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Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: Isolation of two gene regions essential for nodulation

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Summary

Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod-) or nitrogen fixation (Fix-). Seven Nod- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod- mutants and 14 Fix- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His- Nod- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod- mutants and one His-Nod- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.

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Author notes

  1. L. M. Hodgson

    Present address: Commonwealth Serum Laboratories, Molecular Biology R & D, 45, Poplar Rd., 3052, Parkville, Australia

  2. Richard Haugland

    Present address: U.S. Environmental Protection Agency, 26 West Street Clair Street, 45268, Cincinnati, OH, USA

  3. Zsofia Banfalvi

    Present address: Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, P.O. Box 521, H-6701, Szeged, Hungary

Authors and Affiliations

  1. Department of Microbiology and Graduate Program of Ecology, University of Tennessee, 37996, Knoxville, TN, USA

    Jae-Seong So, L. M. Hodgson, Maureen Leavitt, Zsofia Banfalvi, Anthony J. Nieuwkoop & Gary Stacey

  2. Syracuse Research Labotatory, Allied Corporation, 13209, Solvay, NY, USA

    Richard Haugland

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  1. Jae-Seong So
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Communicated by J. Schell

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So, JS., Hodgson, L.M., Haugland, R. et al. Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: Isolation of two gene regions essential for nodulation. Mol Gen Genet 207, 15–23 (1987). https://doi.org/10.1007/BF00331485

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  • DOI: https://doi.org/10.1007/BF00331485

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