Summary
A molecular map was constructed linking the nitrogenase structural genes (nif) and nodulation genes (nod) in the white clover symbiont, Rhizobium trifolii. In R. trifolii strain ANU843 these two genetic regions are located some 16 kilobases (kb) apart on the 180 kb symbiotic (Sym) plasmid. The molecular linkage of nod and nif genetic regions was established by hybridization analysis using recombinant plasmids containing overlapping cloned sequences. Nodulation genes were located by means of a Tn5-induced nodulation-defective mutant that failed to induce clover root hair curling (Hac- phenotype). A cloned wild-type DNA fragment was shown to phenotypically correct the Hac- mutation by complementation. The nifHDK genes were cloned by positive hybridization to another R. trifolii nif-specific probe. Location of the nif genes relative to the nod genes was established by analysis of a Sym plasmid deletion derivative.
Similar content being viewed by others
References
Auerswald E-A, Ludwig G, Schaller H (1981) Structural analysis of Tn5. Cold Spring Harbor Sym Quant Biol 45:107–113
Banfalvi Z, Sakanyan V, Koncz C, Kiss A, Dusha I, Kondorosi A (1981) Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of Rhizobium meliloti. Mol Gen Genet 184:318–325
Banfalvi Z, Randhawa GS, Kondorosi E, Kiss A, Kondorosi A (1983) Construction and characterization of R-prime plasmids carrying symbiotic genes of R. meliloti. Mol Gen Genet 189:129–135
Barth PT, Grinter NJ (1977) Map of plasmid RP4 by insertion of transposon C. J Mol Biol 113:455–474
Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198
Bolivar F Rodriguez R, Greene PJ, Betlach M, Heyneker HL, Boyer HW, Crosa J, Falkow S (1977) Construction and characterisation of new cloning vehicles II A multipurpose cloning system. Gene 2:95–113
Bossi L, Ciampi MS (1981) DNA sequences at the sites of three insertions of the transposable element Tn5 in the histidine operon of Salmonella. Mol Gen Genet 183:406–408
Djordjevic MA, Zurkowski W, Rolfe BG (1982) Plasmids and stability of symbiotic properties of Rhizobium trifolii. J Bacteriol 151:560–568
Gresshoff PM, Skotnicki ML, Eadie JF, Rolfe BG (1977) Viability of Rhizobium trifolii bacteroids from clover root nodules. Plant Sci Lett 10:299–304
Grunstein M, Hogness DS (1975) Colony hybridization: a method for the isolation of cloned DNA's that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965
Hirsch PR, Van Montagu M, Johnston AWB, Brewin NJ, Schell J (1980) Physical identification of bacteriocinogenic, nodulation and other plasmids in strains of Rhizobium leguminosarum. J Gen Microbiol 120:403–412
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 Gen 182:133–136
Hooykaas PJJ, Van Brussel AAN, den Dulk-Ras H, van Slogteren GMS, Schilperoort RA (1981) Sym plasmid of Rhizobium trifolii expressed in different rhizobial species and Agrobacterium tumefaciens. Nature 291:351–353
Jorgensen RA, Rothstein SJ, Reznikoff WS (1979) A restriction enzyme cleavage map of Tn5 and location of a region encoding neomyin resistance. Mol Gen Genet 177:65–72
Lamb JW, Hombrecher G, Johnston AWB (1982) Plasmid-determined nodulation and nitrogen-fixaton abilities in Rhizobium phaseoli. Mol Gen Genet 186:449–452
Long SR, Buikema WJ, Ausubel FM (1982) Cloning of Rhizobium meliloti nodulation genes by direct complementation of Nod- mutants. Nature 298:485–488
Messing J, Vieira J (1982) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276
Miller JM (ed) (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Morrison DA (1977) Transformation in Escherichia coli: cryogenic preservation of competent cells. J Bacteriol 132:349–351
