Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 May;169(5):2177–2186. doi: 10.1128/jb.169.5.2177-2186.1987

Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

K J Wilson, V Anjaiah, P T Nambiar, F M Ausubel
PMCID: PMC212124  PMID: 3032910

Abstract

Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported.

Full text

PDF
2177

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Appelbaum E. R., McLoughlin T. J., O'Connell M., Chartrain N. Expression of symbiotic genes of Rhizobium japonicum USDA 191 in other rhizobia. J Bacteriol. 1985 Jul;163(1):385–388. doi: 10.1128/jb.163.1.385-388.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atkins C. A., Pate J. S., Shelp B. J. Effects of short-term n(2) deficiency on N metabolism in legume nodules. Plant Physiol. 1984 Nov;76(3):705–710. doi: 10.1104/pp.76.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhuvaneswari T. V., Turgeon B. G., Bauer W. D. Early Events in the Infection of Soybean (Glycine max L. Merr) by Rhizobium japonicum: I. LOCALIZATION OF INFECTIBLE ROOT CELLS. Plant Physiol. 1980 Dec;66(6):1027–1031. doi: 10.1104/pp.66.6.1027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broughton W. J., Dilworth M. J. Control of leghaemoglobin synthesis in snake beans. Biochem J. 1971 Dec;125(4):1075–1080. doi: 10.1042/bj1251075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broughton W. J., Wong C. H., Lewin A., Samrey U., Myint H., Meyer H., Dowling D. N., Simon R. Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes. J Cell Biol. 1986 Apr;102(4):1173–1182. doi: 10.1083/jcb.102.4.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cappel R., Klastersky J. Bacteriologic and clinical evaluation of minocycline, a new tetracycline. Curr Ther Res Clin Exp. 1971 Apr;13(4):227–233. [PubMed] [Google Scholar]
  7. Carlson T. A., Guerinot M. L., Chelm B. K. Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum. J Bacteriol. 1985 May;162(2):698–703. doi: 10.1128/jb.162.2.698-703.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cen Y., Bender G. L., Trinick M. J., Morrison N. A., Scott K. F., Gresshoff P. M., Shine J., Rolfe B. G. Transposon mutagenesis in rhizobia which can nodulate both legumes and the nonlegume parasponia. Appl Environ Microbiol. 1982 Jan;43(1):233–236. doi: 10.1128/aem.43.1.233-236.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Vos G. F., Finan T. M., Signer E. R., Walker G. C. Host-dependent transposon Tn5-mediated streptomycin resistance. J Bacteriol. 1984 Jul;159(1):395–399. doi: 10.1128/jb.159.1.395-399.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dylan T., Ielpi L., Stanfield S., Kashyap L., Douglas C., Yanofsky M., Nester E., Helinski D. R., Ditta G. Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4403–4407. doi: 10.1073/pnas.83.12.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Finan T. M., Hirsch A. M., Leigh J. A., Johansen E., Kuldau G. A., Deegan S., Walker G. C., Signer E. R. Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell. 1985 Apr;40(4):869–877. doi: 10.1016/0092-8674(85)90346-0. [DOI] [PubMed] [Google Scholar]
  14. Hawkes T. R., McLean P. A., Smith B. E. Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor. Biochem J. 1984 Jan 1;217(1):317–321. doi: 10.1042/bj2170317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hirsch A. M., Bang M., Ausubel F. M. Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti. J Bacteriol. 1983 Jul;155(1):367–380. doi: 10.1128/jb.155.1.367-380.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hom S. S., Uratsu S. L., Hoang F. Transposon Tn5-induced mutagenesis of Rhizobium japonicum yielding a wide variety of mutants. J Bacteriol. 1984 Jul;159(1):335–340. doi: 10.1128/jb.159.1.335-340.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kneen B. E., Larue T. A. Congo Red Absorption by Rhizobium leguminosarum. Appl Environ Microbiol. 1983 Jan;45(1):340–342. doi: 10.1128/aem.45.1.340-342.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maier R. J., Brill W. J. Ineffective and non-nodulating mutant strains of Rhizobium japonicum. J Bacteriol. 1976 Aug;127(2):763–769. doi: 10.1128/jb.127.2.763-769.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marvel D. J., Kuldau G., Hirsch A., Richards E., Torrey J. G., Ausubel F. M. Conservation of nodulation genes between Rhizobium meliloti and a slow-growing Rhizobium strain that nodulates a nonlegume host. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5841–5845. doi: 10.1073/pnas.82.17.5841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marvel D. J., Torrey J. G., Ausubel F. M. Rhizobium symbiotic genes required for nodulation of legume and nonlegume hosts. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1319–1323. doi: 10.1073/pnas.84.5.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McLean P. A., Dixon R. A. Requirement of nifV gene for production of wild-type nitrogenase enzyme in Klebsiella pneumoniae. Nature. 1981 Aug 13;292(5824):655–656. doi: 10.1038/292655a0. [DOI] [PubMed] [Google Scholar]
  22. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Noel K. D., Stacey G., Tandon S. R., Silver L. E., Brill W. J. Rhizobium japonicum mutants defective in symbiotic nitrogen fixation. J Bacteriol. 1982 Oct;152(1):485–494. doi: 10.1128/jb.152.1.485-494.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Noti J. D., Dudas B., Szalay A. A. Isolation and characterization of nodulation genes from Bradyrhizobium sp. (Vigna) strain IRc 78. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7379–7383. doi: 10.1073/pnas.82.21.7379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pankhurst C. E. Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species. Can J Microbiol. 1977 Aug;23(8):1026–1033. doi: 10.1139/m77-152. [DOI] [PubMed] [Google Scholar]
  26. Putnoky P., Kiss G. B., Ott I., Kondorosi A. Tn5 carries a streptomycin resistance determinant downstream from the kanamycin resistance gene. Mol Gen Genet. 1983;191(2):288–294. doi: 10.1007/BF00334828. [DOI] [PubMed] [Google Scholar]
  27. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  28. Ruvkun G. B., Ausubel F. M. Interspecies homology of nitrogenase genes. Proc Natl Acad Sci U S A. 1980 Jan;77(1):191–195. doi: 10.1073/pnas.77.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ruvkun G. B., Long S. R., Meade H. M., van den Bos R. C., Ausubel F. M. ISRm1: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes. J Mol Appl Genet. 1982;1(5):405–418. [PubMed] [Google Scholar]
  30. Scott K. F. Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp. (Parasponia). Nucleic Acids Res. 1986 Apr 11;14(7):2905–2919. doi: 10.1093/nar/14.7.2905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scott K. F., Rolfe B. G., Shine J. Nitrogenase structural genes are unlinked in the nonlegume symbiont Parasponia rhizobium. DNA. 1983;2(2):141–148. doi: 10.1089/dna.1983.2.141. [DOI] [PubMed] [Google Scholar]
  32. Selvaraj G., Iyer V. N. Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. J Bacteriol. 1983 Dec;156(3):1292–1300. doi: 10.1128/jb.156.3.1292-1300.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Selvaraj G., Iyer V. N. Transposon Tn5 specifies streptomycin resistance in Rhizobium spp. J Bacteriol. 1984 May;158(2):580–589. doi: 10.1128/jb.158.2.580-589.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sherwood M. T. Improved synthetic medium for the growth of Rhizobium. J Appl Bacteriol. 1970 Dec;33(4):708–713. doi: 10.1111/j.1365-2672.1970.tb02253.x. [DOI] [PubMed] [Google Scholar]
  35. Vandenbosch K. A., Noel K. D., Kaneko Y., Newcomb E. H. Nodule initiation elicited by noninfective mutants of Rhizobium phaseoli. J Bacteriol. 1985 Jun;162(3):950–959. doi: 10.1128/jb.162.3.950-959.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES