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The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.


Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and Lupinus angustifolius. A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild-type (wt) strain and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26-kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidity than the wt strain in free-living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free-living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

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  1. 1.

    Hernandez RE, Gallegos‐Monterrosa R, Coulthurst SJ (2020) Type secretion system effector proteins: effective weapons for bacterial competitiveness. Cell Microbiol.22(9).

  2. 2.

    Monjarás Feria J, Valvano MA (2020) An overview of anti-eukaryotic T6SS effectors. Front Cell Infect Microbiol. 10

  3. 3.

    Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I (2009) Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 10:104.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Silverman JM, Brunet YR, Cascales E, Mougous JD (2012) Structure and regulation of the type VI secretion system. Annu Rev Microbiol 66:453–472.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Mougous JD et al (2006) A Virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science (80) 312:1526–1530.

    CAS  Article  Google Scholar 

  6. 6.

    Pukatzki S, Ma AT, Sturtevant D et al (2006) Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci 103:1528–1533.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Bladergroen MR, Badelt K, Spaink HP (2003) Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol Plant-Microbe Interact 16:53–64.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Srinivasa Rao PS, Yamada Y, Tan YP, Leung KY (2004) Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol Microbiol 53:573–586.

    CAS  Article  Google Scholar 

  9. 9.

    Wang J, Brodmann M, Basler M (2019) Assembly and subcellular localization of bacterial type VI secretion systems. Annu Rev Microbiol 73:621–638.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Dix SR, Owen HJ, Sun R et al (2018) Structural insights into the function of type VI secretion system TssA subunits. Nat Commun 9:4765.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Shalom G, Shaw JG, Thomas MS (2007) In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages. Microbiology 153:2689–2699.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Yang X, Long M, Shen X (2018) Effector–immunity pairs provide the T6SS nanomachine its offensive and defensive capabilities. Molecules 23:1009.

    CAS  Article  PubMed Central  Google Scholar 

  13. 13.

    Yu K-W, Xue P, Fu Y, Yang L (2021) T6SS mediated stress responses for bacterial environmental survival and host adaptation. Int J Mol Sci 22:478.

    CAS  Article  PubMed Central  Google Scholar 

  14. 14.

    Böck D, Medeiros JM, Tsao H-F et al (2017) In situ architecture, function, and evolution of a contractile injection system. Science (80) 357:713–717.

    CAS  Article  Google Scholar 

  15. 15.

    Ryu C-M (2015) Against friend and foe: type 6 effectors in plant-associated bacteria. J Microbiol 53:201–208.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Bernal P, Llamas MA, Filloux A (2018) Type VI secretion systems in plant-associated bacteria. Environ Microbiol 20(1):1–15.

    Article  PubMed  Google Scholar 

  17. 17.

    Stępkowski T, Banasiewicz J, Granada C, Andrews M, Passaglia L (2018) Phylogeny and phylogeography of rhizobial symbionts nodulating legumes of the tribe Genisteae. Genes (Basel) 9:163.

    CAS  Article  Google Scholar 

  18. 18.

    Bourebaba Y, Durán D, Boulila F et al (2016) Diversity of Bradyrhizobium strains nodulating Lupinus micranthus on both sides of the Western Mediterranean: Algeria and Spain. Syst Appl Microbiol 39:266–274.

    Article  PubMed  Google Scholar 

  19. 19.

    Sambrook J, Fritsc E, Maniatis T (1989) Molecular cloning: a laboratory manual. New York, NY: Cold Spring Harbor Laboratory Press MacKenzie SL, Lapp MS, Child JJ (1979) Fatty acid composition of Rhizobium spp. Can J Microbiol 25:68–74.

    Article  Google Scholar 

  20. 20.

    Vincent JM (1972) J. M. Vincent, A manual for the practical study of the root-nodule bacteria (IBP Handbuch No. 15 des International Biology Program, London). XI u. 164 S., 10 Abb., 17 Tab., 7 Taf. Oxford-Edinburgh 1970: Blackwell Scientific Publ., 45 s. Z Allg Mikrobiol 12:440–440.

    Article  Google Scholar 

  21. 21.

    O’Gara F, Shanmugan KT (1976) Regulation of nitrogen fixation by rhizobia: export of fixed nitrogen as NH4. Biochim Biophys Acta 437:313–321

    Article  Google Scholar 

  22. 22.

    Ruiz-Argueso T, Hanus J, Evans HJ (1978) Hydrogen production and uptake by pea nodules as affected by strains of Rhizobium leguminosarum. Arch Microbiol 116:113–118.

    CAS  Article  Google Scholar 

  23. 23.

    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72:157–165.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Hartig SM (2013) Basic image analysis and manipulation in ImageJ. Curr Protoc Mol Biol.102

  26. 26.

    Lin J-S, Pissaridou P, Wu H-H, Tsai M-D, Filloux A, Lai E-M (2018) TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha. J Biol Chem 293:8829–8842.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Salinero-Lanzarote A, Pacheco-Moreno A, Domingo-Serrano L, et al (2019) The type VI secretion system of Rhizobium etli Mim1 has a positive effect in symbiosis. FEMS Microbiol Ecol.95

  28. 28.

    Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Abeykoon AH, Noinaj N, Choi B-E et al (2016) Structural insights into substrate recognition and catalysis in outer membrane protein B (OmpB) by protein-lysine methyltransferases from Rickettsia. J Biol Chem 291:19962–19974.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kaneko T (2002) Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res 9:189–197.

    Article  PubMed  Google Scholar 

  31. 31.

