Gene characterization of Bradyrhizobium spp. strains contrasting in biological nitrogen fixation efficiency in soybean

Camila de Medeiros, Gilberto Aguiar Pereira, Janyeli Dorini Silva de Freitas, Olavo Bilac Quaresma de Oliveira Filho, Juliana Silveira do Valle, Giani Andrea Linde, Luzia Doretto Paccola-Meirelles, Nelson Barros Colauto, Fernando Gomes Barcellos


Bacteria from genus Bradyrhizobium can establish symbiosis with soybean and supply the plant nitrogen demands via biological nitrogen fixation (BNF). This study aimed to characterize genes related to BNF efficiency in B. japonicum strains contrasting in BNF efficiency. These gene sequences were previously identified in B. japonicum (strain S370) as probably related to the BNF efficiency in soybean using a DNA subtractive technique. These genes were amplified with primers based on B. japonicum USDA110 genome. The PCR products were digested with restriction endonucleases and the RFLP products were analyzed by horizontal electrophoresis. Among the four genes, only blr3208 and blr4511 amplified for most of the strains. Neither polymorphism of the restriction profile of blr3208 and blr4511 genes nor with endonuclease for PCR-RFLP was observed. The contrasting strains had blr3208 and blr4511 genes sequenced and the multiple alignment analysis of nucleotide sequences showed the presence of preserved internal regions, confirming the analysis with PCR-RFLP. The blr3208 and blr4511 genes are highly conserved among B. japonicum strains, which may be related to adaptive function during the evolutionary process of Bradyrhizobium genus.


Bradyrhizobium; Rhizobacteria; PCR-RFLP; Restriction Polymorphisms.

Full Text:



Barcellos, F. G., Batista, J. S. S., Menna, P., & Hungria, M. (2009). Genetic differences between Bradyrhizobium japonicum variant strains contrasting N2-fixation efficiency revealed by representational difference analysis. Archives of Microbiology, 191(2), 113-122. doi: 10.1007/s00203-008-0432-0

Batista, J. S. S., & Hungria, M. (2012). Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains. Journal of Proteomics, 75(4), 1211-1219. doi: 10.1016/j.jprot.2011.10.032

Bortolan, S., Barcellos, F. G., Marcelino, F. C., & Hungria, M. (2009). Expressão dos genes nod C, node nopP em Bradyrhizobium japonicum estirpe CPAC 15 avaliada por RT-Qpcr. Pesquisa Agropecuária Brasileira, 44(11), 1491-1498. doi: 10.1590/S0100-204X2009001100017

Brencic, A., & Winans, S. C. (2005). Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiology and Molecular Biology Review, 69(1), 155-194. doi: 10.1128/MMBR.69.1.155-194.2005

Caetano-Anollés, G., & Gresshoff, P. M. (1991). Plant genetic control of nodulation. Annual Review of Microbiology, 45, 345-382. doi: 10.1146/annurev.mi.45.100191.002021

Delamuta, J. R. M., Ribeiro, R. A., Ormeño-Orrillo, E., Melo, I. S., Martínez-Romero, E., & Hungria, M., (2013). Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov. International Journal of Systematic and Evolutionary Microbiology, 63(9), 3342-3351. doi: 10.1099/ijs.0.049130-0

Eitinger, T., Rodionov, D. A., Grote, M., & Schneider, E. (2011). Canonical and ECF type ATP binding cassette importers in prokaryotes: diversity in modular organization and cellular functions. FEMS Microbiology Reviews, 35(1), 3-67. doi: 10.1111/j.1574-6976.2010.00230.x

Geddes, B. A., & Oresnik, I. J. (2016). The mechanism of symbiotic nitrogen fixation. In: Hurst C. (ed), The mechanistic benefits of microbial symbionts - advances in environmental microbiology 2 (pp. 69-97). Cham, CH: Springer International Publishing. doi: 10.1007/978-3-319-28068-4_4

Godoy, L. P., Vasconcelos, A. T. R., Chueire, L. M. O., Souza, R. C., Nicolás, M. F., Barcellos, F. G., & Hungria, M. (2008). Genomic panorama of Bradyrhizobium japonicum CPAC 15, a commercial inoculant strain largely established in Brazilian soils and belonging to the same serogroup as USDA 123. Soil Biology and Biochemistry, 40(11), 2743-2753. doi: 10.1016/j.soilbio.2008.07.016

Göttfert, M. (1993). Regulation and function of rhizobial nodulation genes. FEMS Microbiology Reviews, 104(1-2), 39-64. doi: 10.1111/j.1574-6968.1993.tb05863

Guo, M., Huang, Z., & Yang, J. (2017). Is there any crosstalk between the chemotaxis and virulence induction signaling in Agrobacterium tumefaciens? Biotechnology Advances, 35(4), 505-511. doi: 10. 1016/j.biotechadv.2017.03.008

Hu, X., Zhao, J., DeGrado, W. F., & Binns, A. N. (2013). Agrobacterium tumefaciens recognizes its host plant environment using ChvE to bind diverse plant sugars as virulence signals. Proceedings of the National Academy of Sciences of the United States of America, 110(2), 678-683. doi: 10.1073/pnas.121 5033110

