Summary
The expression of a translational Azospirillum brasilense nifH- uidA fusion was studied in A. brasilense and in Rhizobium meliloti strains with mutations in nifA, ntrA and ntrC. Induction of the fusion was observed in the R. meliloti wild-type and NtrC− strains on incubation under microaerobic conditions but not in the NifA− and NtrA− strains, showing the absolute requirement of both σ54 and NifA for activation of the nifH promoter. Histochemical analysis of the root nodules elicited by R. meliloti wild-type showed expression of the fusion in the late symbiotic zone but not in the meristematic and the early symbiotic zones. No induction of the nifH-uidA fusion was observed in the R. meliloti wildtype or NifA− strains incubated aerobically in nitrogen-free medium, indicating that, in contrast to R. meliloti nifH, A. brasilense nifH cannot be activated directly by NtrC. Expression of the nifH gene in A. brasilense only occurs under nitrogen-limiting, microaerobic conditions, suggesting the presence of a nitrogen-dependent control system for nif gene expression.
Similar content being viewed by others
References
Baldani VLD, Alvarez MA de B, Baldani JI, Dobereiner J (1986) Establishment of inoculated Azospirillum spp in the rhizosphere and in roots of field grown wheat and sorghum. Plant and Soil 90:35–46
Better M, Ditta G, Helinski DR (1985) Deletion analysis of Rhizobium meliloti symbiotic promoters. EMBO J 4:2419–2424
Beynon JL, Williams MK, Cannon FC (1988) Expression and functional analysis of the Rhizobium meliloti nifA gene. EMBO J 7:7–14
Buck M, Cannon W (1989) Mutations in the RNA polymerase recognition sequence of the Klebsiella pneumoniae nifH promoter permitting transcriptional activation in the absence of NifA binding to upstream activator sequences. Nucleic Acids Res 17:2597–2612
David M, Daveran ML, 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
de Zamaroczy M, Delorme F, Elmerich C (1989) Regulation of transcription and promoter mapping of the structural genes for nitrogenase (nifHDK) of Azospirillum brasilense Sp7. Mol Gen Genet 220:88–99
Ditta G, Stanfield 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
Ditta G, Virts E, Palomares A, Choong-Hyun K (1987) The nifA gene of Rhizobium meliloti is oxygen regulated. J Bacteriol 169:3217–3223
Dixon R, Eady R, Espin G, Hill S, Iaccarino M, Kahn D, Merrick M (1980) Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature 286:128–132
Drummond M, Clements J, Merrick M, Dixon R (1983) Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae. Nature 301:302–307
Espin G, Alvarez-Morales A, Merrick M (1982) Cloning of the glnA, ntrB and ntrC genes of Klebsiella pneumoniae and studies of their role in regulation of the nitrogen fixation in Klebsiella pneumoniae. Mol Gen Genet 186:518–524
Fari R, Alleta G, Bazzicalupo M, Ricci F, Schipani C, Polsinelli M (1989) Nucleotide sequence of the gene encoding the nitrogenase ion protein (nifH) of Azospirillum brasilense and identification of a region controlling nifH transcription. Mol Gen Genet 220:81–87
Galimand M, Perroud B, Delorme F, Paquelin A, Vieille C, Bozouklian H, Elmerich C (1989) Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7. J Gen Microbiol 135:1047–1059
Gallori E, Bazzicalupo M (1985) Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense. FEMS Microbiol Lett 28:35–38
Hartmann A, Horian FW, Burris RH (1986) Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp. J Bacteriol 165:864–870
Hill S, Kennedy C, Kavanagh E, Goldberg RB, Hanau R (1981) Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in Klebsiella pneumoniae. Nature 290:424–426
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Reports 5:387–405
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
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 354–358
Ow DW, Ausubel FM (1983) Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae. Nature 301:307–313
Peleman J, Boerjan W, Engler G, Seurinck J, Botterman J, Alliotte T, Van Montagu M, Inzé D (1989) Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. The Plant Cell 1:81–93
Quiviger B, Franche C, Lutfalla G, Rice D, Haselkorn R, Elmerich C (1982) Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense. Biochimie 64:495–502
Reitzer LJ, Magasanik B (1986) Transcription of g1nA in E. coli is activator bound to sites far from the promoter. Cell 45:785–792
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
Sharma SB, Signer ER (1990) Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in planta revealed by tranposon Tn5-gusA. Genes Dev 4:344–356
Sundaresan V, Ow DW, Ausubel FM (1983) Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins. Proc Natl Acad Sci USA 80:4030–4034
Szeto WW, Zimmerman JL, Sundaresan V, Ausubel FM (1984) A Rhizobium meliloti symbiotic regulatory gene. Cell 36:1035–1043
Szeto WW, Nixon BT, Ronson CW, Ausubel FM (1987) Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells. J Bacteriol 169:1423–1432
Tarrand JJ, Krieg NR, Döbereiner J (1978) A taxonomic study of the Spirillum lipoferum group, with description of a new genus, Azospirillum gen. nov., and two species Azospirillum lipoferum (Beijerinck) sp. nov. and Azospirillum brasilense sp. nov. Can J Microbiol 24:967–980
Thöny B, Hennecke H (1989) The −24/−12 promoter comes of age. FEMS Microbiol Rev 63:341–358
Vanstockem M, Michiels K, Vanderleyden J, Van Gool A (1987) Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physical analysis of Tn5 and Tn5-mob insertion mutants. Appl Environ Microbiol 53:1387–1405
Vasse J, de Billy F, Camut S, Tnuchet G (1990) Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. J Bacteriol 172:4259–4306
Vincent JM (1970) A manual for the practical study of root-nodule bacteria. International Biological Programme handbook no 15. Blackwell Scientific Publications, Oxford
Author information
Authors and Affiliations
Additional information
Communicated by J. Schell
Rights and permissions
About this article
Cite this article
Broek, A.V., Michiels, J., de Faria, S.M. et al. Transcription of the Azospirillum brasilense nifH gene is positively regulated by NifA and NtrA and is negatively controlled by the cellular nitrogen status. Molec. Gen. Genet. 232, 279–283 (1992). https://doi.org/10.1007/BF00280007
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00280007