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J Bacteriol. Sep 1997; 179(17): 5264–5270.
PMCID: PMC179391

FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum biovar viciae UPM791.


Rhizobium leguminosarum bv. viciae UPM791 contains a second copy of the fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr. This second copy (fnrN2) is located in the symbiotic plasmid, while fnrN1 is in the chromosome. Isolation and sequencing of the fnrN2 gene revealed that the deduced amino acid sequence of FnrN2 is 87.5% identical to the sequence of FnrN1, including a conserved cysteine-rich motif characteristic of Fnr-like proteins. Individual R. leguminosarum fnrN1 and fnrN2 mutants exhibited a Fix+ phenotype and near wild-type levels of nitrogenase and hydrogenase activities in pea (Pisum sativum L.) nodules. In contrast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking both nitrogenase and hydrogenase activities. Unlike the wild-type strain and single fnrN1 or fnrN2 mutants, the fnrN1 fnrN2 double mutant was unable to induce micro-oxic or bacteroid activation of the hypBFCDEX operon, which encodes proteins essential for hydrogenase synthesis. In the search for symbiotic genes that could be controlled by FnrN, a fixNOQP operon, putatively encoding a micro-oxically induced, bacteroid-specific cbb3-type terminal cytochrome oxidase, was isolated from strain UPM791 and partially sequenced. The fixNOQP operon was present in a single copy located in the symbiotic plasmid, and an anaerobox was identified in the fixN promoter region. Consistent with this, a fixNOQP'-lacZ fusion was shown to be highly induced in micro-oxic cells of the wild-type strain. A high level of micro-oxic induction was also observed in single fnrN1 and fnrN2 mutants, but no detectable induction was observed in the fnrN1 fnrN2 double mutant. The lack of expression of fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the observed Fix- phenotype. These data demonstrate that, contrary to the situation in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R. leguminosarum bv. viciae UPM791 in the nodule and suggest that this strain lacks a functional fixK gene.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anthamatten D, Scherb B, Hennecke H. Characterization of a fixLJ-regulated Bradyrhizobium japonicum gene sharing similarity with the Escherichia coli fnr and Rhizobium meliloti fixK genes. J Bacteriol. 1992 Apr;174(7):2111–2120. [PMC free article] [PubMed]
  • Batut J, Boistard P. Oxygen control in Rhizobium. Antonie Van Leeuwenhoek. 1994;66(1-3):129–150. [PubMed]
  • Batut J, Daveran-Mingot ML, David M, Jacobs J, Garnerone AM, Kahn D. fixK, a gene homologous with fnr and crp from Escherichia coli, regulates nitrogen fixation genes both positively and negatively in Rhizobium meliloti. EMBO J. 1989 Apr;8(4):1279–1286. [PMC free article] [PubMed]
  • Castresana J, Lübben M, Saraste M, Higgins DG. Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen. EMBO J. 1994 Jun 1;13(11):2516–2525. [PMC free article] [PubMed]
  • Cherfils J, Gibrat JF, Levin J, Batut J, Kahn D. Model-building of Fnr and FixK DNA-binding domains suggests a basis for specific DNA recognition. J Mol Recognit. 1989 Nov;2(3):114–121. [PubMed]
  • Colonna-Romano S, Arnold W, Schlüter A, Boistard P, Pühler A, Priefer UB. An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK. Mol Gen Genet. 1990 Aug;223(1):138–147. [PubMed]
  • David M, Daveran ML, Batut J, Dedieu A, Domergue O, Ghai J, Hertig C, Boistard P, Kahn D. Cascade regulation of nif gene expression in Rhizobium meliloti. Cell. 1988 Aug 26;54(5):671–683. [PubMed]
  • Dodd IB, Egan JB. Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. Nucleic Acids Res. 1990 Sep 11;18(17):5019–5026. [PMC free article] [PubMed]
  • Eckhardt T. A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid. 1978 Sep;1(4):584–588. [PubMed]
  • Fischer HM. Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev. 1994 Sep;58(3):352–386. [PMC free article] [PubMed]
  • García-Horsman JA, Barquera B, Rumbley J, Ma J, Gennis RB. The superfamily of heme-copper respiratory oxidases. J Bacteriol. 1994 Sep;176(18):5587–5600. [PMC free article] [PubMed]
  • Gilles-Gonzalez MA, Ditta GS, Helinski DR. A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti. Nature. 1991 Mar 14;350(6314):170–172. [PubMed]
  • Green J, Bennett B, Jordan P, Ralph ET, Thomson AJ, Guest JR. Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro. Biochem J. 1996 Jun 15;316(Pt 3):887–892. [PMC free article] [PubMed]
  • Green J, Sharrocks AD, Green B, Geisow M, Guest JR. Properties of FNR proteins substituted at each of the five cysteine residues. Mol Microbiol. 1993 Apr;8(1):61–68. [PubMed]
  • Hernando Y, Palacios JM, Imperial J, Ruiz-Argüeso T. The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene. J Bacteriol. 1995 Oct;177(19):5661–5669. [PMC free article] [PubMed]
  • Kahn D, David M, Domergue O, Daveran ML, Ghai J, Hirsch PR, Batut J. Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation. J Bacteriol. 1989 Feb;171(2):929–939. [PMC free article] [PubMed]
  • Kaminski PA, Desnoues N, Elmerich C. The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Q beta RNA. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4663–4667. [PMC free article] [PubMed]
  • Kaminski PA, Mandon K, Arigoni F, Desnoues N, Elmerich C. Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK-like gene, a positive regulator of nifA. Mol Microbiol. 1991 Aug;5(8):1983–1991. [PubMed]
  • Khoroshilova N, Beinert H, Kiley PJ. Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2499–2503. [PMC free article] [PubMed]
  • Lazazzera BA, Beinert H, Khoroshilova N, Kennedy MC, Kiley PJ. DNA binding and dimerization of the Fe-S-containing FNR protein from Escherichia coli are regulated by oxygen. J Biol Chem. 1996 Feb 2;271(5):2762–2768. [PubMed]
  • Leyva A, Palacios JM, Mozo T, Ruiz-Argüeso T. Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum. J Bacteriol. 1987 Nov;169(11):4929–4934. [PMC free article] [PubMed]
  • Leyva A, Palacios JM, Murillo J, Ruiz-Argüeso T. Genetic organization of the hydrogen uptake (hup) cluster from Rhizobium leguminosarum. J Bacteriol. 1990 Mar;172(3):1647–1655. [PMC free article] [PubMed]
  • Leyva A, Palacios JM, Ruiz-Argüeso T. Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains. Appl Environ Microbiol. 1987 Oct;53(10):2539–2543. [PMC free article] [PubMed]
  • Lutz S, Böhm R, Beier A, Böck A. Characterization of divergent NtrA-dependent promoters in the anaerobically expressed gene cluster coding for hydrogenase 3 components of Escherichia coli. Mol Microbiol. 1990 Jan;4(1):13–20. [PubMed]
  • Mandon K, Kaminski PA, Elmerich C. Functional analysis of the fixNOQP region of Azorhizobium caulinodans. J Bacteriol. 1994 May;176(9):2560–2568. [PMC free article] [PubMed]
  • Melville SB, Gunsalus RP. Mutations in fnr that alter anaerobic regulation of electron transport-associated genes in Escherichia coli. J Biol Chem. 1990 Nov 5;265(31):18733–18736. [PubMed]
  • Morett E, Segovia L. The sigma 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains. J Bacteriol. 1993 Oct;175(19):6067–6074. [PMC free article] [PubMed]
  • Nees DW, Stein PA, Ludwig RA. The Azorhizobium caulinodans nifA gene: identification of upstream-activating sequences including a new element, the 'anaerobox'. Nucleic Acids Res. 1988 Oct 25;16(20):9839–9853. [PMC free article] [PubMed]
  • Palacios JM, Murillo J, Leyva A, Ditta G, Ruiz-Argüeso T. Differential expression of hydrogen uptake (hup) genes in vegetative and symbiotic cells of Rhizobium leguminosarum. Mol Gen Genet. 1990 May;221(3):363–370. [PubMed]
  • Patschkowski T, Schlüter A, Priefer UB. Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue. Mol Microbiol. 1996 Jul;21(2):267–280. [PubMed]
  • Preisig O, Anthamatten D, Hennecke H. Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen-fixing endosymbiosis. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3309–3313. [PMC free article] [PubMed]
  • Preisig O, Zufferey R, Hennecke H. The Bradyrhizobium japonicum fixGHIS genes are required for the formation of the high-affinity cbb3-type cytochrome oxidase. Arch Microbiol. 1996 May;165(5):297–305. [PubMed]
  • Preisig O, Zufferey R, Thöny-Meyer L, Appleby CA, Hennecke H. A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum. J Bacteriol. 1996 Mar;178(6):1532–1538. [PMC free article] [PubMed]
  • Prentki P, Krisch HM. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. [PubMed]
  • Rey L, Fernández D, Brito B, Hernando Y, Palacios JM, Imperial J, Ruiz-Argüeso T. The hydrogenase gene cluster of Rhizobium leguminosarum bv. viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity. Mol Gen Genet. 1996 Sep 13;252(3):237–248. [PubMed]
  • Rey L, Imperial J, Palacios JM, Ruiz-Argüeso T. Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis. J Bacteriol. 1994 Oct;176(19):6066–6073. [PMC free article] [PubMed]
  • Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene. 1994 Jul 22;145(1):69–73. [PubMed]
  • Schlüter A, Patschkowski T, Unden G, Priefer UB. The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen-dependent activation of transcription. Mol Microbiol. 1992 Nov;6(22):3395–3404. [PubMed]
  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. [PubMed]
  • Soupène E, Foussard M, Boistard P, Truchet G, Batut J. Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3759–3763. [PMC free article] [PubMed]
  • Spiro S. The FNR family of transcriptional regulators. Antonie Van Leeuwenhoek. 1994;66(1-3):23–36. [PubMed]
  • Stigter J, Schneider M, de Bruijn FJ. Azorhizobium caulinodans nitrogen fixation (nif/fix) gene regulation: mutagenesis of the nifA -24/-12 promoter element, characterization of a ntrA(rpoN) gene, and derivation of a model. Mol Plant Microbe Interact. 1993 Mar-Apr;6(2):238–252. [PubMed]
  • Thöny B, Anthamatten D, Hennecke H. Dual control of the Bradyrhizobium japonicum symbiotic nitrogen fixation regulatory operon fixR nifA: analysis of cis- and trans-acting elements. J Bacteriol. 1989 Aug;171(8):4162–4169. [PMC free article] [PubMed]
  • Virts EL, Stanfield SW, Helinski DR, Ditta GS. Common regulatory elements control symbiotic and microaerobic induction of nifA in Rhizobium meliloti. Proc Natl Acad Sci U S A. 1988 May;85(9):3062–3065. [PMC free article] [PubMed]

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