Summary
Some primitive pea lines, e.g. cultivar Afghanistan, are resistant to nodulation by most strains of Rhizobium leguminosarum. However the Turkish strain TOM can nodulate cv. Afghanistan in addition to commercial pea varieties, and this extended host range is a property of its symbiotic plasmid, pRL5JI. A gene bank was constructed using DNA from a strain of R. leguminosarum containing pRL5JI. Following transfer to a strain of R. leguminosarum that had been cured of its symbiotic plasmid, two derivatives were isolated that contained cloned nodulation determinants, and were able to nodulate both cv. Afghanistan and a commercial pea variety. In addition, these clones conferred the ability to nodulate peas to a strain of R. phaseoli that had been previously cured of its symbiotic plasmid. One of these clones was subjected to mutagenesis with transposon Tn5, and 11 mutants were identified that were affected in nodulation ability. The sites of Tn5 insertions were mapped using restriction endonucleases and all were found to be within a region of 5 kb. The mutants fell into three classes on the basis of their map positions and their phenotypes on the two different pea lines tested. One class of mutants was affected in gene functions that were common to the nodulation of both pea hosts; a second class was impaired specifically in the nodulation of the commercial pea variety; a third class of mutant failed to confer on a ‘normal’ strain of R. leguminosarum the supplementary ability to nodulate cv. Afghanistan.
Similar content being viewed by others
References
Bagdasarian M, Lurz R, Rückert B, Franklin FCH, Bagdasarian MM, Frey J, Timmis KN (1981) Specific purpose plasmid cloning vectors II broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247
Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198
Beynon JL, Beringer JE, Johnston AWB (1980) Plasmids and host range in Rhizobium leguminosarum and Rhizobium phaseolt. J Gen Microbiol 120:421–429
Brewin NJ, Beringer JE, Johnston AWB (1980) Plasmid mediated transfer of host-range specificity between two strains of Rhizobium leguminosarum. J Gen Microbiol 120:413–420
Brewin NJ, Wood EA, Johnston AWB, Dibb NJ, Hombrecher G (1982) Recombinant nodulation plasmids in R. leguminosarum. J Gen Microbiol 128:1817–1827
Buchanan-Wollaston V, Cannon MC, Beynon JL, Cannon FC (1981) Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature 294:776–778
Dibb NJ, Sorensen GM, Downie JA, Hombrecher G, Brewin NJ (1983) 24K protein — A genetic marker for the symbiotic region of Rhizobium leguminosarum. In: Veeger C, Newton W (eds) Advances in nitrogen fixation research. Nyhoff Publ, Amsterdam, p 677
Ditta G, Stanfield S, Corbin D, Helsinki DR (1980) Broad host range DNA cloning system for gram-negative bacteria; construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351
Downie JA, Hombrecher G, Ma Q-S, Knight CD, Wells B, Johnston AWB (1983a) Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity. Mol Gen Genet 190:359–365
Downie JA, Ma Q-S, Knight CD, Hombrecher G, Johnston AWB (1983b) Cloning of the symbiotic region of Rhizobium leguminosarum: the nodulation genes are between the nitrogenase genes and a nif-A-like gene. EMBO J 2:947–952
Friedman AM, Long SR, Brown SE, Buikema WJ, Ausubel FM (1982) Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296
Grosveld FG, Dal HHM, de Boer E, Flavell RA (1981) Isolation of β-globin-related genes from a human cosmid library. Gene 13:227–237
Hawkes R, Nidag E, Gordon J (1982) A dot immuno-binding assay for monoclonal and other antibodies. Anal Biochem 119:142–147
Holl FB (1975) Host plant control of the inheritance of dinitrogen fixation in the Pisum-Rhizobium symbiosis. Euphytica 24:767–770
Hombrecher G, Brewin NJ, Johnston AWB (1981) Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli. Mol Gen Genet 182:133–136
Johnston AWB, Beynon JL, Buchanan-Wollaston AV, Setchell SM, Hirsch PR, Beringer JE (1978) High frequency transfer of nodulation ability between strains and species of Rhizobium. Nature 276:634–636
Lamb JW, Hombrecher G, Johnston AWB (1982) Plasmid-determined nodulation and nitrogen-fixation abilities in Rhizobium phaseoli. Mol Gen Genet 186:449–452
Lie TA, Hille D, Lambers R, Houwers A (1976) Symbiotic specialisation in pea plants: some environmental effects on nodulation and nitrogen fixation. In: Nutman PS (ed) Symbiotic nitrogen fixation in plants. International Biological Programme, vol 7. Cambridge University Press, p 319–333
Lie TA (1978) Symbiotic specialisation in pea plants: the requirement of specific Rhizobium strains for peas from Afghanistan. Ann Appl Biol 88:462–465
Long SR, Buikema WJ, Ausubel FM (1982) Cloning of Rhizobium meliloti nodulation genes by direct complementation of Nodmutants. Nature 298:485–488
Ma Q-S, Johnston AWB, Hombrecher G, Downie JA (1982) Molecular genetics of mutants of Rhizobium leguminosarum which fail to fix nitrogen. Mol Gen Genet 187:166–171
Merrick M, Filser M, Kennedy C, Dixon R (1978) Polarity of mutations induced by Tn5, Tn7, and Tn10 into the nif gene cluster of Klebsiella pneumoniae. Mol Gen Genet 165:103–111
Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–88
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Winarno R, Lie TA (1979) Competition between Rhizobium strains in nodule formation: interactions between nodulating and nonnodulating strains. Plant and Soil 51:135–142
Young JPW, Johnston AWB, Brewin NJ (1982) A search for peas showing strain specificity for symbiotic Rhizobium leguminosarum. Heredity 48:197–201
Author information
Authors and Affiliations
Additional information
Communicated by Ch. Auerbach
Rights and permissions
About this article
Cite this article
Hombrecher, G., Götz, R., Dibb, N.J. et al. Cloning and mutagenesis of nodulation genes from Rhizobium leguminosarum TOM, a strain with extended host range. Molec. Gen. Genet. 194, 293–298 (1984). https://doi.org/10.1007/BF00383530
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00383530