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EMBO J. 1982; 1(4): 499–503.
PMCID: PMC553074

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

Abstract

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N2 as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.

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Selected References

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  • Ditta G, Stanfield S, Corbin D, Helinski DR. 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. [PMC free article] [PubMed]
  • Eady RR, Smith BE, Cook KA, Postgate JR. Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins. Biochem J. 1972 Jul;128(3):655–675. [PMC free article] [PubMed]
  • Elmerich C, Houmard J, Sibold L, Manheimer I, Charpin N. Genetic and biochemical analysis of mutants induced by bacteriophage Mu DNA integration into Klebsiella pneumoniae nitrogen fixation genes. Mol Gen Genet. 1978 Oct 4;165(2):181–189. [PubMed]
  • Hohn B, Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. [PubMed]
  • Humphreys GO, Willshaw GA, Anderson ES. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta. 1975 Apr 2;383(4):457–463. [PubMed]
  • Loenen WA, Brammar WJ. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene. 1980 Aug;10(3):249–259. [PubMed]
  • Maier RJ, Brill WJ. Ineffective and non-nodulating mutant strains of Rhizobium japonicum. J Bacteriol. 1976 Aug;127(2):763–769. [PMC free article] [PubMed]
  • Mazur BJ, Rice D, Haselkorn R. Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proc Natl Acad Sci U S A. 1980 Jan;77(1):186–190. [PMC free article] [PubMed]
  • Merrick M, Filser M, Dixon R, Elmerich C, Sibold L, Houmard J. The use of translocatable genetic elements to construct a fine-structure map of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster. J Gen Microbiol. 1980 Apr;117(2):509–520. [PubMed]
  • Prakash RK, Schilperoort RA, Nuti MP. Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes. J Bacteriol. 1981 Mar;145(3):1129–1136. [PMC free article] [PubMed]
  • Riedel GE, Ausubel FM, Cannon FC. Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2866–2870. [PMC free article] [PubMed]
  • Rosenberg C, Boistard P, Dénarié J, Casse-Delbart F. Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet. 1981;184(2):326–333. [PubMed]
  • Ruvkun GB, Ausubel FM. Interspecies homology of nitrogenase genes. Proc Natl Acad Sci U S A. 1980 Jan;77(1):191–195. [PMC free article] [PubMed]
  • Ruvkun GB, Ausubel FM. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. [PubMed]
  • Scott KF, Rolfe BG, Shine J. Biological nitrogen fixation: primary structure of the Klebsiella pneumoniae nifH and nifD genes. J Mol Appl Genet. 1981;1(1):71–81. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Sundaresan V, Ausubel FM. Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae. J Biol Chem. 1981 Mar 25;256(6):2808–2812. [PubMed]
  • Bánfalvi Z, Sakanyan V, Koncz C, Kiss A, Dusha I, Kondorosi A. Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Mol Gen Genet. 1981;184(2):318–325. [PubMed]
  • Benton WD, Davis RW. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. [PubMed]
  • Bergersen FJ, Turner GL, Gibson AH, Dudman WF. Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentrations of dissolved oxygen. Biochim Biophys Acta. 1976 Aug 24;444(1):164–174. [PubMed]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [PMC free article] [PubMed]
  • Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed]
  • Buchanan-Wollaston V, Cannon MC, Cannon FC. The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae. Mol Gen Genet. 1981;184(1):102–106. [PubMed]
  • Cannon FC, Riedel GE, Ausubel FM. Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized. Mol Gen Genet. 1979 Jul 2;174(1):59–66. [PubMed]
  • Cohen SN, Chang AC, Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. [PMC free article] [PubMed]
  • Corbin D, Ditta G, Helinski DR. Clustering of nitrogen fixation (nif) genes in Rhizobium meliloti. J Bacteriol. 1982 Jan;149(1):221–228. [PMC free article] [PubMed]

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