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Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

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Summary

A cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.

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References

  • Alvarez-Morales A, Hennecke H (1985) Expression of Rhizobium japonicum nifH and nifDK operons can be activated by the Klebsiella pneumoniae nifA protein but not by the product of ntrC. Mol Gen Genet 199:306–314

    Google Scholar 

  • Ausubel FM (1984) Regulation of nitrogen fixation genes. Cell 37:5–6

    Google Scholar 

  • Ausubel FM, Buikema WJ, Earl CD, Klingensmith JA, Nixon BT, Szeto WW (1985) Organization and regulation of Rhizobium meliloti and Parasponia Bradyrhizobium nitrogen fixation genes. In: Evans HJ, Bottomley PJ, Newton WE (eds) Nitrogen fixation research progress. Martinus Nijhoff, Dordrecht Boston Lancaster, pp 165–171

    Google Scholar 

  • Backman K, Chen YM, Magasanik B (1981) Physical and genetic characterization of the glnA-glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci USA 78:3743–4747

    Google Scholar 

  • Bender RA, Janssen KA, Resnick AD, Blumenberg M, Foor F, Magasanik B (1977) Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol 129:1001–1009

    Google Scholar 

  • Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–189

    Google Scholar 

  • Beringer JE (1978) Transfer of the drug-resistant transposon Tn5 to Rhizobium. Nature 276:633–634

    Google Scholar 

  • Beynon J, Cannon M, Buchanan-Wollaston V, Cannon F (1983) The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell 34:665–671

    Google Scholar 

  • Bolivar F, Rodrigues RL, Greene PJ, Betlach H, Heynecker HL, Boyer HW, Crossa JH, Falkow S (1977) Construction and characterisation of new cloning vehicles. A multipurpose cloning system. Gene 2:95–100

    Google Scholar 

  • Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in E. coli. J Mol Biol 41:459–472

    Google Scholar 

  • Broughton WJ, Dilworth MY (1971) Control of leghaemoglobin synthesis in snake beans. Biochem J 125:1075–1080

    Google Scholar 

  • Broughton WJ, John CK (1979) Rhizobia in tropical legumes. III Experimentation and supply in Malaysia 1927–1976. In: Broughton WJ, John CK, Rajarao JC, Lin B (eds) Soil microbiology and plant nutrition. University of Malaysia Press. Kuala Lumpur, Malaysia, pp 113–136

    Google Scholar 

  • de Bruijn FJ (1987) Tn5 mutagenesis to map genes. Methods Enzymol (in press)

  • de Bruijn FJ, Ausubel fM (1983) The cloning and characterization of the glnF (ntrA) gene of Klebsiella pneumoniae: Role of glnF (ntrA) in the regulation of nitrogen fixation (nif) and other nitrogen assimilation genes. Mol Gen Genet 192:342–353

    Google Scholar 

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

    Google Scholar 

  • de Bruijn FJ, Sundaresan V, Szeto WW, Ow DW, Ausubel FM (1984) Regulation of the nitrogen fixation (nif) genes of Klebsiella pneumoniae and Rhizobium meliloti: role of nitrogen regulation (ntr) genes. In: Veeger C, Newton WE (eds) Advances in nitrogen fixation research. Nijhoff/Junk, The Hague, pp 627–633

    Google Scholar 

  • Buck M, Khan H, Dixon R (1985) Site-directed mutagenesis of the Klebsiella pneumoniae nifL and nifH promoters and in vivo analysis of promoter activity. Nucleic Acids Res 13:7621–7637

    Google Scholar 

  • Buikema WJ, Szeto WW, Lemley PV, Orme-Johnson WH, Ausubel FM (1985) Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae. Nucleic Acids Res 13:4539–4555

    Google Scholar 

  • Casadaban M (1975) Fusion of the E. coli lac genes to the ara promoter: a general technique using bacteriophage Mu-1 insertions. Proc Natl Acad Sci USA 72:4399–4403

    Google Scholar 

  • Casadaban M (1976) Transposition and fusion of the lac genes to selected promoters in E. coli using bacteriophages lambda and Mu. J Mol Biol 104:541–555

    Google Scholar 

  • Castilho B, Olfson P, Casadaban M (1984) Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposon. J Bacteriol 158:488–495

    Google Scholar 

  • Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156

    Google Scholar 

  • Chen YM, Backman K, Magasanik B (1981) Characterization of a gene, glnL, the product of which is involved in the regulation of nitrogen utilization in Escherichia coli. J Bacteriol 150:214–220

    Google Scholar 

  • Ditta G, Stanfield S, Corbin D, Helinski D (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

    Google Scholar 

  • Dixon R (1984a) The genetic complexity of nitrogen fixation. The ninth Fleming lecture. J Gen Microbiol 130:2745–2755

    Google Scholar 

  • Dixon R (1984b) Tandem promoters determine regulation of the Klebsiella pneumoniae glutamine synthetase (glnA) gene. Nucleic Acids Res 12:7811–7830

    Google Scholar 

  • Donald RGK, Ludwig RA (1984) Rhizobium sp. strain ORS571 ammonium assimilation and nitrogen fixation. J Bacteriol 158:1144–1151

    Google Scholar 

  • Donald RGK, Nees, DW, Raymond CK, Loroch AL, Ludwig RA (1986) Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes. J Bacteriol 165:72–81

    Google Scholar 

  • Dreyfus BL, Dommergues YR (1981) Nitrogen-fixing nodules induced by Rhizobium on the stem of the tropical legume Sesbania rostrata. FEMS Microbiol Lett 10:313–317

    Google Scholar 

  • Dreyfus BL, Elmerich C, Dommergues YR (1983) Free-living Rhizobium strain able to grow under N2 as a sole nitrogen source. Appl Environ Microbiol 45:711–713

