogen fixation in tropical rain forests: results from observational and experimental analyses. Biogeochemistry. 2013;114:135–47.

    CAS  Article  Google Scholar 

  15. 15.

    Matson AL, Corre MD, Burneo JI, Veldkamp E. Free-living nitrogen fixation responds to elevated nutrient inputs in tropical montane forest floor and canopy soils of southern Ecuador. Biogeochemistry. 2015;122:281–94.

    CAS  Article  Google Scholar 

  16. 16.

    Lee KB, De Backer P, Aono T, Liu CT, Suzuki S, Suzuki T, et al. The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571. BMC Genomics. 2008;9:1–14.

    Article  CAS  Google Scholar 

  17. 17.

    Dreyfus B, Garcia JL, Gillis M. Characterization of Azorhizobium Caulinodans gen. nov,, sp. nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int J Syst Bacteriol. 1988;38:89–98.

    CAS  Article  Google Scholar 

  18. 18.

    Dreyfus BL, Elmerich C, Dommergues YR. Free-living Rhizobium strain able to grow on N2 as the sole nitrogen source. Appl Environ Microbiol. 1983;45:711–3.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Alazard D. Nitrogen fixation in pure culture by rhizobia isolated from stem nodules of tropical Aeschynomene species. FEMS Microbiol Lett. 1990;68:177–82.

    CAS  Article  Google Scholar 

  20. 20.

    Terakado-Tonooka J, Fujihara S, Ohwaki Y. Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes. Plant Soil. 2013;367:639–50.

    CAS  Article  Google Scholar 

  21. 21.

    Wongdee J, Boonkerd N, Teaumroong N, Tittabutr P, Giraud E. Regulation of nitrogen fixation in Bradyrhizobium sp. strain DOA9 involves two distinct NifA regulatory proteins that are functionally redundant during symbiosis but not during free-living growth. Front Microbiol. 2018;9:1644.

    PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Hunt S, Layzell DB. Gas exchange of legume nodules and the regulation of nitrogenase activity. Annu Rev Plant Phys. 1993;44:483–511.

    CAS  Article  Google Scholar 

  23. 23.

    Gallon JR. Reconciling the incompatible: N2 fixation and O2. N. Phytol. 1992;122:571–609.

    CAS  Article  Google Scholar 

  24. 24.

    Coombs JT, Franco CMM. Isolation and identification of actinobacteria from surface-sterilized wheat roots. Appl Environ Microbiol. 2003;69:5603–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Sachs JL, Kembel SW, Lau AH, Simms EL. In situ phylogenetic structure and diversity of wild Bradyrhizobium communities. Appl Environ Microbiol. 2009;75:4727–35.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2017;67:1613–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–77.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–9.

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25:1043–55.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32:268–74.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019;20:1–14.

    Article  Google Scholar 

  32. 32.

    Maddison WP, Maddison DR Mesquite: a modular system for evolutionary analysis. Version 3.61. 2019.

  33. 33.

    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Berger SA, Stamatakis A. Aligning short reads to reference alignments and trees. Bioinformatics. 2011;27:2068–75.

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Barbera P, Kozlov AM, Czech L, Morel B, Darriba D, Flouri T, et al. EPA-ng: massively parallel evolutionary placement of genetic sequences. Syst Biol. 2019;68:365–9.

    PubMed  Article  Google Scholar 

  36. 36.

    Durrant MG, Li MM, Siranosian BA, Montgomery SB, Bhatt AS. A bioinformatic analysis of integrative mobile genetic elements highlights their role in bacterial adaptation. Cell Host Microbe. 2020;27:140–53.

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Starikova EV, Tikhonova PO, Prianichnikov NA, Rands CM, Zdobnov EM, Ilina EN, et al. Phigaro: high-throughput prophage sequence annotation. Bioinformatics. 2020;36:3882–4.

    PubMed  Article  Google Scholar 

  38. 38.

    Giraud E, Moulin L, Vallenet D, Barbe V, Cytryn E, Avarre JC, et al. Legumes symbioses: absence of nod genes in photosynthetic bradyrhizobia. Science. 2007;316:1307–12.

    PubMed  Article  Google Scholar 

  39. 39.

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

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Outten FW, Djaman O, Storz G. A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli. Mol Microbiol. 2004;52:861–72.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Ledbetter RN, Costas AMG, Lubner CE, Mulder DW, Tokmina-Lukaszewska M, Artz JH, et al. The electron bifurcating FixABCX protein complex from Azotobacter vinelandii: generation of low-potential reducing equivalents for nitrogenase catalysis. Biochemistry. 2017;56:4177–90.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Okubo T, Piromyou P, Tittabutr P, Teaumroong N, Minamisawa K. Origin and evolution of nitrogen fixation genes on symbiosis islands and plasmid in Bradyrhizobium. Microbes Environ. 2016;31:260–7.

    PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Okubo T, Tsukui T, Maita H, Okamoto S, Oshima K, Fujisawa T, et al. Complete genome sequence of Bradyrhizobium sp. S23321: insights into symbiosis evolution in soil oligotrophs. Microbes Environ. 2012;27:306–15.

    PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Nouwen N, Arrighi JF, Cartieaux F, Chaintreuil C, Gully D, Klopp C, et al. The role of rhizobial (NifV) and plant (FEN1) homocitrate synthases in Aeschynomene/photosynthetic Bradyrhizobium symbiosis. Sci Rep. 2017;7:1–10.

    CAS  Article  Google Scholar 

  46. 46.

    Hakoyama T, Niimi K, Watanabe H, Tabata R, Matsubara J, Sato S, et al. Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation. Nature. 2009;462:514–7.

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Liu C, He Y, Chang Z. Truncated hemoglobin o of Mycobacterium tuberculosis: the oligomeric state change and the interaction with membrane components. Biochem Bioph Res Co. 2004;316:1163–72.

    CAS  Article  Google Scholar 

  48. 48.

    Pathania R, Navani NK, Rajomohan G, Dikshit KL. Mycobacterium tuberculosis Hemoglobin HbO associates with membranes and stimulates cellular respiration of recombinant Escherichia coli. J Biol Chem. 2002;277:15293–302.

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Ascenzi P, De Marinis E, Coletta M, Visca P. H2O2 and NO scavenging by Mycobacterium leprae truncated hemoglobin O. Biochem Bioph Res Co. 2008;373:197–201.

    CAS  Article  Google Scholar 

  50. 50.

    Shimuta T, Nakano K, Yamaguchi Y, Ozaki S, Fujimitsu K, Matsunaga C, et al. Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli. Genes Cells. 2004;9:1151–66.

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Jimenez-Vicente E, Yang ZY, del Campo JSM, Cash VL, Seefeldt LC, Dean DR. The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters. J Biol Chem. 2019;294:6204–13.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Langille MGI, Hsiao WWL, Brinkman FSL. Detecting genomic islands using bioinformatics approaches. Nat Rev Microbiol. 2010;8:372–82.

    Article  CAS  Google Scholar 

  53. 53.

    Hacker J, Kaper JB. Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol. 2000;54:641–79.

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Krupovic M, Gribaldo S, Bamford DH, Forterre P. The evolutionary history of archaeal MCM helicases: a case study of vertical evolution combined with hitchhiking of mobile genetic elements. Mol Biol Evol. 2010;27:2716–32.

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Kaneko T, Nakamura Y, Sato S, Minamisawa K, Uchiumi T, Sasamoto S, et al. Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res. 2002;9:189–97.

    PubMed  Article  Google Scholar 

  56. 56.

    Melnyk RA, Hossain SS, Haney CH. Convergent gain and loss of genomic islands drive lifestyle changes in plant-associated Pseudomonas. ISME J. 2019;13:1575–88.

    PubMed  PubMed Central  Article  Google Scholar 

  57. 57.

    Dupuy P, Sauviac L, Bruand C. Stress-inducible NHEJ in bacteria: function in DNA repair and acquisition of heterologous DNA. Nucleic Acids Res. 2019;47:1335–49.

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Cury J. Evolutionary genomics of conjugative elements and integrons. Paris, France: PhD dissertation, Université Sorbonne Paris Cité; 2017.

  59. 59.

    Gaby JC, Buckley DH. A global census of nitrogenase diversity. Environ Microbiol. 2011;13:1790–9.

    CAS  PubMed  Article  Google Scholar 

  60. 60.

    Han LL, Wang Q, Shen JP, Di HJ, Wang JT, Wei WX, et al. Multiple factors drive the abundance and diversity of the diazotrophic community in typical farmland soils of China. FEMS Microbiol Ecol. 2019;95:fiz113.

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Meng H, Zhou Z, Wu R, Wang Y, Gu J. Diazotrophic microbial community and abundance in acidic subtropical natural and re-vegetated forest soils revealed by high-throughput sequencing of nifH gene. Appl Microbiol Biot. 2019;103:995–1005.

