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Effects of over-expression of the regulatory enzymes DraT and DraG on the ammonium-dependent post-translational regulation of nitrogenase reductase in Azospirillum brasilense

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Abstract

Nitrogen fixation in Azospirillum brasilense is regulated at transcriptional and post-translational levels. Post-translational control occurs through the reversible ADP-ribosylation of dinitrogenase reductase (Fe Protein), mediated by the dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase glycohydrolase (DraG). Although the DraT and DraG activities are regulated in vivo, the molecules responsible for such regulation remain unknown. We have constructed broad-host-range plasmids capable of over-expressing, upon IPTG induction, the regulatory enzymes DraT and DraG as six-histidine-N-terminal fused proteins (His). Both DraT-His and DraG-His are functional in vivo. We have analyzed the effects of DraT-His and DraG-His over-expression on the post-translational modification of Fe Protein. The DraT-His over-expression led to Fe Protein modification in the absence of ammonium addition, while cells over-expressing DraG-His showed only partial ADP-ribosylation of Fe Protein by adding ammonium. These results suggest that both DraT-His and DraG-His lose their regulation upon over-expression, possible by titrating out negative regulators.

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References

  • Bashan Y, Holguin G (1997) Azospirillum—plant relationships: environmental and physiological advances (1990–1996). Can J Microbiol 43:103–121

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Burnette WN (1981) “Western Blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203

    Google Scholar 

  • Burris RH (1991) Nitrogenases. J Biol Chem 266:9339–9342

    Google Scholar 

  • Drepper T, Gross S, Yakunin AF, Hallenbeck PC, Masepohl B, Klipp W (2003) Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus. Microbiology 149:2203–2212

    Google Scholar 

  • Grunwald SK, Lies DP, Roberts GP, Ludden PW (1995) Post-translational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase. J Bacteriol 177:628–635

    Google Scholar 

  • Halbleid CM, Ludden PW (1999) Characterization of the interaction of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum with bacterial membranes. Arch Microbiol 172:51–58

    Google Scholar 

  • Halbleid CM, Zhang Y, Ludden PW (2000) Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein. J Biol Chem 275:3493–3500

    Google Scholar 

  • Harlow E, Lane D (1988) Antibody, a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • HartmannA,Fu H, Burris RH (1986) Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp. J Bacteriol 165:864–870

    Google Scholar 

  • Kim K, Zhang Y, Roberts GP (2004) Characterization of altered regulation variants of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum. FEBS Lett 559:84–88

    Google Scholar 

  • Klassen G, Souza EM, Yates MG, Rigo LU, Inaba J, Pedrosa FO (2001) Control of nitrogenase reactivation by the GlnZ protein in Azospirillum brasilense. J Bacteriol 183:6710–6713

    Google Scholar 

  • Kleiner D, Paul W, Merrick MJ (1988) Construction of multicopy expression vectors for regulated over-production of proteins in Klebsiella pneumoniae and other enteric bacteria. J Gen Microbiol 134:1779–1784

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685

    Google Scholar 

  • Ljungstrom E, Yates MG, Nordlund S (1989) Purification of the activating enzyme for the Fe-Protein of nitrogenase from Azospirillum brasilense. Biochim Biophys Acta 994:210–214

    Google Scholar 

  • Lowery RG, Ludden PW (1988) Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum. J Biol Chem 263:16714–16719

    CAS  PubMed  Google Scholar 

  • Machado HB, Funayama S, Rigo LU, Pedrosa FO (1991) Excretion of ammonium by Azospirillum brasilense mutants resistant to ethylenediamine. Can J Microbiol 37:549–553

    Google Scholar 

  • Martin DE, Reinhold-Hurek B (2002) Distinct roles of PII-like signal transmitter proteins and amtB in regulation of nif gene expression, nitrogenase activity, and posttranslational modification of NifH in Azoarcus sp. strain BH72. J Bacteriol 184:2251–2259

    Google Scholar 

  • Nordlund S, Eriksson U, Baltscheffsky H (1977) Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum. Biochim Biophys Acta 462:187–195

    Google Scholar 

  • Pawlowski A, Riedel KU, Klipp W, Dreiskemper P, Gross S, Bierhoff H, Drepper T, Masepohl B (2003) Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus. J Bacteriol 185:5240–5247

    Google Scholar 

  • Pedrosa FO, Yates MG (1984) Regulation of nitrogen fixation (nif) genes of Azospirillum brasilense by nifA and ntrC (glnG) type genes. FEMS Microbiol Lett 55:95–101

    Google Scholar 

  • Saari LL, Triplett EW, Ludden PW (1984) Purification and properties of the activating enzyme for iron protein of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum. J Biol Chem 259:15502–15508

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1:784–791

    CAS  Google Scholar 

  • Spaink HP, Okker RJH, Wijffelman CA, Pees E, Lugtenberg BJJ (1987) Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRLIJI. Plant Mol Biol 9:27–39

    Google Scholar 

  • Steenhout O, Vanderleyden J (2000) Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 24:487–506

    Google Scholar 

  • Yakunin AF, Hallenbeck PC (2002) AmtB is necessary for NH +4 induced nitrogenase switch-off and ADP-ribosylation in Rhodobacter capsulatus. J Bacteriol 184:4081–4088

    Google Scholar 

  • Zhang Y, Burris RH, Roberts GP (1992) Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense. J Bacteriol 174:3364–3369

    Google Scholar 

  • Zhang Y, Burris RH, Ludden PW, Roberts GP (1993) Post-translational regulation of nitrogenase activity by anaerobiosis and ammonium in Azospirillum brasilense. J Bacteriol 175:6781–6788

    Google Scholar 

  • Zhang Y, Burris RH, Ludden PW, Roberts GP (1997) Regulation of nitrogen fixation in Azospirillum brasilense. FEMS Microbiol Lett 152:195–204

    Google Scholar 

  • Zhang Y, Pohlmann EL, Halbleib CM, Ludden PW, Roberts GP (2001a) Effect of PII and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase activating glycohydrolase regulatory system in Klebsiella pneumoniae. J Bacteriol 183:1610–1620

    Google Scholar 

  • Zhang Y, Pohlmann EL, Ludden PW, Roberts GP (2001b) Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status. J Bacteriol 183:6159–6168

    Google Scholar 

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Acknowledgements

We are grateful to Yaoping Zhang, Gary Roberts, and Paul Ludden for providing the A. brasilense UB2 and UB4 strains. We thank Cândido J.T. Pereira and Antônio L. Andrade for assistance in antibody production. We also thank Valter A. de Baura, Roseli Prado, and Julieta Pie for technical assistance. This work was supported by PRONEX, CNPq, CAPES, Fundação Araucária and Paraná Tecnologia.

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  1. Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, 81531-990, Curitiba, PR, Brazil

    Luciano F. Huergo, Emanuel M. Souza, Maria B. R. Steffens, M. Geoffrey. Yates, Fábio O. Pedrosa & Leda S. Chubatsu

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  1. Luciano F. Huergo
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  2. Emanuel M. Souza
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  3. Maria B. R. Steffens
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  4. M. Geoffrey. Yates
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  5. Fábio O. Pedrosa
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  6. Leda S. Chubatsu
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Correspondence to Leda S. Chubatsu.

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Huergo, L.F., Souza, E.M., Steffens, M.B.R. et al. Effects of over-expression of the regulatory enzymes DraT and DraG on the ammonium-dependent post-translational regulation of nitrogenase reductase in Azospirillum brasilense. Arch Microbiol 183, 209–217 (2005). https://doi.org/10.1007/s00203-005-0763-z

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  • DOI: https://doi.org/10.1007/s00203-005-0763-z

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