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Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism

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Summary

Azorhizobium caulinodans ORS571 nifA regulation is partially mediated by the nitrogen regulatory gene ntrC. However, the residual nifA expression in ntrC mutant strains is still modulated by the cellular nitrogen and oxygen status. A second ntrC-homologous region, linked to ntrC, was identified and characterized by site-directed insertion mutagenesis and DNA sequencing. Tn5 insertions in this region cause pleiotropic defects in nitrogen metabolism and affect free-living as well as symbiotic nitrogen fixation. DNA sequencing and complementation studies revealed the existence of a bicistronic operon (ntrYX). NtrY is likely to represent the transmembrane ‘sensor’ protein element in a two-component regulatory system. NtrX shares a high degree of homology with NtrC proteins of other organisms and probably constitutes the regulator protein element. The regulation of the ntrYX and ntrC loci and the effects of ntrYX, ntrY and ntrX mutations on nifA expression were examined using β-galactosidase gene fusions. NtrY/NtrX were found to modulate nifA expression and ntrYX transcription was shown to be partially under the control of NtrC.

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References

  • Albin R, Weber R, Silverman PM (1986) The Cpx proteins of Escherichia coli K12. Immunologic detection of the chromosomal cpxA gene product. J Biol Chem 261:4698–4705

    Google Scholar 

  • Albright LM, Huala E, Ausubel FM (1989) Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu Rev Genet 23:311–336

    Google Scholar 

  • Beck F, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycinphosphotransferase gene from transposon Tn5. Gene 19:327–336

    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 

  • Brown NL, Misra TK, Winnie JN, Schmidt A, Seiff M, Silver S (1986) The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system. Mol Gen Genet 202:143–151

    Google Scholar 

  • de Bruijn FJ (1987) Tn5 mutagenesis to map genes. Meth Enzymol 154:175–196

    Google Scholar 

  • de Bruijn FJ (1989) The unusual symbiosis between the diazotrophic stem-nodulating bacterium Azorhizobium caulinodans ORS571 and its host, the tropical legume Sesbania rostrata. In: Nester E, Kosuge T (eds) Plant-microbe interactions vol 3. McGraw-Hill, New York, pp 457–493

    Google Scholar 

  • de Bruijn FJ, Downie JA (1991) Biochemical and molecular studies of symbiotic nitrogen fixation. Curr Opin in Biotechnology 2:184–192

    Google Scholar 

  • de Bruijn FJ, Pawlowski K, Ratet P, Hilgert U, Schell J (1987) The unusual symbiosis between the nitrogen-fixing bacterium ORS571 and its host Sesbania rostrata: Regulation of nitrogen fixation and assimilation genes in the free-living versus symbiotic state. In: Verma DPS, Brisson N (eds) Molecular genetics of plant-microbe interactions. Martinus Nijhoff, Dordrecht, pp 266–271

    Google Scholar 

  • de Bruijn FJ, Pawlowski K, Ratet P, Hilgert U, Wong Ch, Meyer zA H, Schell J (1988) Molecular genetics of nitrogen fixation by Azorhizobium caulinodans ORS571, the diazotrophic stem nodulating symbiont of Sesbania rostrata. In: Bothe H, de Bruijn FJ, Newton WE (eds) Nitrogen fixation: Hundred years after. Gustav Fischer, Stuttgart-New York, pp 351–355

    Google Scholar 

  • de Bruijn FJ, Rossbach S, Schneider M, Ratet P, Messmer S, Szeto WW, Ausubel FM, Schell J (1989) Rhizobium meliloti has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation. J Bacteriol 171:1673–1682

    Google Scholar 

  • de Bruijn FJ, Hilgert U, Stigter J, Schneider M, Meyer zA H, Klosse U, Pawlowski K (1990) Regulation of nitrogen fixation and assimilation genes in the free-living versus symbiotic state. In: Gresshoff P, Roth E, Stacey G, Newton W (eds) Nitrogen fixation: achievements and objectives. Chapman and Hall, New York-London, pp 33–44

    Google Scholar 

  • Buck M (1990) Transcriptional activation of nitrogen fixation genes in Klebsiella pneumoniae. In: Gresshoff P, Roth E, Stacey G, Newton W (eds) Nitrogen fixation: achievements and objectives. Chapman and Hall, New-London, pp 451–457

    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 MJ (1975) Fusion of the Escherichia coli lac genes to the ara promoter: A general technique using bacteriophage Mu-1 insertions. Proc Natl Acad Sci USA 72:809–813

    Google Scholar 

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

    Google Scholar 

  • Comeau DE, Ikenaka K, Tsung K, Inouye M (1985) Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ protein. J Bacteriol 164:578–584