    Lin H-H, Huang H-M, Yu M, Lai E-M, Chien H-L, Liu C-T (2018) Functional exploration of the bacterial type VI secretion system in mutualism: Azorhizobium caulinodans ORS571– Sesbania rostrata as a research model. Mol Plant-Microbe Interact 31:856–867.

    Article  PubMed  Google Scholar 

  32. 32.

    Lin J-S, Ma L-S, Lai E-M (2013) Systematic dissection of the Agrobacterium type VI secretion system reveals machinery and secreted components for subcomplex formation. Roujeinikova A, ed. PLoS One 8:e67647.

  33. 33.

    Santos MS, Nogueira MA, Hungria M (2019) Microbial inoculants: reviewing the past, discussing the present and previewing an outstanding future for the use of beneficial bacteria in agriculture. AMB Express 9:205.

    Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Walker L, Lagunas B, Gifford ML (2020) Determinants of host range specificity in legume-rhizobia symbiosis. Front Microbiol.11

  35. 35.

    Nelson MS, Sadowsky MJ (2015) Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes. Front Plant Sci.6

  36. 36.

    Sugawara M, Epstein B, Badgley BD et al (2013) Comparative genomics of the core and accessory genomes of 48 Sinorhizobium strains comprising five genospecies. Genome Biol 14:R17.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    De Campos SB, Lardi M, Gandolfi A, Eberl L, Pessi G (2017) Mutations in two Paraburkholderia phymatum type VI secretion systems cause reduced fitness in interbacterial competition. Front Microbiol.8

  38. 38.

    Berrabah F, Ratet P, Gourion B (2019) Legume nodules: massive infection in the absence of defense induction. Mol Plant-Microbe Interact 32:35–44.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Ratu STN, Teulet A, Miwa H et al (2021) Rhizobia use a pathogenic-like effector to hijack leguminous nodulation signalling. Sci Rep 11:2034.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Jiménez-Guerrero I, Pérez-Montaño F, Medina C, Ollero FJ, López-Baena FJ (2015) NopC is a rhizobium-specific type 3 secretion system effector secreted by Sinorhizobium (Ensifer) fredii HH103 Mergaert P, ed. PLoS One 10:e0142866.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Songwattana P, Chaintreuil C, Wongdee J et al (2021) Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species. Sci Rep 11:4874.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Xin D-W, Liao S, Xie Z-P et al (2012) Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL) domain effector of Rhizobium sp. strain NGR234 Chang J, ed. PLoS Pathog 8:e1002707.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Yasuda M, Miwa H, Masuda S, Takebayashi Y, Sakakibara H, Okazaki S (2016) Effector-triggered immunity determines host genotype-specific incompatibility in legume–rhizobium symbiosis. Plant Cell Physiol 57:1791–1800.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Jiménez-Guerrero I, Acosta-Jurado S, Medina C et al (2020) The Sinorhizobium fredii HH103 type III secretion system effector NopC blocks nodulation with Lotus japonicus Gifu. Gifford M, ed. J Exp Bot 71:6043–6056.

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Engström P, Burke TP, Tran CJ, Iavarone AT, Welch MD (2021) Lysine methylation shields an intracellular pathogen from ubiquitylation and autophagy. Sci Adv 7:eabg2517.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Sun Q, Huang M, Wei Y (2021) Diversity of the reaction mechanisms of SAM-dependent enzymes. Acta Pharm Sin B 11:632–650.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Durán D, Bernal P, Vazquez-Arias D et al (2021) Pseudomonas fluorescens F113 type VI secretion systems mediate bacterial killing and adaption to the rhizosphere microbiome. Sci Rep 11:5772.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Trunk K, Peltier J, Liu Y-C et al (2018) The type VI secretion system deploys antifungal effectors against microbial competitors. Nat Microbiol 3:920–931.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Chen L, Zou Y, She P, Wu Y (2015) Composition, function, and regulation of T6SS in Pseudomonas aeruginosa. Microbiol Res 172:19–25.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Hug S, Liu Y, Heiniger B, et al (2021) Differential expression of Paraburkholderia phymatum type VI secretion systems (T6SS) suggests a role of T6SS-b in early symbiotic interaction. Front Plant Sci 12

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We thank Carlos González Carneros for their assistance with microscopy work and Rodrigo Gómez Pellicer for technical support. We also thank the reviewers for their comments to substantially improve the manuscript.


This research was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain (RTI2018-094985-B-100). BFSS is supported by a scholarship from CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico-GDE-204842/2018–2. L.T. is supported by scholarships from the National Exceptional Program (PNE), Ministry of Higher Education and Scientific research of Algeria and from the Erasmus + Program project 2019–1-ES01-KA107-063778. EG is supported by the ANR grants “SymEffectors” and “ET-Nod” (ANR-16-CE20-0013 and ANR-20-CE20-0012, respectively).

Author information




LR, FB, and LT designed the study. EG hosted Lilia in his lab to perform the tsb1 mutant. TRA led the first collaborative project Algeria-Spain and provided funding for scientific meetings between groups. JI funded the sequencing and assembly of the A16 strain. JMP leads the Spanish project that has funded most of the work. The experiments were performed by LT. LT, BFSS, and LR analyzed the data. LT and LR wrote the initial draft and all authors contributed to manuscript revision and approved it to be published.

Corresponding author

Correspondence to L. Rey.

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The authors declare no competing interests.


The funders had no role in the design of the study, the collection, analyses, or interpretation of data, writing of the manuscript, or the decision to publish the results.

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In memory of T. Ruiz-Argüeso

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Tighilt, L., Boulila, F., De Sousa, B.F.S. et al. The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.. Microb Ecol (2021).

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  • Type VI secretion
  • Rhizobium–legume symbiosis
  • Bradyrhizobium
  • Lupinus
  • Effector
  • Methyltransferase