Skip to main content
Log in

Rhizobium etli cytochrome mutants with derepressed expression of cytochrome terminal oxidases and enhanced symbiotic nitrogen accumulation

  • Original Paper
  • Applied Microbial and Cell Physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

A method to isolate mutants with derepressed expression of cytochrome oxidases and better symbiotic performance is presented. A mutant of Rhizobium etli, CFN030, isolated by its azide-resistant phenotype, was obtained by transposon Tn5 -mob mutagenesis. This mutant has a derepressed expression of cytochrome aa3, higher respiratory activities when cultured microaerobically and an improved symbiotic nitrogen fixation capacity. This phenotype was similar to the previously described mutant CFN037, which was isolated by its increased capacity to oxidize N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) [Soberón M et al. (1990) J Bacteriol 172:1676–1680]. We show here that although both mutants have a similar symbiotic phenotype, they are affected in different genes. Strain CFN030 has the Tn5 inserted in the chromosome while in strain CFN037 the transposon was located in plasmid b. Cytochrome spectral analysis of both mutant strains in the post-exponential phase of growth, showed the expression of an additional terminal oxidase (cbb3) that is not expressed in the wild-type strain.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Appleby CA (1969) Electron transport systems of Rhizobium japonicum. II. Rhizobium haemoproteins, cytochromes and oxidases in free living (cultured) cells. Biochim Biophys Acta 172: 88–105

    Article  CAS  Google Scholar 

  • Barquera B, García-Horsman A, Escamilla EJ (1991) Cytochrome d expression and regulation pattern in free living Rhizobium phaseoli. Arch Microbiol 115: 114–119

    Article  Google Scholar 

  • Brewin NJ, Beringer JE, Johnston AWB (1980) Plasmid-mediated transfer of host-range specificity between two strains of Rhizobium leguminosarum. J Gen Microbiol 120: 413–420

    Google Scholar 

  • Brom S, García de los Santos A, Stepkowsky T, Flores M, Dávila G. Romero D, Palacios R (1992) Different plasmids of Rhizobium leguminosarum bv. phaseoli are required for optimal symbiotic performance. J Bacteriol 174: 5183–5189

    CAS  Google Scholar 

  • Cotter PA, Chepuri V, Gennis RB and Gunsalus RP (1990) Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen. pH, and the fnr gene product. J Bacteriol 172: 6333–6338

    CAS  Google Scholar 

  • DeHollander JA, Stouthamer AH (1980) The electron transport chain of Rhizobium trifolii. Eur J Biochem 111: 473–478

    Article  CAS  Google Scholar 

  • Fortin Y, Phoenix P, Drapeau GR (1990) Mutations conferring resistance to azide in Escherichia coli occur primarily in the secA gene. J Bacteriol 172: 6607–6610

    CAS  Google Scholar 

  • de Gier J-WL, Lübben M, Reijnders WNM, Tipker CA, Slotboom D-J, van Spanning RJM, Stouthamer AH, van der Oost J (1994) The terminal oxidases of Paracoccus denitrificans. Mol Microbiol 13: 183–196

    Article  Google Scholar 

  • Gray KA, Grooms M, Myllykallio H, Moomaw C, Slaughter C, Daldal F 1994 Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center. Biochemistry 33: 3120–3127

    Article  CAS  Google Scholar 

  • Green GN, Kranz RG, Lorence RM, Gennis RB (1984) Identification of subunit I as the cytochrome b558 component of the cytocrome d terminal oxidase complex of Escherichia coli. J Biol Chem 59: 7994–7997

    Google Scholar 

  • Iuchi S, Cameron DC, Lin ECC (1989) A second global regulator gene (arcB) mediating repression of enzymes in aerobic pathways of Escherichia coli. J Bacteriol 171: 868–873

    CAS  Google Scholar 

  • Iuchi S, Chepuri V, Fu H-A, Gennis RB, and Lin ECC (1990) Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd. J Bacteriol 172: 6020–6025

    CAS  Google Scholar 

  • Kahn D, Batut J, Daveran ML, Fourment J (1993) Structure and regulation of the fixNOQP operon from Rhizobium meliloti. In: Palacios R. Mora J, Newton WE (eds) New horizons in nitrogen fixation. Kluwer Academic Publishers, Dordrecht, The Netherlands, p 474

    Google Scholar 

  • Keefe RG, Maier RJ (1993) Purification and characterization of an Cvutilizing cytochrome c oxidase complex from Bradyrhizobium japonicum bacteroid membranes. Biochim Biophys Acta 1183: 91–104

    Article  CAS  Google Scholar 

  • Kretovich WL, Romanov VI, Korolyov AU (1973) Rhizobium leguminosarum cytochromes (Vicia faba). Plant Soil 39: 614–634

    Article  Google Scholar 

  • Mandon K, Kaminski PA, Elmerich C (1994) Functional analysis of the fixNOQP region of Azorhizobium caulinodans. J Bacteriol 176: 2560–2568

    CAS  Google Scholar 

  • Markwell MAK, Haas SM, Vieber ZZ, and Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206–210

    Article  CAS  Google Scholar 

  • Martinez E, Pardo MA, Palacios R, Cevallos MA (1985) Reiteration of nitrogen fixation gene sequences and specificity of Rhizobium inoculation and nitrogen fixation in Phaseolus vulgaris. J Gen Microbiol 13: 1779–1786

    Google Scholar 

  • Noel KD, Sánchez A, Fernández L, Leemans J, Cevallos MA (1984) Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions. J Bacteriol 158: 148–155

    CAS  Google Scholar 

  • O’Brian MR, Maier RJ (1989) Molecular aspects of the energetics of nitrogen fixation in Rhizobium-legume symbiosis. Biochim Biophys Acta 974: 229–246

    Article  Google Scholar 

  • Oliver DB, Cabelli RJ, Dolan M, Jarosik GP (1990) Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-component of the protein export machinery. Proc Natl Acad Sci USA 87: 8227–8231

    Article  CAS  Google Scholar 

  • Poole RK (1983) Bacterial cytochrome oxidases: a structurally and functional diverse group of electron transfer proteins. Biochim Biophys Acta 726: 205–243

    CAS  Google Scholar 

  • Preisig O, Anthamatten D, Hennecke H (1993) Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen fixing endosymbiosis. Proc Natl Acad Sci USA 90: 3309–3313

    Article  CAS  Google Scholar 

  • Quinto C, de la Vega H, Flores M, Fernández L, Ballado R, Soberón G, Palacios R (1982) Reiteration of nitrogen fixation gene sequences in Rhizobium phaseoli. Nature 224: 724–726

    Article  Google Scholar 

  • Schlüter A, Patschkowski T, Weidner S, Unden G, Hynes MF, Priefer UB (1993) Functional and regulatory characteristics of FnrN, an oxygen-responsive transcriptional activator in Rhizobium leguminosarum bv. viciae. In: Palacios R, Mora J, Newton WE (eds) New horizons in nitrogen fixation. Kluwer Academic Publishers. Dordrecht, The Netherlands, p 493

    Google Scholar 

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

    Article  CAS  Google Scholar 

  • Soberón M, Williams HD, Poole RK, Escamilla E (1989) Isolation of a Rhizobium phaseoli cytochrome mutant with enhanced respiration and symbiotic nitrogen fixation. J Bacteriol 171: 465–472

    Google Scholar 

  • Soberón M, Membrillo-Hernández J, Aguilar GR, Sánchez F (1990) Isolation of Rhizobium phaseoli Tn5-induced mutants with altered expression of cytochrome terminal oxidases o and aa3. J Bacteriol 172: 1676–1680

    Google Scholar 

  • Stam H, Van Verseveld WH, de Vries W, Stouthamer AH (1984) Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited cultures of Rhizobium ORS 571. Arch Microbiol 139: 53–60

    Article  CAS  Google Scholar 

  • Wacek T, Brill WJ (1976) Simple rapid assay for screening nitrogen fixing ability in soybean. Crop Sci 16: 519–522

    CAS  Google Scholar 

  • Witty JF, Minchin FR (1990) In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: achievements and objectives. Chapman and Hall, New York, pp 285–292

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to M. Soberón.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Miranda, J., Membrillo-Hernández, J., Tabche, M.L. et al. Rhizobium etli cytochrome mutants with derepressed expression of cytochrome terminal oxidases and enhanced symbiotic nitrogen accumulation. Appl Microbiol Biotechnol 45, 182–188 (1996). https://doi.org/10.1007/s002530050668

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s002530050668

Keywords

Navigation