Morrison NA, Hau CY, Trinick MJ, Shine J, Rolfe BG (1983) Heat curing of a sym plasmid in a fast-growing Rhizobium sp. that is able to nodulate legumes and the non-legume Parasponia sp. J Bacteriol 153:527–531
Nuti MP, Ledeboer AM, Lepidi AA, Schilperoort RA (1977) Large plasmids in different Rhizobium species. J Gen Microbiol 100:242–248
Nuti MP, Lepidi AA, Prakash RK, Schilperoort RA, Cannon FC (1979) Evidence for nitrogen fixing (nif) genes on indigenous Rhizobium plasmids. Nature 282:533–535
O'Farrell PH, Kutter E, Nakanishi M (1980) A restriction map of the bacteriophage T4 genome. Mol Gen Genet 179:421–435
Rolfe BG, Gresshoff PM, Shine J (1980) Rapid screening for symbiotic mutants of Rhizobium and white clover. Plant Sci Lett 19:277–284
Rolfe BG, Djordjevic MA, Scott KF, Hughes JE, Badenoch-Jones J, Gresshoff PM, Cen Y, Dudman WF, Zurkowski W, Shine J (1981) Analysis of the nodule forming ability of fast-growing Rhizobium strains. In: Gibson AH, Newton WE, (Eds) Current perspectives in nitrogen fixation. Australian Academy of Science, Canberra, pp 142–145
Rosenberg C, Boistard P, Dénarié J, Casse-Delbart F (1981) Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet 184:326–333
Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–88
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Scott DB, Farnden KJF, Robertson JG (1976) Ammonia assimilation in lupin nodules. Nature 263:703–705
Scott KF, Hughes JE, Gresshoff PM, BeringerJE, Rolfe BG, Shine J (1982) Molecular cloning of Rhizobium trifolii genes involved in symbiotic nitrogen fixation. J Mol Appl Genet 1:315–326
Scott KF, Rolfe BG, Shine J (1983) Biological nitrogen fixation: Primary structure of the Rhizobium trifolii iron protein gene. DNA 2:147–153
Shine J, Seeburg PH, Martial JA, Baxter JD, Goodman HM (1977) Construction and analysis of recombinant DNA for human chorionic somatomammotropin. Nature 270:494–499
Soberon X, Covarrubias L, Bolivar F (1980) Construction and characterization of new cloning vehicles IV Deletion derivatives of pBR322 and pBR325. Gene 9:287–305
Southern EM (1975) Detection of pecific sequences among fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Timmis KN, Cabello F, Cohen SN (1974) Utilization of two distinct modes of replication by a hybrid replicon constructed in vitro from separate replicons. Proc Natl Acad Sci USA 71:4556–4560
Vincent JM (1974) Root-nodule symbiosis with Rhizobium. In: Quispel A (ed) The biology of nitrogen fixation. North-Holland Publ Co, Amsterdam, pp 265–341
Vincent JM (1980) Factors controlling the legume Rhizobium symbiosis. In: Newton WE, Orme-Johnson WH (eds) Nitrogen fixation 2. Baltimore University Park Press, pp 103–129
Watson JM, Schmidt L, Willetts NS (1980) Cloning the Tra1 region of RP1. Plasmid 4:175–183
Whitfeld PL, Seeburg PH, Shine J (1982) The human proopiomelanocortin gene: Organization, sequence, and interspersion with repetitive DNA. DNA 1:133–143
Zurkowski W, Lorkiewicz Z (1978) Effective method for the isolation of non-nodulating mutants of Rhizobium trifolii. Genet Res 32:311–314
Author information
Authors and Affiliations
Additional information
Communicated by D. Sherratt
Rights and permissions
About this article
Cite this article
Schofield, P.R., Djordjevic, M.A., Rolfe, B.G. et al. A molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii . Mol Gen Genet 192, 459–465 (1983). https://doi.org/10.1007/BF00392191
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00392191