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. 1985 Mar;161(3):882–887. doi: 10.1128/jb.161.3.882-887.1985

Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

S S Hom, L A Graham, R J Maier
PMCID: PMC214979  PMID: 3882669

Abstract

Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.

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Selected References

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  1. Agarwal A. K., Keister D. L. Physiology of Ex Planta Nitrogenase Activity in Rhizobium japonicum. Appl Environ Microbiol. 1983 May;45(5):1592–1601. doi: 10.1128/aem.45.5.1592-1601.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Dixon R. O. Hydrogenase in legume root nodule bacteroids: occurrence and properties. Arch Mikrobiol. 1972;85(3):193–201. doi: 10.1007/BF00408844. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Fuhrmann M., Hennecke H. Rhizobium japonicum nitrogenase Fe protein gene (nifH). J Bacteriol. 1984 Jun;158(3):1005–1011. doi: 10.1128/jb.158.3.1005-1011.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Hom S. S., Uratsu S. L., Hoang F. Transposon Tn5-induced mutagenesis of Rhizobium japonicum yielding a wide variety of mutants. J Bacteriol. 1984 Jul;159(1):335–340. doi: 10.1128/jb.159.1.335-340.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lepo J. E., Hanus F. J., Evans H. J. Chemoautotrophic growth of hydrogen-uptake-positive strains of Rhizobium japonicum. J Bacteriol. 1980 Feb;141(2):664–670. doi: 10.1128/jb.141.2.664-670.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Lim S. T., Shanmugam K. T. Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP. Biochim Biophys Acta. 1979 May 16;584(3):479–492. doi: 10.1016/0304-4165(79)90121-1. [DOI] [PubMed] [Google Scholar]
  15. Maier R. J., Brill W. J. Mutant Strains of Rhizobium japonicum with Increased Ability to Fix Nitrogen for Soybean. Science. 1978 Aug 4;201(4354):448–450. doi: 10.1126/science.201.4354.448. [DOI] [PubMed] [Google Scholar]
  16. Maier R. J., Hanus F. J., Evans H. J. Regulation of hydrogenase in Rhizobium japonicum. J Bacteriol. 1979 Feb;137(2):825–829. doi: 10.1128/jb.137.2.825-829.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Maier R. J., Mutaftschiev S. Reconstitution of H2 oxidation activity from H2 uptake-negative mutants of Rhizobium japonicum bacteroids. J Biol Chem. 1982 Feb 25;257(4):2092–2096. [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. 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]
  21. Schubert K. R., Evans H. J. Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1207–1211. doi: 10.1073/pnas.73.4.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stults L. W., O'Hara E. B., Maier R. J. Nickel is a component of hydrogenase in Rhizobium japonicum. J Bacteriol. 1984 Jul;159(1):153–158. doi: 10.1128/jb.159.1.153-158.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang R., Healey F. P., Myers J. Amperometric measurement of hydrogen evolution in chlamydomonas. Plant Physiol. 1971 Jul;48(1):108–110. doi: 10.1104/pp.48.1.108. [DOI] [PMC free article] [PubMed] [Google Scholar]

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