    CAS  Article  Google Scholar 

  62. 62.

    Gano-Cohen KA, Wendlandt CE, Stokes PJ, Blanton MA, Quides KW, Zomorrodian A, et al. Interspecific conflict and the evolution of ineffective rhizobia. Ecol Lett. 2019;22:914–24.

    PubMed  Article  Google Scholar 

  63. 63.

    Helene LCF, Delamuta JRM, Ribeiro RA, Hungria M. Bradyrhizobium mercantei sp nov., a nitrogen-fixing symbiont isolated from nodules of Deguelia costata (syn. Lonchocarpus costatus). Int J Syst Evol Micr. 2017;67:1827–34.

    CAS  Article  Google Scholar 

  64. 64.

    Toniutti MA, Fornasero LV, Albicoro FJ, Martini M, Draghi W, Alvarez F, et al. Nitrogen-fixing rhizobial strains isolated from Desmodium incanum DC in Argentina: Phylogeny, biodiversity and symbiotic ability. Syst Appl Microbiol. 2017;40:297–307.

    CAS  PubMed  Article  Google Scholar 

  65. 65.

    Okazaki S, Tittabutr P, Teulet A, Thouin J, Fardoux J, Chaintreuil C, et al. Rhizobium-legume symbiosis in the absence of Nod factors: two possible scenarios with or without the T3SS. ISME J. 2016;10:64–74.

    CAS  PubMed  Article  Google Scholar 

  66. 66.

    Nelson MB, Martiny AC, Martiny JBH. Global biogeography of microbial nitrogen-cycling traits in soil. Proc Natl Acad Sci USA. 2016;113:8033–40.

    CAS  PubMed  Article  Google Scholar 

  67. 67.

    Remigi P, Zhu J, Young JPW, Masson-Boivin C. Symbiosis within symbiosis: Evolving nitrogen-fixing legume symbionts. Trends Microbiol. 2016;24:63–75.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  68. 68.

    Novick RP, Ram G. The Floating (Pathogenicity) Island: A Genomic Dessert. Trends Genet. 2016;32:114–26.

    CAS  PubMed  Article  Google Scholar 

  69. 69.

    Gal-Mor O, Finlay BB. Pathogenicity islands: a molecular toolbox for bacterial virulence. Cell Microbiol. 2006;8:1707–19.

    CAS  PubMed  Article  Google Scholar 

  70. 70.

    Soumare A, Diedhiou AG, Thuita M, Hafidi M, Ouhdouch Y, Gopalakrishnan S, et al. Exploiting biological nitrogen fixation: a route towards a sustainable agriculture. Plants-Basel. 2020;9:1011.

    CAS  PubMed Central  Article  PubMed  Google Scholar 

  71. 71.

    Santi C, Bogusz D, Franche C. Biological nitrogen fixation in non-legume plants. Ann Bot-Lond. 2013;111:743–67.

    CAS  Article  Google Scholar 

  72. 72.

    Cock PJA, Antao T, Chang JT, Chapman BA, Cox CJ, Dalke A, et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009;25:1422–3.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. 73.

    Goto N, Prins P, Nakao M, Bonnal R, Aerts J, Katayama T. BioRuby: bioinformatics software for the Ruby programming language. Bioinformatics. 2010;26:2617–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. 74.

    Zulkower V, Rosser S. DNA Features Viewer: a sequence annotation formatting and plotting library for Python. Bioinformatics. 2020;36:4350–2.

    CAS  PubMed  Article  Google Scholar 

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We thank Xiaoyuan Feng for help with genome assembly. We appreciate Shuang Wang and Jie Liu for providing soil samples and Jianjun Xu for assisting in sample collection. We are grateful to Xingqin Lin for her helpful suggestions in medium design. We also thank Qin Li, Yan Li, Xiaojun Wang, Xiao Chu, Hao Zhang, Danli Luo and Mei Xie for their helpful discussion. This work was supported by the National Natural Science Foundation of China (92051113), the Hong Kong Research Grants Council Area of Excellence Scheme (AoE/M-403/16), the Direct Grant of CUHK (4053495), and The CUHK Impact Postdoctoral Fellowship Scheme to SW.

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Tao, J., Wang, S., Liao, T. et al. Evolutionary origin and ecological implication of a unique nif island in free-living Bradyrhizobium lineages. ISME J (2021).

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