Abstract
Azospirillum brasilense NifA, which is synthesized under all physiological conditions, exists in an active or inactive from depending on the availability of ammonia. The activity also depends on the presence of PII, as NifA is inactive in a glnB mutant. To investigate further the mechanism that regulates NifA activity, several deletions of the nifA coding sequence covering the amino-terminal domain of NifA were constructed. The ability of these truncated NifA proteins to activate the nifH promoter in the absence or presence of ammonia was assayed in A. brasilense wild-type and mutant strains. Our results suggest that the N-terminal domain is not essential for NifA activity. This domain plays an inhibitory role which prevents NifA activity in the presence of ammonia. The truncated proteins were also able to restore nif gene expression to a glnB mutant, suggesting that PII is required to activate NifA by preventing the inhibitory effect of its N-terminal domain under conditions of nitrogen fixation. Low levels of nitrogenase activity in the presence of ammonia were also observed when the truncated gene was introduced into a strain devoid of the ADP-ribosylation control of nitrogenase. We propose a model for the regulation of NifA activity in A. brasilense.
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- Beynon J. L., Williams M. K., Cannon F. C. Expression and functional analysis of the Rhizobium meliloti nifA gene. EMBO J. 1988 Jan;7(1):7–14. doi: 10.1002/j.1460-2075.1988.tb02777.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Buck M., Khan H., Dixon R. Site-directed mutagenesis of the Klebsiella pneumoniae nifL and nifH promoters and in vivo analysis of promoter activity. Nucleic Acids Res. 1985 Nov 11;13(21):7621–7638. doi: 10.1093/nar/13.21.7621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cannon W., Buck M. Central domain of the positive control protein NifA and its role in transcriptional activation. J Mol Biol. 1992 May 20;225(2):271–286. doi: 10.1016/0022-2836(92)90921-6. [DOI] [PubMed] [Google Scholar]
- Contreras A., Drummond M., Bali A., Blanco G., Garcia E., Bush G., Kennedy C., Merrick M. The product of the nitrogen fixation regulatory gene nfrX of Azotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded by glnD in enteric bacteria. J Bacteriol. 1991 Dec;173(24):7741–7749. doi: 10.1128/jb.173.24.7741-7749.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Da Re S., Bertagnoli S., Fourment J., Reyrat J. M., Kahn D. Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory domain. Nucleic Acids Res. 1994 May 11;22(9):1555–1561. doi: 10.1093/nar/22.9.1555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drummond M. H., Contreras A., Mitchenall L. A. The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of Klebsiella pneumoniae. Mol Microbiol. 1990 Jan;4(1):29–37. doi: 10.1111/j.1365-2958.1990.tb02012.x. [DOI] [PubMed] [Google Scholar]
- Drummond M., Whitty P., Wootton J. Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J. 1986 Feb;5(2):441–447. doi: 10.1002/j.1460-2075.1986.tb04230.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eydmann T., Söderbäck E., Jones T., Hill S., Austin S., Dixon R. Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL. J Bacteriol. 1995 Mar;177(5):1186–1195. doi: 10.1128/jb.177.5.1186-1195.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fernández S., de Lorenzo V., Pérez-Martín J. Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains. Mol Microbiol. 1995 Apr;16(2):205–213. doi: 10.1111/j.1365-2958.1995.tb02293.x. [DOI] [PubMed] [Google Scholar]
- Fischer H. M., Bruderer T., Hennecke H. Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding. Nucleic Acids Res. 1988 Mar 25;16(5):2207–2224. doi: 10.1093/nar/16.5.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischer H. M., Fritsche S., Herzog B., Hennecke H. Critical spacing between two essential cysteine residues in the interdomain linker of the Bradyrhizobium japonicum NifA protein. FEBS Lett. 1989 Sep 11;255(1):167–171. doi: 10.1016/0014-5793(89)81083-x. [DOI] [PubMed] [Google Scholar]
- Fischer H. M. Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev. 1994 Sep;58(3):352–386. doi: 10.1128/mr.58.3.352-386.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frise E., Green A., Drummond M. Chimeric transcriptional activators generated in vivo from VnfA and AnfA of Azotobacter vinelandii: N-terminal domain of AnfA is responsible for dependence on nitrogenase Fe protein. J Bacteriol. 1994 Nov;176(21):6545–6549. doi: 10.1128/jb.176.21.6545-6549.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Furthmayr H., Timpl R. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem. 1971 Jun;41(2):510–516. doi: 10.1016/0003-2697(71)90173-4. [DOI] [PubMed] [Google Scholar]
- Galimand M., Perroud B., Delorme F., Paquelin A., Vieille C., Bozouklian H., Elmerich C. Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7. J Gen Microbiol. 1989 May;135(5):1047–1059. doi: 10.1099/00221287-135-5-1047. [DOI] [PubMed] [Google Scholar]
- Gross R., Aricò B., Rappuoli R. Families of bacterial signal-transducing proteins. Mol Microbiol. 1989 Nov;3(11):1661–1667. doi: 10.1111/j.1365-2958.1989.tb00152.x. [DOI] [PubMed] [Google Scholar]
- Huala E., Ausubel F. M. The central domain of Rhizobium meliloti NifA is sufficient to activate transcription from the R. meliloti nifH promoter. J Bacteriol. 1989 Jun;171(6):3354–3365. doi: 10.1128/jb.171.6.3354-3365.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huala E., Stigter J., Ausubel F. M. The central domain of Rhizobium leguminosarum DctD functions independently to activate transcription. J Bacteriol. 1992 Feb;174(4):1428–1431. doi: 10.1128/jb.174.4.1428-1431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hübner P., Masepohl B., Klipp W., Bickle T. A. nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations. Mol Microbiol. 1993 Oct;10(1):123–132. doi: 10.1111/j.1365-2958.1993.tb00909.x. [DOI] [PubMed] [Google Scholar]
- Iismaa S. E., Watson J. M. The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N-terminal domain found in other NifA proteins. Mol Microbiol. 1989 Jul;3(7):943–955. doi: 10.1111/j.1365-2958.1989.tb00244.x. [DOI] [PubMed] [Google Scholar]
- Kamberov E. S., Atkinson M. R., Ninfa A. J. The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J Biol Chem. 1995 Jul 28;270(30):17797–17807. doi: 10.1074/jbc.270.30.17797. [DOI] [PubMed] [Google Scholar]
- Kay R., McPherson J. Hybrid pUC vectors for addition of new restriction enzyme sites to the ends of DNA fragments. Nucleic Acids Res. 1987 Mar 25;15(6):2778–2778. doi: 10.1093/nar/15.6.2778. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee J. H., Scholl D., Nixon B. T., Hoover T. R. Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a sigma 54-dependent transcriptional activator. J Biol Chem. 1994 Aug 12;269(32):20401–20409. [PubMed] [Google Scholar]
- Liang Y. Y., Arsène F., Elmerich C. Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity. Mol Gen Genet. 1993 Aug;240(2):188–196. doi: 10.1007/BF00277056. [DOI] [PubMed] [Google Scholar]
- Liang Y. Y., Kaminski P. A., Elmerich C. Identification of a nifA-like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia. Mol Microbiol. 1991 Nov;5(11):2735–2744. doi: 10.1111/j.1365-2958.1991.tb01982.x. [DOI] [PubMed] [Google Scholar]
- Liang Y. Y., de Zamaroczy M., Arsène F., Paquelin A., Elmerich C. Regulation of nitrogen fixation in Azospirillum brasilense Sp7: involvement of nifA, glnA and glnB gene products. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):113–119. doi: 10.1111/j.1574-6968.1992.tb14028.x. [DOI] [PubMed] [Google Scholar]
- Ludden P. W. Reversible ADP-ribosylation as a mechanism of enzyme regulation in procaryotes. Mol Cell Biochem. 1994 Sep;138(1-2):123–129. doi: 10.1007/BF00928453. [DOI] [PubMed] [Google Scholar]
- Morett E., Buck M. In vivo studies on the interaction of RNA polymerase-sigma 54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters. The role of NifA in the formation of an open promoter complex. J Mol Biol. 1989 Nov 5;210(1):65–77. doi: 10.1016/0022-2836(89)90291-x. [DOI] [PubMed] [Google Scholar]
- Morett E., Buck M. NifA-dependent in vivo protection demonstrates that the upstream activator sequence of nif promoters is a protein binding site. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9401–9405. doi: 10.1073/pnas.85.24.9401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morett E., Fischer H. M., Hennecke H. Influence of oxygen on DNA binding, positive control, and stability of the Bradyrhizobium japonicum NifA regulatory protein. J Bacteriol. 1991 Jun;173(11):3478–3487. doi: 10.1128/jb.173.11.3478-3487.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. doi: 10.1128/jb.175.19.6067-6074.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Narberhaus F., Lee H. S., Schmitz R. A., He L., Kustu S. The C-terminal domain of NifL is sufficient to inhibit NifA activity. J Bacteriol. 1995 Sep;177(17):5078–5087. doi: 10.1128/jb.177.17.5078-5087.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shingler V. Signal sensing by sigma 54-dependent regulators: derepression as a control mechanism. Mol Microbiol. 1996 Feb;19(3):409–416. doi: 10.1046/j.1365-2958.1996.388920.x. [DOI] [PubMed] [Google Scholar]
- Tarrand J. J., Krieg N. R., Döbereiner J. A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol. 1978 Aug;24(8):967–980. doi: 10.1139/m78-160. [DOI] [PubMed] [Google Scholar]
- Thöny B., Hennecke H. The -24/-12 promoter comes of age. FEMS Microbiol Rev. 1989 Dec;5(4):341–357. doi: 10.1016/0168-6445(89)90028-4. [DOI] [PubMed] [Google Scholar]
- Wootton J. C., Drummond M. H. The Q-linker: a class of interdomain sequences found in bacterial multidomain regulatory proteins. Protein Eng. 1989 May;2(7):535–543. doi: 10.1093/protein/2.7.535. [DOI] [PubMed] [Google Scholar]
- Zhang Y., Burris R. H., Ludden P. W., Roberts G. P. Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium. J Bacteriol. 1996 May;178(10):2948–2953. doi: 10.1128/jb.178.10.2948-2953.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang Y., Burris R. H., Roberts G. P. Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense. J Bacteriol. 1992 May;174(10):3364–3369. doi: 10.1128/jb.174.10.3364-3369.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Zamaroczy M., Paquelin A., Elmerich C. Functional organization of the glnB-glnA cluster of Azospirillum brasilense. J Bacteriol. 1993 May;175(9):2507–2515. doi: 10.1128/jb.175.9.2507-2515.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]