Hungria, M., & Mendes, I. C. (2015). Nitrogen fixation with soybean: the perfect symbiosis? In F. J. de Bruijn (Ed.), Biological nitrogen fixation (pp. 1009-1024). New Jersey: John Wiley & Sons, Inc. Retrieved from

Hungria, M., & Stacey, G. (1997). Molecular signals exchanged between host plants and rhizobia: basic aspects and potential application in agriculture. Soil Biology and Biochemistry, 29(1-2), 819-830. doi: 10.1016/S0038-0717(96)00239-8

Hungria, M., Boddey, L. H., Santos, M. A., & Vargas, M. A. T. (1998). Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains. Biology and Fertility of Soils, 27(4), 393-399. doi: 10.1007/s003740050449

Hungria, M., Campo, R. J., & Mendes, I. C. (2007). A importância do processo de fixação biológica do nitrogênio para a cultura da soja: componente essencial para a competitividade do produto brasileiro. Londrina, PR: EMBRAPA Soja.

Jordan, D. C. (1982). Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. International Journal of Systematic Bacteriology, 32(1), 136-139. doi: 10.1099/00207713-32-1-136

Kalendar, R., Lee, D., & Schulman, A. H. (2009). Fast PCR software for PCR primer and probe design and repeat search. Genes, Genomes and Genomics, 3(1), 1-14. Retrieved from http://www.globalscience

Kaminski, P. A., Batut, J., & Boistard, P. (1998). A survey of symbiotic nitrogen fixation by rhizobia. In H. P. Spaink, A. Kondorosi, P. J. J. Hooykaas (eds) The Rhizobiaceae (pp. 431-460). The Netherlands: Springer, Dordrecht. doi: 10.1007/978-94-011-5060-6_23

Kaneko, T., Nakamura, Y., Sato, S., Minamisawa, K., Uchiumi, T., Sasamoto, S.,… Tabata, S. (2002). Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA 110. DNA Research, 9(6), 189-197. doi: 10.1093/dnares/9.6.189

Kemner, J. M., Liang, X., & Nester, E. W. (1997). The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-Type sugar transport operon. Journal of Bacteriology, 179(7), 2452-2458. doi: 10.1128/jb.179.7.2452-2458.1997

Kijne, J. W. (1992). The Rhizobium infection process. In G. Stacey, R.H. Burris, & H. J. Evans (Eds.), Biological nitrogen fixation (pp. 349-398). New York, NY: Chapman & Hall.

Lisitsyn, N., Lisitsyn, N., & Wigler, M. (1993). Cloning the differences between two complex genomes. Science, 259(5097), 946-951. doi: 10.1126/science.8438152

Liu, C. W., & Murray, J. D. (2016). The role of flavonoids in nodulation host-range specificity: an update. Plants, 5(3), 33. doi: 10.3390/plants5030033

Mahmud, K., Makaju, S., Ibrahim, R., & Missaoui, A. (2020). Current progress in nitrogen fixing plants and microbiome research. Plants, 9(1), 97. doi: 10.3390/plants9010097

Nester, E. W. (2015) Agrobacterium: nature’s genetic engineer. Frontiers in Plant Science, 5, 730. doi: 10.3389/fpls.2014.00730

Orrell, T., & Nicolson, D. (2020). ITIS: The integrated taxonomic information system. In Y. Roskov, G. Ower, T. Orrell, D. Nicolson, N. Bailly, P. M. Kirk, T. Bourgoin; R. E. DeWalt, W. Decock, E. van Nieukerken, & L. Penev (Eds.), Species 2000 & ITIS catalogue of life, 2020-08-01 Beta Naturalis, Leiden, The Netherlands. Recovered from

Passaglia, L. M. P. (2017). Bradyrhizobium elkanii nod regulon: insights through genomic analysis. Genetics and Molecular Biology, 40(3), 703-716. doi: 10.1590/1678-4685-gmb-2016-0228

Perin, J. G., Bertéli, M. B. D., Valle, J. S. do, Linde, G. A., Paccola-Meirelles, L. D., Colauto, N. B., & Barcellos, F. G. (2018). Characterization of aapP and nopP genes related to the biological nitrogen fixation efficiency with soybean in contrasting strains of Bradyrhizobium japonicum. Genetics Molecular Research 17(1), gmr16039867. doi: 10.4238/gmr16039867

Reis, F. B. dos, Jr., Mendes, I. C., Reis, V. M., & Hungria, M. (2011). Fixação biológica de nitrogênio uma revolução na agricultura. In F. G. Faleiro, S. R. M. de Andrade, & F. B. dos Reis Jr. (Eds.), Biotecnologia: estado da arte e aplicações na agropecuária (pp. 247-281). Planaltina, DF: EMBRAPA Cerrado.

Ribeiro, R. A., Barcellos, F. G., Thompson, F. L., & Hungria, M. (2009). Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common beans (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity. Research in Microbiology, 160(4), 297-306. doi: 10.1016/j.resmic.2009.03.009

Santos, M. A., Nicolás, M. F., & Hungria, M. (2006). Identificação de QTL associados à simbiose entre Bradyrhizobium japonicum, B. elkanii e soja. Pesquisa Agropecuária Brasileira, 41(1), 67-75. doi: 10. 1590/S0100-204X2006000100010


Semina: Ciênc. Agrár.
Londrina - PR
E-ISSN 1679-0359
DOI: 10.5433/1679-0359
Este obra está licenciado com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional