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Localization of a symbiotic fix region on Rhizobium meliloti pSym megaplasmid more than 200 kilobases from the nod-nif region

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Summary

We have established the HindIII physical map of a cloned 290 kilobase fragment of the Rhizobium meliloti 2011 pSym megaplasmid. The cloned fragment, which contains nodulation genes as well as the nitrogenase structural genes (nifHDK), has been shown to be colinear with the corresponding genomic region. Using transposon mutagenesis we have demonstrated that a region which is located more than 200 kb from the nifHDK operon on pSym is essential for symbiotic nitrogen fixation.

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References

  • 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 R. meliloti. Mol Gen Genet 184:318–325

    Google Scholar 

  • Barth PT, Grinter NJ (1977) Map of plasmid RP4 derived by insertion of transposon C. J Mol Biol 113:455–474

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Google Scholar 

  • Bolivar F, Rodriguez R, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa J, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113

    Google Scholar 

  • Broughton WJ, Heycke N, Heiner Meyer ZA, Pankhurst CE (1984) Plasmid-linked nif and “nod” genes in fast-growing rhizobia that nodulate Glycine max, Psophocarpus tetragonolobus and Vigna unguiculata. Proc Natl Acad Sci USA 81:3093–3097

    Google Scholar 

  • Burkardt B, Burkardt HJ (1984) Visualization and exact molecular weight determination of a Rhizobium meliloti megaplasmid. J Mol Biol 175:213–218

    Google Scholar 

  • Cami B, Kourilsky P (1978) Screening of cloned recombinant DNA in bacteria by in situ colony hybridization. Nucleic Acids Res 5:2381–2390

    Google Scholar 

  • Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 139:1141–1156

    Google Scholar 

  • Clewell DB, Helinski DR (1969) Supercoiled circular DNA-protein complex in E. coli: purification and induced conversion to an open circular DNA form. Proc Natl Acad Sci USA 62:1159–1163

    Google Scholar 

  • Cohen SN, Chang ACY, Hsu L (1972) Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of E. coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114

    Google Scholar 

  • Corbin D, Barran L, Ditta G (1983) Organization and expression of Rhizobium meliloti nitrogen fixation genes. Proc Natl Acad Sci USA 80:3005–3009

    Google Scholar 

  • Currier TC, Nester EW (1976) Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal Biochem 76:431–441

    Google Scholar 

  • Dagert M, Ehrlich SD (1979) Prolonged incubation in calcium chloride improves the competence of E. coli. Gene 6:23–28

    Google Scholar 

  • Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH (1971) Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol 108:1244–1249

    Google Scholar 

  • de Bruijn FJ, Lupski JR (1984) The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments into multicopy plasmids — a review. Gene 27:131–149

    Google Scholar 

  • Downie JA, Ma QS, Knight CD, Hombrecher G, Johnston AWB (1983) Cloning of the symbiotic region of Rhizobium leguminosarum: the nodulation genes are between the nitrogenase genes and a nifA-like gene. EMBO J 2:947–952

    Google Scholar 

  • Eckhardt T (1978) A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid 1:584–588

    Google Scholar 

  • Finan TM, Wood JM, Jordan DC (1981) Succinate transport in Rhizobium leguminosarum. J Bacteriol 148:193–202

    Google Scholar 

  • Finan TM, Wood JM, Jordan DC (1983) Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum. J Bacteriol 154:1403–1413

    Google Scholar 

  • Forrai T, Vincze E, Banfalvi Z, Kiss GB, Randhawa GS, Kondorosi A (1983) Localization of symbiotic mutations in Rhizobium meliloti. J Bacteriol 153:635–643

    Google Scholar 

  • 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

    Google Scholar 

  • Gergen JP, Stern RH, Wensink PC (1979) Filter replicas and permanent collections of recombinant DNA plasmids. Nucleic Acids Res 7:2115–2135

    Google Scholar 

  • Hombrecher G, Brewin NJ, Johnston AWB (1981) Linkage of genes for nitrogenase and nodulation ability on plasmids in R. leguminosarum and R. phaseoli. Mol Gen Genet 182:133–136

    Google Scholar 

  • 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 A. tumefaciens. Nature 291:351–354

    Google Scholar 

  • Hooykaas PJJ, Snijdewint FGM, Schilperoort RA (1982) Identification of the Sym-plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other rhizobia and Agrobacterium tumefaciens. Plasmid 8:73–82

    Google Scholar 

  • Huguet T, Rosenberg C, Casse-Delbart F, de Lajudie P, Jouanin L, Batut J, Boistard P, Julliot JS, Dénarié J (1983) Studies on Rhizobium meliloti plasmids and their role in the control of nodule formation and nitrogen fixation. I. The pSym megaplasmids and the other large plasmids. In: Pühler A (ed) Molecular genetics of the bacteria-plant interaction. Springer-Verlag, Berlin, Heidelberg, New York, pp 35–45

    Google Scholar 

  • Jacoby GA, Jacob AE, Hedges RW (1976) Recombination between plasmids of incompatibility groups P1 and P2. J Bacteriol 127:1278–1285

    Google Scholar 

  • Jouanin L, de Lajudie P, Bazetoux S, Huguet T (1981) DNA sequence homology in Rhizobium meliloti plasmids. Mol Gen Genet 182:189–195

    Google Scholar 

  • Julliot JS, Boistard P (1979) Use of RP4-prime plasmids constructed in vitro to promote a polarized transfer of the chromosome in Escherichia coli and Rhizobium meliloti. Mol Gen Genet 173:289–298

    Google Scholar 

  • Julliot JS, Dusha I, Renalier MH, Terzaghi B, Garnerone AM, Boistard P (1984) An RP4-prime containing a 285 kb fragment of Rhizobium meliloti pSym megaplasmid: Structural characterization and utilization for genetic studies of symbiotic functions controlled by pSym. Mol Gen Genet 193:17–26

    Google Scholar 

  • Kennedy C, Cannon F, Cannon M, Dixon R, Hill S, Jensen J, Kumar S, McLean P, Merrick M, Robson R, Postgate J (1981) Recent advances in the genetics and regulation of nitrogen fixation. In: Gibson AH, Newton WE (eds) Current perspectives in nitrogen fixation. Australian Academy of Sciences, Canberra, pp 146–156

    Google Scholar 

  • Kondorosi E, Banfalvi Z, Kondorosi A (1984) Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: Identification of nodulation genes. Mol Gen Genet 193:445–452

    Google Scholar 

  • Lamb JW, Hombrecher G, Johnston AWB (1982) Plasmid-determined nodulation and nitrogen-fixation abilities in Rhizobium phaseoli. Mol Gen Genet 186:449–452

    Google Scholar 

  • Long SR, Buikema WJ, Ausubel FM (1982) Cloning of R. meliloti nodulation genes by direct complementation of Nod− mutants. Nature 298:485–488

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Matzke AJM, Chilton MD (1981) Site-specific insertion of genes into T-DNA of the Agrobacterium tumor-inducing plasmid: An approach to genetic engineering of higher plant cells. J Mol Appl Genet 1:39–49

    Google Scholar 

  • Mazur BJ, Rice D, Haselkorn R (1980) Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proc Natl Acad Sci USA 77:186–190

    Google Scholar 

  • Meade HR, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122

    Google Scholar 

  • 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 nonlegume Parasponia sp. J Bacteriol 153:527–531

    Google Scholar 

  • Noel KD, Stacey G, Tandon SR, Silver LE, Brill MJ (1982) Rhizobium japonicum mutants defective in symbiotic nitrogen fixation. J Bacteriol 152:485–494

    Google Scholar 

  • Nuti MP, Lepidi AA, Prakash RK, Schilperoort RA, Cannon FC (1979) Evidence for nitrogen fixation (nif) genes on indigenous Rhizobium plasmids. Nature 282:533–535

    Google Scholar 

  • Prakash RK, Schilperoort RA, Nuti MP (1981) Large plasmids of fast-growing rhizobia: Homology studies and location of structural nitrogen fixation (nif) genes. J Bacteriol 145:1129–1136

    Google Scholar 

  • Pühler A, Aguilar MO, Hynes P, Müller P, Klipp W, Priefer U, Simon R, Weber G (1984) Advances in the genetics of free-living and symbiotic nitrogen fixing bacteria. In: Veeger C, Newton WE (eds) Advances in nitrogen fixation research. Nijhoff-Junk/Pudoc, The Hague/Wageningen, pp 609–619

