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Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Nov;169(11):4929–4934. doi: 10.1128/jb.169.11.4929-4934.1987

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A Leyva 1, J M Palacios 1, T Mozo 1, T Ruiz-Argüeso 1
PMCID: PMC213888  PMID: 2822654

Abstract

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.

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Selected References

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  1. Beringer J. E. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974 Sep;84(1):188–198. doi: 10.1099/00221287-84-1-188. [DOI] [PubMed] [Google Scholar]
  2. Cantrell M. A., Haugland R. A., Evans H. J. 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. doi: 10.1073/pnas.80.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clewell D. B., Helinski D. R. Effect of growth conditions on the formation of the relaxation complex of supercoiled ColE1 deoxyribonucleic acid and protein in Escherichia coli. J Bacteriol. 1972 Jun;110(3):1135–1146. doi: 10.1128/jb.110.3.1135-1146.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins J. Escherichia coli plasmids packageable in vitro in lambda bacteriophage particles. Methods Enzymol. 1979;68:309–326. doi: 10.1016/0076-6879(79)68022-9. [DOI] [PubMed] [Google Scholar]
  6. Corbin D., Ditta G., Helinski D. R. Clustering of nitrogen fixation (nif) genes in Rhizobium meliloti. J Bacteriol. 1982 Jan;149(1):221–228. doi: 10.1128/jb.149.1.221-228.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ditta G., Stanfield S., Corbin D., Helinski D. R. 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. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Drevon J. J., Frazier L., Russell S. A., Evans H. J. Respiratory and Nitrogenase Activities of Soybean Nodules Formed by Hydrogen Uptake Negative (Hup) Mutant and Revertant Strains of Rhizobium japonicum Characterized by Protein Patterns. Plant Physiol. 1982 Nov;70(5):1341–1346. doi: 10.1104/pp.70.5.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  10. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanus F. J., Maier R. J., Evans H. J. 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. doi: 10.1073/pnas.76.4.1788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haugland R. A., Cantrell M. A., Beaty J. S., Hanus F. J., Russell S. A., Evans H. J. Characterization of Rhizobium japonicum hydrogen uptake genes. J Bacteriol. 1984 Sep;159(3):1006–1012. doi: 10.1128/jb.159.3.1006-1012.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hom S. S., Graham L. A., Maier R. J. Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum. J Bacteriol. 1985 Mar;161(3):882–887. doi: 10.1128/jb.161.3.882-887.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lambert G. R., Cantrell M. A., Hanus F. J., Russell S. A., Haddad K. R., Evans H. J. 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. doi: 10.1073/pnas.82.10.3232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Langridge J., Langridge P., Bergquist P. L. Extraction of nucleic acids from agarose gels. Anal Biochem. 1980 Apr;103(2):264–271. doi: 10.1016/0003-2697(80)90266-3. [DOI] [PubMed] [Google Scholar]
  16. Lepo J. E., Hickok R. E., Cantrell M. A., Russell S. A., Evans H. J. Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum. J Bacteriol. 1981 May;146(2):614–620. doi: 10.1128/jb.146.2.614-620.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maier R. J., Campbell N. E., Hanus F. J., Simpson F. B., Russell S. A., Evans H. J. Expression of hydrogenase activity in free-living Rhizobium japonicum. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3258–3262. doi: 10.1073/pnas.75.7.3258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maier R. J., Merberg D. M. Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen. J Bacteriol. 1982 Apr;150(1):161–167. doi: 10.1128/jb.150.1.161-167.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maier R. J. Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2. J Bacteriol. 1981 Jan;145(1):533–540. doi: 10.1128/jb.145.1.533-540.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Merberg D., O'Hara E. B., Maier R. J. Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen. J Bacteriol. 1983 Dec;156(3):1236–1242. doi: 10.1128/jb.156.3.1236-1242.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  22. Moshiri F., Stults L., Novak P., Maier R. J. Nif- Hup- mutants of Rhizobium japonicum. J Bacteriol. 1983 Aug;155(2):926–929. doi: 10.1128/jb.155.2.926-929.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. O'Gara F., Shanmugam K. T. Regulation of nitrogen fixation by Rhizobia. Export of fixed N2 as NH+4. Biochim Biophys Acta. 1976 Jul 21;437(2):313–321. doi: 10.1016/0304-4165(76)90001-5. [DOI] [PubMed] [Google Scholar]
  24. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  25. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  26. Sternberg N., Tiemeier D., Enquist L. In vitro packaging of a lambda Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1. Gene. 1977 May;1(3-4):255–280. doi: 10.1016/0378-1119(77)90049-x. [DOI] [PubMed] [Google Scholar]
  27. Takeda Y., Harding N. E., Smith D. W., Zyskind J. W. The chromosomal origin of replication (oriC) of Erwinia carotovora. Nucleic Acids Res. 1982 Apr 24;10(8):2639–2650. doi: 10.1093/nar/10.8.2639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zuber M., Harker A. R., Sultana M. A., Evans H. J. Cloning and expression of Bradyrhizobium japonicum uptake hydrogenase structural genes in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7668–7672. doi: 10.1073/pnas.83.20.7668. [DOI] [PMC free article] [PubMed] [Google Scholar]

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