• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Mar 1990; 172(3): 1647–1655.
PMCID: PMC208644

Genetic organization of the hydrogen uptake (hup) cluster from Rhizobium leguminosarum.


In symbiosis with peas, Rhizobium leguminosarum UPM791 induces the synthesis of a hydrogen uptake (Hup) system that recycles hydrogen generated in nodules by nitrogenase. A cosmid (pAL618) containing hup genes from this strain on a 20-kilobase-pair (kb) DNA insert has previously been isolated in our laboratory (A. Leyva, J. M. Palacios, T. Mozo, and T.Ruiz-Argüeso, J. Bacteriol. 169:4929-4934, 1987). Here we show that cosmid pAL618 contains all of the genetic information required to confer high levels of hydrogenase activity on the naturally Hup- strains R. leguminosarum UML2 and Rhizobium phaseoli CFN42, and we also describe in detail the organization of hup genes on pAL618. To study hup gene organization, site-directed transposon mutagenesis and complementation analysis were carried out. According to the Hup phenotype associated with the transposon insertions, hup genes were found to span a 15-kilobase-pair region within pAL618 insert DNA. Complementation analysis revealed that Hup- mutants fell into six distinct complementation groups that define six transcriptional units, designated regions hupI to hupVI. Region hupI was subcloned and expressed in Escherichia coli cells under the control of a bacteriophage T7 promoter. A polypeptide of ca. 65 kilodaltons that was cross-reactive with antiserum against the large subunit of Bradyrhizobium japonicum hydrogenase was detected both in E. coli cells carrying the cloned hupI region and in pea bacteroids from strain UPM791, indicating that region hupI codes for structural genes of R. leguminosarum hydrogenase.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Arp DJ. Rhizobium japonicum hydrogenase: purification to homogeneity from soybean nodules, and molecular characterization. Arch Biochem Biophys. 1985 Mar;237(2):504–512. [PubMed]
  • Arp DJ, McCollum LC, Seefeldt LC. Molecular and immunological comparison of membrane-bound, H2-oxidizing hydrogenases of Bradyrhizobium japonicum, Alcaligenes eutrophus, Alcaligenes latus, and Azotobacter vinelandii. J Bacteriol. 1985 Jul;163(1):15–20. [PMC free article] [PubMed]
  • Boursier P, Hanus FJ, Papen H, Becker MM, Russell SA, Evans HJ. Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme. J Bacteriol. 1988 Dec;170(12):5594–5600. [PMC free article] [PubMed]
  • Cantrell MA, Haugland RA, Evans HJ. Construction of a Rhizobium japonicum gene bank and use in isolation of a hydrogen uptake gene. Proc Natl Acad Sci U S A. 1983 Jan;80(1):181–185. [PMC free article] [PubMed]
  • Chaudhuri A, Krasna AI. Isolation of genes required for hydrogenase synthesis in Escherichia coli. J Gen Microbiol. 1987 Dec;133(12):3289–3298. [PubMed]
  • Ditta G, Stanfield S, Corbin D, Helinski DR. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. [PMC free article] [PubMed]
  • Eberz G, Eitinger T, Friedrich B. Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus. J Bacteriol. 1989 Mar;171(3):1340–1345. [PMC free article] [PubMed]
  • Emerich DW, Ruiz-Argüeso T, Ching TM, Evans HJ. Hydrogen-dependent nitrogenase activity and ATP formation in Rhizobium japonicum bacteroids. J Bacteriol. 1979 Jan;137(1):153–160. [PMC free article] [PubMed]
  • Hanus FJ, Maier RJ, Evans HJ. Autotrophic growth of H2-uptake-positive strains of Rhizobium japonicum in an atmosphere supplied with hydrogen gas. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1788–1792. [PMC free article] [PubMed]
  • Harker AR, Xu LS, Hanus FJ, Evans HJ. Some properties of the nickel-containing hydrogenase of chemolithotrophically grown Rhizobium japonicum. J Bacteriol. 1984 Sep;159(3):850–856. [PMC free article] [PubMed]
  • Harker AR, Zuber M, Evans HJ. Immunological homology between the membrane-bound uptake hydrogenases of Rhizobium japonicum and Escherichia coli. J Bacteriol. 1986 Feb;165(2):579–584. [PMC free article] [PubMed]
  • Haugland RA, Cantrell MA, Beaty JS, Hanus FJ, Russell SA, Evans HJ. Characterization of Rhizobium japonicum hydrogen uptake genes. J Bacteriol. 1984 Sep;159(3):1006–1012. [PMC free article] [PubMed]
  • Hom SS, Graham LA, Maier RJ. Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum. J Bacteriol. 1985 Mar;161(3):882–887. [PMC free article] [PubMed]
  • Jacobs TW, Egelhoff TT, Long SR. Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC. J Bacteriol. 1985 May;162(2):469–476. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lambert GR, Cantrell MA, Hanus FJ, Russell SA, Haddad KR, Evans HJ. Intra- and interspecies transfer and expression of Rhizobium japonicum hydrogen uptake genes and autotrophic growth capability. Proc Natl Acad Sci U S A. 1985 May;82(10):3232–3236. [PMC free article] [PubMed]
  • Lambert GR, Harker AR, Cantrell MA, Hanus FJ, Russell SA, Haugland RA, Evans HJ. Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes. Appl Environ Microbiol. 1987 Feb;53(2):422–428. [PMC free article] [PubMed]
  • Leclerc M, Colbeau A, Cauvin B, Vignais PM. 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. 1988 Sep;214(1):97–107. [PubMed]
  • Leyva A, Palacios JM, Mozo T, Ruiz-Argüeso T. Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum. J Bacteriol. 1987 Nov;169(11):4929–4934. [PMC free article] [PubMed]
  • Leyva A, Palacios JM, Ruiz-Argüeso T. Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains. Appl Environ Microbiol. 1987 Oct;53(10):2539–2543. [PMC free article] [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Maier RJ. Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2. J Bacteriol. 1981 Jan;145(1):533–540. [PMC free article] [PubMed]
  • O'Gara F, Shanmugam KT. Regulation of nitrogen fixation by Rhizobia. Export of fixed N2 as NH+4. Biochim Biophys Acta. 1976 Jul 21;437(2):313–321. [PubMed]
  • Seefeldt LC, Arp DJ. Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing alpha beta dimer. Biochimie. 1986 Jan;68(1):25–34. [PubMed]
  • Stachel SE, An G, Flores C, Nester EW. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. [PMC free article] [PubMed]
  • Stults LW, Moshiri F, Maier RJ. Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography. J Bacteriol. 1986 Jun;166(3):795–800. [PMC free article] [PubMed]
  • Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Waugh R, Boxer DH. Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity. Biochimie. 1986 Jan;68(1):157–166. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...