    Google Scholar 

  • David M, Daveran M-L, Batut J, Dedieu A, Domergue O, Ghai J, Hertig C, Boistard P, Kahn D (1988) Cascade regulation of nif gene expression in Rhizobium meliloti. Cell 54:671–683

    Google Scholar 

  • Devereux J, Haeberli J, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395

    Google Scholar 

  • Ditta G, Stanfields S, Corbin D, Helinski 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

    Google Scholar 

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

    Google Scholar 

  • Donald RGK, LaPointe J, Ludwig RA (1988) Characterization of the Azorhizobium sesbaniae genomic locus encoding NADPH-glutamate synthase. J Bacteriol 170:1197–1204

    Google Scholar 

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

    Google Scholar 

  • Dreyfus B, Garcia JL, Gillis M (1988) Characterization of Azorhizobium caulinodans gen. nov., sp. nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int J Bacteriol 38:89–98

    Google Scholar 

  • Elmerich C, Dreyfus B, Reysset G, Aubert JP (1982) Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium. EMBO J 5:441–447

    Google Scholar 

  • Fickett JW (1982) Recognition of protein coding regions in DNA sequences. Nucleic Acids Res 10:5303–5318

    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 

  • Gebhardt C, Turner GL, Gibson AH, Dreyfus BL, Bergersen FJ (1984) Nitrogen-fixing growth in continuous culture of a strain of Rhizobium sp. isolated from stem nodules of Sesbania rostrata. J Gen Microbiol 130:843–848

    Google Scholar 

  • Genilloud O, Garrido MC, Moreno F (1984) The transposon Tn5 carries a bleomycin-resistance determinant. Gene 32:225–233

    Google Scholar 

  • Gillis-Gonzales MA, Ditta GS, Helinski DR (1991) A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti. Nature 15:170–172

    Google Scholar 

  • Gribskov M, Burgess RR (1986) Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res 14:6745–6763

    Google Scholar 

  • Gribskov M, Devereux J, Burgess RR (1984) The codonpreference plot: Graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res 12:539–549

    Google Scholar 

  • Gussin GN, Ronson CW, Ausubel FM (1986) Regulation of nitrogen fixation genes. Annu Rev Genet 15:31–49

    Google Scholar 

  • Henikoff S (1984) Unidirectional digestion with exonuclease III creates target breakpoints for DNA sequencing. Gene 28:351–359

    Google Scholar 

  • Hertig C, Li RY, Louarn A-M, Garnerone A-M, David M, Batut J, Kahn D, Boistard P (1989) Rhizobium meliloti regulatory gene fixJ activates transcription of R. meliloti nifA and fixK genes in E. coli. J Bacteriol 171:1736–1738

    Google Scholar 

  • Heusterspreute M, Ha-Thi V, Emery S, Tournis-Gamble S, Kennedy N, Davidson J (1985) Vectors with restriction site banks IV. pJRD184, a 3793-bp plasmid vector having 43 unique cloning sites. Gene 39:299–304

    Google Scholar 

  • Hilgert U, Schell J, de Bruijn FG (1987) Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT−) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus. Mol Gen Genet 210:195–202

    Google Scholar 

  • Hyde DR, Tu C-PD (1985) tnpM: A novel regulatory gene that enhances Tn21 transposition and suppresses cointegrate resolution. Cell 42:629–638

    Google Scholar 

  • Iuchi S, Cameron DC, Lin ECC (1989a) A second global regulator gene arcB mediating repression of enzymes in aerobic pathways of Escherichia coli. J Bacteriol 171:868–873

    Google Scholar 

  • Iuchi S, Furlong D, Lin ECC (1989b) Differentiation of arcA, arcB, and cpxA mutant phenotypes of Escherichia coli by sex pilus formation and enzyme regulation. J Bacteriol 171:2899–2893

    Google Scholar 

  • Kaminski PA, Elmerich C (1991) Involvement of fixLJ in the regulation of nitrogen fixation in Azorhizobium caulinodans. Mol Microbiol 5:665–673

    Google Scholar 

  • Kondorosi A, Svab Z, Kiss GB, Dixon RA (1977) Ammonium assimilation and nitrogen fixation in Rhizobium meliloti. Mol Gen Genet 151:221–226

    Google Scholar 

  • Kranz RG, Haselkorn R (1988) Ammonia-constitutive nitrogen fixation mutants of Rhodobacter capsulatus. Gene 71:65–74

    Google Scholar 

  • Krikos A, Mutoh N, Boyd A, Simon MI (1983) Sensory transducers of E. coli are composed of discrete structural and functional domains. Cell 33:615–622