    Google Scholar 

  • Ronson CW, Lyttleton P, Robertson JG (1981) C4-dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc Natl Acad Sci USA 78:4284–4288

    Google Scholar 

  • Rosenberg C, Boistard P, Dénarié J, Casse-Delbart F (1981) Genes controlling early and late functions in symbiosis are located on a megaplasmid in R. meliloti. Mol Gen Genet 184:326–333

    Google Scholar 

  • Rosenberg C, Casse-Delbart F, Dusha I, David M, Boucher C (1982) Megaplasmids in the plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum. J Bacteriol 150:402–406

    Google Scholar 

  • Rosenberg C, Huguet T (1984) The pAtC58 plasmid of Agrobacterium tumefaciens is not essential for tumour induction. Mol Gen Genet 196:533–536

    Google Scholar 

  • Ruvkun GB, Ausubel FM (1980) Interspecies homology of nitrogenase genes. Proc Natl Acad Sci USA 77:191–195

    Google Scholar 

  • Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–89

    Google Scholar 

  • Ruvkun GB, Sundaresan V, Ausubel FM (1982) Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes. Cell 29:551–559

    Google Scholar 

  • Schofield PR, Djordjevic MA, Rolfe BG, Shine J, Watson JM (1983) A molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii. Mol Gen Genet 192:459–465

    Google Scholar 

  • Scott KF, Hughes JE, Gresshoff PM, Beringer JE, Rolfe BG, Shine J (1982) Molecular cloning of Rhizobium trifolii genes involved in symbiotic nitrogen fixation. J Mol Appl Genet 1:315–326

    Google Scholar 

  • Sundaresan V, Jones JDG, Ow DW, Ausubel FM (1983) Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter. Nature 301:728–732

    Google Scholar 

  • Truchet G, Michel M, Dénarié J (1980) Sequential analysis of the organogenesis of lucerne (Medicago sativa) root nodules using symbiotically-defective mutants of R. meliloti. Differentiation 16:163–172

    Google Scholar 

  • Truchet G, Rosenberg C, Vasse J, Julliot JS, Camut S, Dénarié J (1984) Transfer of Rhizobium meliloti pSym genes into A. tumefaciens: Host-specific nodulation by atypical infection. J Bacteriol 157:134–142

    Google Scholar 

  • Verma DPS, Long SR (1983) The molecular biology of Rhizobium-legume symbiosis. Int Rev Cytol [Suppl] 14:211–245

    Google Scholar 

  • Verma DPS, Nadler K (1984) Legume-Rhizobium symbiosis: Host's point of view. In: Verma DPS, Hohn Th (eds) Genes involved in microbe-plant interactions. Springer-Verlag, Berlin, Heidelberg, New York, pp 57–93

    Google Scholar 

  • Vincent JM (1970) A manual for the practical study of root-nodule bacteria. (IBP Handbook 15). Blackwell Scientific Publications, Oxford, p 6

    Google Scholar 

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Author notes

  1. B. Terzaghi

    Present address: Plant Physiology Division, DSIR, Private Bag, Palmerston North, New Zealand

  2. E. Terzaghi

    Present address: Massey University, Palmerston North, New Zealand

Authors and Affiliations

  1. Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, CNRS-INRA, Groupement Scientifique Microbiologie Toulouse, BP 27, F-31326, Castanet-Tolosan Cedex, France

    J. Batut, B. Terzaghi, M. Ghérardi, M. Huguet, E. Terzaghi, A. M. Garnerone, P. Boistard & T. Huguet

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  1. J. Batut
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  2. B. Terzaghi
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  4. M. Huguet
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Communicated by J. Schell

Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal

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Batut, J., Terzaghi, B., Ghérardi, M. et al. Localization of a symbiotic fix region on Rhizobium meliloti pSym megaplasmid more than 200 kilobases from the nod-nif region. Molec Gen Genet 199, 232–239 (1985). https://doi.org/10.1007/BF00330264

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  • DOI: https://doi.org/10.1007/BF00330264

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