    Google Scholar 

  • Drummond M, Whitty P, Wooton J (1986) Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J 5:441–447

    Google Scholar 

  • Elmerich C, Dreyfus B, Reysset G, Aubert JP (1982) Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium. EMBO J 1:499–503

    Google Scholar 

  • Fischer H-M, Alvarez-Morales A, Hennecke H (1986) The pleiotropic nature of symbiotic regulatory mutants: Bradyrhizobium japonicum nifA is involved in control of nif gene expression and formation of determinate symbiosis. EMBO J 5:1165–1173

    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 

  • Fuchs RL, Keister DL (1980) Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp. J Bacteriol 144:641–648

    Google Scholar 

  • Hirschmann J, Wong PK, Sei K, Keener J, Kustu S (1985) Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci USA 82:7525–7529

    Google Scholar 

  • Hunt TP, Magasanik B (1985) Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG and glnL. Proc Natl Acad Sci USA 82:8453–8357

    Google Scholar 

  • Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998

    Google Scholar 

  • Jarvis BDW, Gillis M, De Ley J (1986) Intra-and intergeneric similarities between the ribosomal ribonucleic acid cistrons of Rhizobium and Bradyrhizobium species and some related bacteria. Int J Syst Bacteriol 36:129–138

    Google Scholar 

  • Kush A, Elmerich C, Aubert JP (1985) Nitrogenase of Sesbania Rhizobium strain ORS571: purification, properties and ‘switch off’ by ammonia. J Gen Microbiol 131:1765–1777

    Google Scholar 

  • Legocki RP, Yun AC, Szalay AA (1984) Expression of β-galactosidase controlled by a nitrogenase promoter in stem nodules of Aeschynomene scabra. Proc Natl Acad Sci USA 81:5806–5810

    Google Scholar 

  • Ludwig RA (1986) Rhizobium sp. strain ORS571 grows synergistically on N2 and Nicotinate as N sources. J Bacteriol 165:304–307

    Google Scholar 

  • Maas R (1983) An improved colony hybridization method with significantly increased sensitivity for detection of single genes. Plasmid 10:296–298

    Google Scholar 

  • Magasanik B (1982) Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet 16:135–168

    Google Scholar 

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

    Google Scholar 

  • Meade HM, 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 

  • Merrick M (1983) Nitrogen control of the nif regulon in Klebsiella pneumoniae: involvement of the ntrA gene and analogies between ntrC and nifA. EMBO J 2:39–44

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Norel F, Desnoues N, Elmerich C (1985) Characterization of DNA sequences homologous to Klebsiella pneumoniae nifH, D, K and E in the tropical Rhizobium ORS571. Mol Gen Genet 199:352–356

    Google Scholar 

  • Ow DW, Ausubel FM (1983) Regulation of nitrogen metabolism genes by the nifA gene product in Klebsiella pneumoniae. Nature 301:307–313

    Google Scholar 

  • Ow DW, Sundaresan V, Rothstein DM, Brown SE, Ausubel FM (1983) Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the-15 region. Proc Natl Acad Sci USA 80:2524–2528

    Google Scholar 

  • Ratet P and Richaud F (1986) Construction and uses of a new transposable element whose insertion is able to produce gene fusions with the neomycin-phosphotransferase-coding region of Tn903. Gene 42:185–192

    Google Scholar 

  • Riedel GE, Brown SE, Ausubel FM (1983) Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids. J Bacteriol 153:45–46

    Google Scholar 

  • Rossen L, Ma QS, Mudd EA, Johnston AWB, Downie JA (1984) Identification and DnA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum. Nucleic Acids Res 12:7123–7134

    Google Scholar 

  • Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–88

    Google Scholar 

  • Schetgens R, Hontelez J, van den Bos R, van Kammen A (1985) Identification and phenotypical characterization of a cluster of fix genes, including a nif regulatory gene, from Rhizobium leguminosarum PRE. Mol Gen Genet 200:368–374

    Google Scholar 

  • Scott DB, Court CB, Ronson CW, Scott KF, Watson JM, Schofield PR, Shine J (1984) Organization of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid. Arch Microbiol 139:151–157

    Google Scholar 

  • Shapira SK, Chou J, Richaud F, Casadaban MJ (1983) New versatile hybrid protein β-galactosidase gene fusion plasmid vectors. Gene 25:71–82

    Google Scholar 

  • Sundaresan V, Jones JDG, Ow DW, Ausubel FM (1983a) Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter. Nature 301:728–731

    Google Scholar 

  • Sundaresan V, Ow DW, Ausubel FM (1983b) Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins. Proc Natl Acad Sci USA 80:4030–4034

    Google Scholar 

  • Szeto W, Zimmermann L, Sundaresan V, Ausubel FM (1984) A Rhizobium meliloti symbiotic regulatory gene. Cell 36:1035–1043

    Google Scholar 

  • Weber G, Reilander H, Pühler A (1985) Mapping and expression of a regulatory nitrogen fixation gene (fixD) of Rhizobium meliloti. EMBO J 4:2751–2756

    Google Scholar 

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Authors and Affiliations

  1. Max-Planck-Institut für Züchtungsforschung, D-5000, Köln 30, Federal Republic of Germany

    K. Pawlowski, P. Ratet, J. Schell & F. J. de Bruijn

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  1. K. Pawlowski
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  2. P. Ratet
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  3. J. Schell
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  4. F. J. de Bruijn
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Communicated by H. Saedler

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Pawlowski, K., Ratet, P., Schell, J. et al. Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state. Mol Gen Genet 206, 207–219 (1987). https://doi.org/10.1007/BF00333576

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