Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1981 May;146(2):614–620. doi: 10.1128/jb.146.2.614-620.1981

Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.

J E Lepo, R E Hickok, M A Cantrell, S A Russell, H J Evans
PMCID: PMC217004  PMID: 6783623

Abstract

We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.

Full text

PDF
614

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albrecht S. L., Maier R. J., Hanus F. J., Russell S. A., Emerich D. W., Evans H. J. Hydrogenase in Rhizobium japonicum Increases Nitrogen Fixation by Nodulated Soybeans. Science. 1979 Mar 23;203(4386):1255–1257. doi: 10.1126/science.203.4386.1255. [DOI] [PubMed] [Google Scholar]
  2. Arp D. J., Burris R. H. Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules. Biochim Biophys Acta. 1979 Oct 11;570(2):221–230. doi: 10.1016/0005-2744(79)90142-6. [DOI] [PubMed] [Google Scholar]
  3. Carter K. R., Jennings N. T., Hanus J., Evans H. J. Hydrogen evolution and uptake by nodules of soybeans inoculated with different strains of Rhizobium japonicum. Can J Microbiol. 1978 Mar;24(3):307–311. doi: 10.1139/m78-051. [DOI] [PubMed] [Google Scholar]
  4. Cole M. A., Elkan G. H. Transmissible resistance to penicillin G, neomycin, and chloramphenicol in Rhizobium japonicum. Antimicrob Agents Chemother. 1973 Sep;4(3):248–253. doi: 10.1128/aac.4.3.248. [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. Emerich D. W., Ruiz-Argüeso T., Ching T. M., Evans H. J. Hydrogen-dependent nitrogenase activity and ATP formation in Rhizobium japonicum bacteroids. J Bacteriol. 1979 Jan;137(1):153–160. doi: 10.1128/jb.137.1.153-160.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Evans H. J., Koch B., Klucas R. Preparation of nitrogenase from nodules and separation into components. Methods Enzymol. 1972;24:470–476. doi: 10.1016/0076-6879(72)24092-7. [DOI] [PubMed] [Google Scholar]
  8. Evans H. J., Ruiz-Argüeso T., Jennings N., Hanus J. Energy coupling efficiency of symbiotic nitrogen fixation. Basic Life Sci. 1977;9:333–354. doi: 10.1007/978-1-4684-0880-5_21. [DOI] [PubMed] [Google Scholar]
  9. GOA J. A micro biuret method for protein determination; determination of total protein in cerebrospinal fluid. Scand J Clin Lab Invest. 1953;5(3):218–222. doi: 10.3109/00365515309094189. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. KAUDEWITZ F. Production of bacterial mutants with nitrous acid. Nature. 1959 Jun 27;183:1829–1830. doi: 10.1038/1831829a0. [DOI] [PubMed] [Google Scholar]
  12. Klapwijk P. M., de Jonge A. J., Schilperoort R. A., Rörsch A. An enrichment technique for auxotrophs of Agrobacterium tumefaciens using a combination of carbenicillin and lysozyme. J Gen Microbiol. 1975 Nov;91(1):177–182. doi: 10.1099/00221287-91-1-177. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. 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]
  15. Lim S. T. Determination of Hydrogenase in Free-living Cultures of Rhizobium japonicum and Energy Efficiency of Soybean Nodules. Plant Physiol. 1978 Oct;62(4):609–611. doi: 10.1104/pp.62.4.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  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., 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]
  19. McCrae R. E., Hanus J., Evans H. J. Properties of the hydrogenase system in Rhizobium japonicum bacteroids. Biochem Biophys Res Commun. 1978 Jan 30;80(2):384–390. doi: 10.1016/0006-291x(78)90688-5. [DOI] [PubMed] [Google Scholar]
  20. Ruiz-Argüeso T., Emerich D. W., Evans H. J. Hydrogenase system in legume nodules: a mechanism of providing nitrogenase with energy and protection from oxygen damage. Biochem Biophys Res Commun. 1979 Jan 30;86(2):259–264. doi: 10.1016/0006-291x(79)90860-x. [DOI] [PubMed] [Google Scholar]
  21. STICKLAND L. H. The determination of small quantities of bacteria by means of the biuret reaction. J Gen Microbiol. 1951 Oct;5(4):698–703. doi: 10.1099/00221287-5-4-698. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Schubert K. R., Jennings N. T., Evans H. J. Hydrogen Reactions of Nodulated Leguminous Plants: II. Effects on Dry Matter Accumulation and Nitrogen Fixation. Plant Physiol. 1978 Mar;61(3):398–401. doi: 10.1104/pp.61.3.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tabita F. R., Caruso P., Whitman W. Facile assay of enzymes unique to the Calvin cycle in intact cells, with special reference to ribulose 1,5-bisphosphate carboxylase. Anal Biochem. 1978 Feb;84(2):462–472. doi: 10.1016/0003-2697(78)90064-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES