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. 1987 Mar;169(3):1089–1094. doi: 10.1128/jb.169.3.1089-1094.1987

Tn5-induced cytochrome mutants of Bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture.

M R O'Brian, P M Kirshbom, R J Maier
PMCID: PMC211904  PMID: 3029019

Abstract

Two Bradyrhizobium japonicum cytochrome mutants were obtained by Tn5 mutagenesis of strain LO and were characterized in free-living cultures and in symbiosis in soybean root nodules. One mutant strain, LO501, expressed no cytochrome aa3 in culture; it had wild-type levels of succinate oxidase activity but could not oxidize NADH or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The cytochrome content of LO501 root nodule bacteroids was nearly identical to that of the wild type, but the mutant expressed over fourfold more bacteroid cytochrome c oxidase activity than was found in strain LO. The Tn5 insertion of the second mutant, LO505, had a pleiotropic effect; this strain was missing cytochromes c and aa3 in culture and had a diminished amount of cytochrome b as well. The oxidations of TMPD, NADH, and succinate by cultured LO505 cells were very similar to those by the cytochrome aa3 mutant LO501, supporting the conclusion that cytochromes c and aa3 are part of the same branch of the electron transport system. Nodules formed from the symbiosis of strain LO505 with soybean contained no detectable amount of leghemoglobin and had no N2 fixation activity. LO505 bacteroids were cytochrome deficient but contained nearly wild-type levels of bacteroid cytochrome c oxidase activity. The absence of leghemoglobin and the diminished bacterial cytochrome content in nodules from strain LO505 suggest that this mutant may be deficient in some aspect of heme biosynthesis.

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Selected References

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

  1. Appleby C. A. Electron transport systems of Rhizobium japonicum. I. Haemoprotein P-450, other CO-reactive pigments, cytochromes and oxidases in bacteroids from N2-fixing root nodules. Biochim Biophys Acta. 1969 Jan 14;172(1):71–87. doi: 10.1016/0005-2728(69)90093-0. [DOI] [PubMed] [Google Scholar]
  2. Appleby C. A., Turner G. L., Macnicol P. K. Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium. Biochim Biophys Acta. 1975 Jun 17;387(3):461–474. doi: 10.1016/0005-2728(75)90086-9. [DOI] [PubMed] [Google Scholar]
  3. Avissar Y. J., Nadler K. D. Stimulation of tetrapyrrole formation in Rhizobium japonicum by restricted aeration. J Bacteriol. 1978 Sep;135(3):782–789. doi: 10.1128/jb.135.3.782-789.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergersen F. J., Turner G. L. Leghaemoglobin and the supply of O2 to nitrogen-fixing root nodule bacteroids: presence of two oxidase systems and ATP production at low free O2 concentration. J Gen Microbiol. 1975 Dec;91(2):345–354. doi: 10.1099/00221287-91-2-345. [DOI] [PubMed] [Google Scholar]
  5. Bisseling T., van den Bos R. C., van Kammen A. The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum. Biochim Biophys Acta. 1978 Feb 13;539(1):1–11. doi: 10.1016/0304-4165(78)90115-0. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Cutting J. A., Schulman H. M. The site of heme synthesis in soybean root nodules. Biochim Biophys Acta. 1969 Dec 30;192(3):486–493. doi: 10.1016/0304-4165(69)90398-5. [DOI] [PubMed] [Google Scholar]
  8. Daniel R. M., Appleby C. A. Anaerobic-nitrate, symbiotic and aerobic growth of Rhizobium japonicum: effects on cytochrome P 450 , other haemoproteins, nitrate and nitrite reductases. Biochim Biophys Acta. 1972 Sep 20;275(3):347–354. doi: 10.1016/0005-2728(72)90215-0. [DOI] [PubMed] [Google Scholar]
  9. Godfrey C. A., Dilworth M. J. Haem biosynthesis from ( 14 C)- -aminolaevulinic acid in laboratory-grown and root nodule Rhizobium lupini. J Gen Microbiol. 1971 Dec;69(3):385–390. doi: 10.1099/00221287-69-3-385. [DOI] [PubMed] [Google Scholar]
  10. Guerinot M. L., Chelm B. K. Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1837–1841. doi: 10.1073/pnas.83.6.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  12. Leong S. A., Ditta G. S., Helinski D. R. Heme biosynthesis in Rhizobium. Identification of a cloned gene coding for delta-aminolevulinic acid synthetase from Rhizobium meliloti. J Biol Chem. 1982 Aug 10;257(15):8724–8730. [PubMed] [Google Scholar]
  13. 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]
  14. Marrs B., Gest H. Genetic mutations affecting the respiratory electron-transport system of the photosynthetic bacterium Rhodopseudomonas capsulata. J Bacteriol. 1973 Jun;114(3):1045–1051. doi: 10.1128/jb.114.3.1045-1051.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nadler K. D., Avissar Y. J. Heme Synthesis in Soybean Root Nodules: I. On the Role of Bacteroid delta-Aminolevulinic Acid Synthase and delta-Aminolevulinic Acid Dehydrase in the Synthesis of the Heme of Leghemoglobin. Plant Physiol. 1977 Sep;60(3):433–436. doi: 10.1104/pp.60.3.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. O'Brian M. R., Maier R. J. Electron transport components involved in hydrogen oxidation in free-living Rhizobium japonicum. J Bacteriol. 1982 Oct;152(1):422–430. doi: 10.1128/jb.152.1.422-430.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. O'Brian M. R., Maier R. J. Expression of cytochrome o in hydrogen uptake constitutive mutants of Rhizobium japonicum. J Bacteriol. 1985 Feb;161(2):507–514. doi: 10.1128/jb.161.2.507-514.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O'Brian M. R., Maier R. J. Involvement of cytochromes and a flavoprotein in hydrogen oxidation in Rhizobium japonicum bacteroids. J Bacteriol. 1983 Aug;155(2):481–487. doi: 10.1128/jb.155.2.481-487.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Poole R. K. Bacterial cytochrome oxidases. A structurally and functionally diverse group of electron-transfer proteins. Biochim Biophys Acta. 1983 Sep 15;726(3):205–243. doi: 10.1016/0304-4173(83)90006-x. [DOI] [PubMed] [Google Scholar]
  20. Porra R. J. A rapid spectrophotometric assay for ferrochelatase activity in preparations containing much endogenous hemoglobin and its application to soybean root-nodule preparations. Anal Biochem. 1975 Sep;68(1):289–298. doi: 10.1016/0003-2697(75)90707-1. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Wittenberg J. B. Facilitated oxygen diffusion. The role of leghemoglobin in nitrogen fixation by bacteroids isolated from soybean root nodules. J Biol Chem. 1974 Jul 10;249(13):4057–4066. [PubMed] [Google Scholar]

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