Skip to main content
SpringerLink
Log in

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

An hydrogenase-deficient (Hup−) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup− mutant, strain JP91, selected by its inability to grow autotrophically (Aut− phenotype) together with other Hup− mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup− mutants, span 15–20 kb of DNA.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adams MWW, Mortenson LE, Chen JS (1981) Hydrogenase. Biochim Biophys Acta 594:105–176

    Google Scholar 

  • Ahombo G, Willison JC, Vignais PM (1986) The nifHDK genes are contiguous with a nifA-like regulatory gene in Rhodobacter capsulatus. Mol Gen Genet 205:442–445

    Google Scholar 

  • Alias A, Cejudo FJ, Chabert J, Willison JC, Vignais PM (1989) Nucleotide sequence of wild-type and mutant nifR4(ntrA) genes of Rhodobacter capsulatus: identification of an essential glycine residue. Nucleic Acids Res 17:5377

    Google Scholar 

  • Allibert P, Willison JC, Vignais PM (1987) Complementation of nitrogen regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product. J Bacteriol 169:260–271

    Google Scholar 

  • Boyer MW, Roullard-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472

    Google Scholar 

  • Brewin NJ, Dejong TM, Phillips DA, Johnston AWB (1980) Cotransfer of determinants for hydrogenase activity and nodulation ability in Rhizobium leguminosarum. Nature 288:77–79

    Google Scholar 

  • Colbeau A, Kelley BC, Vignais PM (1980) Hydrogenase activity in Rhodopseudomonas capsulata: Relationship with nitrogenase activity. J Bacteriol 144:141–148

    Google Scholar 

  • Colbeau A, Godfroy A, Vignais PM (1986) Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata. Biochimie 68:147–155

    Google Scholar 

  • Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang XW, Finlay DR, Guiney D, Helinski DR (1985) Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13:149–153

    Google Scholar 

  • 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

    Google Scholar 

  • Ford CM, Garg N, Garg RP, Tibelius KH, Yates MG, Arp DJ, Seefeldt LC (1990) The identification, characterization and sequencing of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum. Molecular Microbiology (in press)

  • Friedman AM, Long SR, Brown SE, Buikema WJ, Ausubel FM (1982) Construction of broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296

    Google Scholar 

  • Harayama S, Tsuda M, Iino T (1980) High frequency mobilization of the chromosome of Escherichia coli by a mutant of plasmid RP4 temperature-sensitive for maintenance. Mol Gen Genet 180:46–56

    Google Scholar 

  • Haugland RA, Cantrell MA, Beaty JS, Hanus FJ, Russell SA, Evans HJ (1984) Characterization of Rhizobium japonicum hydrogen uptake genes. J Bacteriol 959:1006–1012

    Google Scholar 

  • Heffron F (1983) Tn3 and its derivatives. In Shapiro JA (ed): Mobile genetic elements. Academic Press, New York, pp 223–260

    Google Scholar 

  • Hillmer P, Gest H (1977) H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures. J Bacteriol 129:724–731

    Google Scholar 

  • Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298

    Google Scholar 

  • Imhoff JF, Trüper HG, Pfennig N (1984) Rearrangement of the species and genera of the phototrophic “purple non sulfur bacteria”. Int J Systematic Bacteriol 34:340–343

    Google Scholar 

  • Jones RW, Haselkorn R (1989) The DNA sequence of the Rhodobacter capsulatus ntrA, ntrB and ntrC gene analogues required for nitrogen fixation. Mol Gen Genet 215:507–516

    Google Scholar 

  • Klipp W, Masepohl B, Pühler A (1988) Identification and mapping of nitrogen fixation genes in Rhodobacter capsulatus: duplication of a nifA-nifB region. J Bacteriol 170:693–699

    Google Scholar 

  • Kortlüke C, Hogrefe C, Eberz G, Pühler A, Friedrich B (1987) Genes of lithoautotrophic metabolism are clustered on the megaplasmid pHG1 in Alcaligenes eutrophus. Mol Gen Genet 210:122–128

    Google Scholar 

  • Kovacs KL, Seefeldt LC, Tigyi G, Doyle CM, Mortenson LE, Arp DJ (1989) Immunological relationship among hydrogenases. J Bacteriol 171:430–435

    Google Scholar 

  • Kranz RG, Haselkorn R (1985) Characterization of nif regulatory genes in Rhodopseudomonas capsulata. Gene 40:203–215

    Google Scholar 

  • Leclerc M (1988) Génétique de l'hydrogénase chez Rhodobacter capsulatus: séquençage de gènes et identification des ARNm. Ph. D Thesis, Université Joseph Fourier Grenoble I, France

    Google Scholar 

  • Leclerc M, Colbeau A, Cauvin B, Vignais PM (1988) Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus. Mol Gen Genet 214:97–107, Erratum 215:368

    Google Scholar 

  • Magnin JP, Willison JC, Vignais PM (1987) Elimination of R plasmids from the photosynthetic bacterium Rhodobacter capsulatus. FEMS Microbiol Lett 41:157–161

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218

    Google Scholar 

  • Marrs B (1974) Genetic recombination in Rhodopseudomonas capsulata. Proc Natl Acad Sci USA 71:971–973

    Google Scholar 

  • Nelson LM, Grosskopf E, Tichy HV, Lotz W (1985) Characterization of hup-specific DNA in Rhizobium leguminosarum strains of different origin. FEMS Microbiol Lett 30:53–58

    Google Scholar 

  • Rella M, Mercenier A, Haas D (1985) Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster. Gene 33:293–303

    Google Scholar 

  • Richaud P, Vignais PM, Colbeau A, Uffen RL, Cauvin B (1990) Molecular biology studies of the uptake hydrogenase of Rhodobacter capsulatus and Rhodocyclus gelatinosus. FEMS Microbiol Rev, in press

  • Sayavedra-Soto LA, Powell GK, Evans HJ, Morris RO (1988) Nucleotide sequence of the genetic loci encoding subunits of Bradyrhizobium japonicum uptake hydrogenase. Proc Natl Acad Sci USA 85:8395–8399

    Google Scholar 

  • Seefeldt LC, McCollum LC, Doyle CM, Arp DJ (1987) Immunological and molecular evidence for a membrane-bound dimeric hydrogenase in Rhodopseudomonas capsulata. Biochim Biophys Acta 914:299–303

    Google Scholar 

  • Uffen RL, Colbeau A., Richaud P, Vignais PM (1990) Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus. Mol Gen Genet (in press)

  • Vignais PM, Colbeau A, Willison JC, Jouanneau Y (1985) Hydrogenase, nitrogenase and hydrogen metabolism in the photosynthetic bacteria. Adv Microbial Physiol 26:155–234

    Google Scholar 

  • Wall JD, Love J, Quinn SP (1984) Spontaneous Nif− mutants of Rhodopseudomonas capsulata. J Bacteriol 159:652–657

    Google Scholar 

  • Weaver PF, Wall JD, Gest H (1975) Characterization of Rhodopseudomonas capsulata. Arch Microbiol 105:207–216

    Google Scholar 

  • Willison JC, Vignais PM (1982) The use of metronidazole to isolate Nif− mutants of Rhodopseudomonas capsulata, and the identification of a mutant with altered regulatory properties of nitrogenase. J Gen Microbiol 128:3001–3010

    Google Scholar 

  • Willison JC, Madern D, Vignais PM (1984) Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata. Biochem J 219:593–600

    Google Scholar 

  • Willison JC, Ahombo G, Chabert J, Magnin JP, Vignais PM (1985) Genetic mapping of the Rhodopseudomonas capsulata chromosome shows non clustering of genes involved in nitrogen fixation. J Gen Microbiol 131:3001–3015

    Google Scholar 

  • Willison JC, Magnin JP, Vignais PM (1987) Isolation and characterization of Rhodobacter capsulatus strains lacking endogenous plasmids. Arch Microbiol 147:1134–1142

    Google Scholar 

  • Xu MW, Love J, Borghese R, Wall JD (1989) Identification and isolation of genes essential for H2 oxidation in Rhodobacter capsulatus. J Bacteriol 171:714–721

    Google Scholar 

Download references

Author information

Author notes

  1. J.-P. Magnin

    Present address: CREMGP/ENSEEG Domaine Universitaire, BP 75, 38402, Saint-Martin-d'Hères Cedex, France

Authors and Affiliations

  1. Biochimie Microbienne/LBIO/DRF (CNRS UA1130), Centre d'Etudes Nucléaires de Grenoble, 85 X, F-38041, Grenoble Cedex, France

    A. Colbeau, J.-P. Magnin, B. Cauvin, T. Champion & P. M. Vignais

Authors
  1. A. Colbeau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J.-P. Magnin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. Cauvin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T. Champion
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P. M. Vignais
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by H. Hennecke

Rights and permissions

Reprints and permissions

About this article

Cite this article

Colbeau, A., Magnin, JP., Cauvin, B. et al. Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus . Mol Gen Genet 220, 393–399 (1990). https://doi.org/10.1007/BF00391744

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00391744

Key words

Search

Navigation