1887

Abstract

NifA, the transcriptional activator of nitrogenase () genes, has up to now been described to be regulated in its activity via the sensor NifL only for members of the γ-subgroup of the . This paper reports a functionally similar NifL-like protein outside this group in sp. strain BH72, a diazotrophic grass endophyte belonging to the β-subgroup of the . Its structural genes for nitrogenase () are regulated in response to combined nitrogen and O and expressed endophytically inside rice roots. In order to characterize nitrogen-regulatory genes, an sp. BH72 genomic library was used to select cosmids that complemented a mutation in . Sequence analysis of the 34 kb genomic region complementing showed two ORFs with sequence identities of 44% to NifL and 61% to NifA of . According to Northern blot and reverse transcriptase PCR analysis, the transcript was more abundant at low combined nitrogen and O levels, results which were corroborated by GUS (β-glucuronidase) assays using a transcriptional :: fusion. N fixation was abolished in a NifLA and a NifA mutant, wild-type fixation being restored by . The NifLA mutant also failed to activate :: expression, indicating that NifA is the obligate transcriptional activator for . A mutant was diazotrophic and did not show repression of :: by ammonium or O, suggesting that NifL of sp. strain BH72 has a similar role in inactivating NifA in response to O and combined nitrogen as NifL in bacteria of the γ-.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-10-3203
2002-10-01
2020-09-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/10/1483203a.html?itemId=/content/journal/micro/10.1099/00221287-148-10-3203&mimeType=html&fmt=ahah

References

  1. Altschul S. F, Gish W, Miller W, Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol215:403–410
    [Google Scholar]
  2. Arcondéguy T, Jack R., Merrick M. 2001; PII signal transduction proteins, pivotal players in microbial nitrogen control. Microbiol Mol Biol Rev65:80–105
    [Google Scholar]
  3. Arsène F, Kaminski P. A., Elmerich C. 1996; Modulation of NifA activity by PII in Azospirillum brasilense : evidence for a regulatory role of the NifA N-terminal domain. J Bacteriol178:4830–4838
    [Google Scholar]
  4. Arsène F, Kaminski P. A., Elmerich C. 1999; Control of Azospirillum brasilense NifA activity by PII: effect of replacing Tyr residues of the NifA N-terminal domain on NifA activity. FEMS Microbiol Lett179:339–343
    [Google Scholar]
  5. Austin S, Buck M, Cannon W, Eydmann T., Dixon R. 1994; Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL. J Bacteriol176:3460–3465
    [Google Scholar]
  6. Ausubel F. M, Brent R, Kingston R. E, Moore D. D, Seidman J. G, Smith J. A., Struhl K. (editors) 1987; Current Protocols in Molecular Biology. New York: Wiley;
    [Google Scholar]
  7. Blanco G, Drummond M, Woodley P., Kennedy C. 1993; Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii . Mol Microbiol9:869–879
    [Google Scholar]
  8. Dixon R. 1998; The oxygen-responsive NifL–NifA complex: a novel two-component regulatory system controlling nitrogenase synthesis in γ- Proteobacteria . Arch Microbiol169:371–380
    [Google Scholar]
  9. Dixon R. A, Henderson N. C., Austin S. 1988; DNA supercoiling and aerobic regulation of transcription from the Klebsiella pneumoniae nifLA promoter. Nucleic Acids Res16:9933–9946
    [Google Scholar]
  10. Drummond M, Clements J, Merrick M., Dixon R. 1983; Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae . Nature301:302–307
    [Google Scholar]
  11. Egener T, Hurek T., Reinhold-Hurek B. 1999; Endophytic expression of nif genes of Azoarcus sp. strain BH72 in rice roots. Mol Plant–Microbe Interact12:813–819
    [Google Scholar]
  12. Egener T, Martin D. E, Sarkar A., Reinhold-Hurek B. 2001; Role of a ferrodoxin gene cotranscribed with the nifHDK operon in N2 fixation and nitrogenase ‘switch off’ of Azoarcus sp. strain BH72. J Bacteriol183:3752–3760
    [Google Scholar]
  13. Fischer H. M, Bruderer T., Hennecke H. 1988; 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 Res16:2207–2224
    [Google Scholar]
  14. Govantes F, Molina-Lopez J. A., Santero E. 1996; Mechanism of coordinated synthesis of the antagonistic regulatory proteins NifL and NifA of Klebsiella pneumoniae . J Bacteriol178:6817–6823
    [Google Scholar]
  15. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol166:557–580
    [Google Scholar]
  16. He L, Soupene E, Ninfa A., Kustu S. 1998; Physiological role for the GlnK protein of enteric bacteria: relief of NifL inhibition under nitrogen-limiting conditions. J Bacteriol180:6661–6667
    [Google Scholar]
  17. Hill S, Austin S, Eydmann T, Jones T., Dixon R. 1996; Azotobacter vinelandii NifL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch. Proc Natl Acad Sci USA93:2143–2148
    [Google Scholar]
  18. Hu B, Zhu J, Shen S. C., Yu G. Q. 2000; A promoter region binding protein and DNA gyrase regulate anaerobic transcription of nifLA in Enterobacter cloacae . J Bacteriol182:3920–3923
    [Google Scholar]
  19. Hurek T, Reinhold B, Fendrik I., Niemann E. G. 1987; Root-zone-specific oxygen tolerance of Azospirillum spp. and diazotrophic rods closely associated with Kallar grass. Appl Environ Microbiol53:163–169
    [Google Scholar]
  20. Hurek T, Burggraf S, Woese C. R., Reinhold-Hurek B. 1993; 16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs. Appl Environ Microbiol59:3816–3824
    [Google Scholar]
  21. Hurek T, Reinhold-Hurek B, Turner G. L., Bergersen F. J. 1994a; Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp. strain BH72. J Bacteriol176:4726–4733
    [Google Scholar]
  22. Hurek T, Reinhold-Hurek B, Van Montagu M., Kellenberger E. 1994b; Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J Bacteriol176:1913–1923
    [Google Scholar]
  23. Hurek T, Van Montagu M, Kellenberger E., Reinhold-Hurek B. 1995; Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72. Mol Microbiol18:225–236
    [Google Scholar]
  24. Hurek T, Wagner B., Reinhold-Hurek B. 1997a; Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR-based genomic fingerprints. Appl Environ Microbiol63:4331–4339
    [Google Scholar]
  25. Hurek T, Egener T., Reinhold-Hurek B. 1997b; Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the β-subclass. J Bacteriol179:4172–4178
    [Google Scholar]
  26. Jack R, De Zamaroczy M., Merrick M. 1999; The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae . J Bacteriol181:1156–1162
    [Google Scholar]
  27. Jefferson R. A, Burgess S. M., Hirsh D. 1986; β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA83:8447–8451
    [Google Scholar]
  28. Karg T., Reinhold-Hurek B. 1996; Global changes in protein composition of N2-fixing Azoarcus sp. strain BH72 upon diazosome formation. J Bacteriol178:5748–5754
    [Google Scholar]
  29. Liang Y. Y, Kaminski P. A., Elmerich C. 1991; 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 Microbiol5:2735–2744
    [Google Scholar]
  30. Little R, Reyes-Ramirez F, Zhang Y, van Heeswijk W. C., Dixon R. 2000; Signal transduction to the Azotobacter vinelandii NifL–NifA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein. EMBO J19:6041–6050
    [Google Scholar]
  31. Martin D, Hurek T., Reinhold-Hurek B. 2000; Occurrence of three PII-like signal transmitter proteins in the diazotroph Azoarcus sp. BH72. Mol Microbiol38:276–288
    [Google Scholar]
  32. Merrick M. 1992; Regulation of nitrogen fixation genes in free-living and symbiotic bacteria. In Biological Nitrogen Fixation pp835–876 Edited by Stacey G., Burris R., Evans H.. New York: Chapman & Hall;
    [Google Scholar]
  33. Minchin S. D, Austin S., Dixon R. A. 1988; The role of activator binding sites in transcriptional control of the divergently transcribed nifF and nifLA promoters from Klebsiella pneumoniae . Mol Microbiol2:433–442
    [Google Scholar]
  34. Newton J. W, Wilson P. W., Burris R. H. 1953; Direct demonstration of ammonia as an intermediate in nitrogen fixation by Azotobacter . J Biol Chem204:445–451
    [Google Scholar]
  35. Ninfa A. J., Atkinson M. R. 2000; PII signal transduction proteins. Trends Microbiol8:172–189
    [Google Scholar]
  36. Page W. J., Sadoff H. L. 1976; Physiological factors affecting transformation of Azotobacter vinelandii . J Bacteriol125:1080–1087
    [Google Scholar]
  37. Parkinson J. S., Kofoid E. C. 1992; Communication modules in bacterial signaling modules. Annu Rev Genet26:71–112
    [Google Scholar]
  38. Prentki P., Krisch H. M. 1984; In vitro insertional mutagenesis with a selectable DNA fragment. Gene29:303–314
    [Google Scholar]
  39. Reinhold B, Hurek T., Fendrik I. 1985; Strain-specific chemotaxis of Azospirillum spp. J Bacteriol162:190–195
    [Google Scholar]
  40. Reinhold B, Hurek T, Niemann E.-G., Fendrik I. 1986; Close association of Azospirillum and diazotrophic rods with different root zones of Kallar grass. Appl Environ Microbiol52:520–526
    [Google Scholar]
  41. Reinhold-Hurek B, Hurek T, Claeyssens M., Van M. M. 1993a; Cloning, expression in Escherichia coli , and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph. J Bacteriol175:7056–7065
    [Google Scholar]
  42. Reinhold-Hurek B, Hurek T, Gillis M, Hoste B, Vancanneyt M, Kersters K., De Ley J. 1993b; Azoarcus gen. nov., nitrogen-fixing proteobacteria associated with roots of Kallar grass ( Leptochloa fusca (L.) Kunth.) and description of two species Azoarcus indigens sp.nov. and Azoarcus communis sp. nov. Int J Syst Bacteriol43:574–584
    [Google Scholar]
  43. Schultz J, Milpetz F, Bork P., Ponting C. P. 1998; SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA95:5857–5864
    [Google Scholar]
  44. Shah V. K, Davis I. C, Gordon J. K, Orme-Johnson W. H., Brill W. J. 1973; Nitrogenaseless mutants of Azotobacter vinelandii : activities, cross-reactions and EPR spectra. Biochim Biophys Acta292:246–255
    [Google Scholar]
  45. Siddavattam D, Steibl H.-D, Kreutzer R., Klingmüller W. 1995; Regulation of nif gene expression in Enterobacter agglomerans : nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription. Mol Gen Genet249:629–636
    [Google Scholar]
  46. Sonnhammer E. L, Eddy S. R., Durbin R. 1997; Pfam: a comprehensive database of protein domain families based on seed alignments. Proteins28:405–420
    [Google Scholar]
  47. Souza E. M, Funayama S, Rigo L. U, Yates M. G., Pedrosa F. O. 1991; Sequence and structural organization of a nifA -like gene and part of a nifB -like gene of Herbaspirillum seropedicae strain Z78. J Gen Microbiol137:1511–1522
    [Google Scholar]
  48. Souza E. M, Pedrosa F. O, Drummond M, Rigo L. U., Yates M. G. 1999; Control of Herbaspirillum seropedicae NifA activity by ammonium ions and oxygen. J Bacteriol181:681–684
    [Google Scholar]
  49. Souza E. M, Pedrosa F. O, Rigo L. U, Machado H. B., Yates M. G. 2000; Expression of the nifA gene of Herbaspirillum seropedicae : role of the NtrC and NifA binding sites and of the −24/−12 promoter element. Microbiology146:1407–1418
    [Google Scholar]
  50. Staskawicz B, Dahlbeck D, Keen N., Napoli C. 1987; Molecular characterisation of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea . J Bacteriol169:5789–5794
    [Google Scholar]
  51. Woodley P., Drummond M. 1994; Redundancy of the conserved His residue in Azotobacter vinelandii NifL, a histidine autokinase homologue which regulates transcription of nitrogen fixation genes. Mol Microbiol13:619–626
    [Google Scholar]
  52. Yanisch-Perron C, Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene33:103–119
    [Google Scholar]
  53. Zhulin I. B, Taylor B. L., Dixon R. 1997; PAS domain S-boxes in archaea, bacteria and sensors for oxygen and redox. Trends Biochem Sci22:331–333
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-10-3203
Loading
/content/journal/micro/10.1099/00221287-148-10-3203
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error