    Google Scholar 

  • Kustu S, Santero E, Keener J, Popham D, Weiss D (1989) Expression of σ54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev 53:367–376

    Google Scholar 

  • Kyte J, Dootlittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    Google Scholar 

  • Leroux B, Yanofsky MF, Winans SW, Ward JE, Ziegler SF, Nester EW (1987) Characterization of the virA locus of Agrobacterium tumefaciens: A transcriptional regulator and host range determinant. EMBO J 6:849–856

    Google Scholar 

  • Magasanik B (1988) Reversible phosphorylation of an enhancer binding protein regulates the transcription of bacterial nitrogen utilization genes. Trends Biochem Sci 13:475–479

    Google Scholar 

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

    Google Scholar 

  • Martin GB, Chapman KA, Chelm BK (1988) Role of the Bradyrhizobium japonicum ntrC gene product in differential regulation of the glutamine synthetase II gene (glnII). J Bacteriol 170:5452–5459

    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 

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

    Google Scholar 

  • Nees DW, Stein PA, Ludwig RA (1988) The Azorhizobium caulinodans nifA gene: Identification of upstream-activating sequences including a new element, the ‘anaerobox’. Nucleic Acids Res 16:9839–9853

    Google Scholar 

  • Ninfa AJ, Gottlin Ninfa E, Lupas AN, Stock A, Magasanik B, Stock J (1988) Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: Evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc Natl Acad Sci USA 83:5492–5496

    Google Scholar 

  • Nixon BT, Ronson CW, Ausubel FM (1986) Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci USA 83:7850–7855

    Google Scholar 

  • Norel F, Elmerich C (1987) Nucleotide sequence and functional analysis of the two nifH copies of Rhizobium ORS571. J Gen Microbiol 133:1563–1576

    Google Scholar 

  • Pawlowski K, Ratet P, Schell J, de Bruijn FJ (1987) 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 freeliving versus symbiotic state. Mol Gen Genet 206:207–219

    Google Scholar 

  • Ratet P, Schell J, de Bruijn FJ (1988a) MiniMu-lac transposons with broad host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae. Gene 63:41–52

    Google Scholar 

  • Ratet P, Pawlowski K, Meyer zA H, Schell J, de Bruijn FJ (1988b) Regulation of nitrogen fixation (nif) genes of Azorhizobium caulinodans in culture and in planta. J Plant Physiol 132:405–411

    Google Scholar 

  • Ratet P, Pawlowski K, Schell J, de Bruijn FJ (1989) The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status. Mol Microbiol 3:825–838

    Google Scholar 

  • Ronson CW, Nixon BT, Albright LM, Ausubel FM (1987) Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions. J Bacteriol 169:2424–2431

    Google Scholar 

  • Russo AF, Koshland DE (1983) Separation of signal transduction and adaptation functions of the aspartate receptor in bacterial sensing. Science 220:1016–1020

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Sanger F, Coulson AR, Barell BG, Smith AJH, Roe BA (1980) Cloning in a single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161–178

    Google Scholar 

  • Shine J, Dalgarno L (1974) The 3′ terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346

    Google Scholar 

  • Spiro S, Guest JR (1990) Fnr and its role in oxygen-regulated gene expression in Escherichia coli. FEMS Microbiol Rev 75:399–428

    Google Scholar 

  • Szeto WW, Nixon T, Ronson CW, Ausubel FM (1987) Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activating nitrogen fixation genes in free-living and symbiotic cells. J Bacteriol 169:1423–1432

    Google Scholar 

  • Thöny B, Hennecke H (1989) The −24/−12 promoter comes of age. FEMS Microbiol Rev 63:341–358

    Google Scholar 

  • Thöny B, Anthamatten D, Hennecke H (1989) Dual control of the Bradyrhizobium japonicum symbiotic nitrogen fixation regulatory operon fixRnifA: Analysis of cis- and trans-acting elements. J Bacteriol 171:4164–4169

    Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 cloning vectors and host strains: Nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–110

    Google Scholar 

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Authors and Affiliations

  1. Max-Planck-Institut für Züchtungsforschung, Carl-Von-Linné-Weg 10, W-5000, Köln 30, Germany

    K. Pawlowski, U. Klosse & F. J. de Bruijn

  2. MSU-DOE Plant Research Laboratory and Department of Microbiology, Michigan State University, 48824, East Lansing, MI, USA

    F. J. de Bruijn

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  1. K. Pawlowski
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  2. U. Klosse
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  3. F. J. de Bruijn
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Communicated by H. Hennecke

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Pawlowski, K., Klosse, U. & de Bruijn, F.J. Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism. Molec. Gen. Genet. 231, 124–138 (1991). https://doi.org/10.1007/BF00293830

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