Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1996 Oct; 178(20): 5989–5994.
PMCID: PMC178456

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.


Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.

Full Text

The Full Text of this article is available as a PDF (231K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Appleby CA. 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. [PubMed]
  • Appleby CA. Electron transport systems of Rhizobium japonicum. II. Rhizobium haemoglobin, cytochromes and oxidases in free-living (cultured) cells. Biochim Biophys Acta. 1969 Jan 14;172(1):88–105. [PubMed]
  • Batut J, Daveran-Mingot ML, David M, Jacobs J, Garnerone AM, Kahn D. fixK, a gene homologous with fnr and crp from Escherichia coli, regulates nitrogen fixation genes both positively and negatively in Rhizobium meliloti. EMBO J. 1989 Apr;8(4):1279–1286. [PMC free article] [PubMed]
  • Bott M, Bolliger M, Hennecke H. Genetic analysis of the cytochrome c-aa3 branch of the Bradyrhizobium japonicum respiratory chain. Mol Microbiol. 1990 Dec;4(12):2147–2157. [PubMed]
  • Castresana J, Lübben M, Saraste M, Higgins DG. Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen. EMBO J. 1994 Jun 1;13(11):2516–2525. [PMC free article] [PubMed]
  • Donald RG, Ludwig RA. Rhizobium sp. strain ORS571 ammonium assimilation and nitrogen fixation. J Bacteriol. 1984 Jun;158(3):1144–1151. [PMC free article] [PubMed]
  • Donald RG, Nees DW, Raymond CK, Loroch AI, Ludwig RA. Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes. J Bacteriol. 1986 Jan;165(1):72–81. [PMC free article] [PubMed]
  • Drozd J, Postgate JR. Effects of oxygen on acetylene reduction, cytochrome content and respiratory activity of Azotobacter chroococcum. J Gen Microbiol. 1970 Sep;63(1):63–73. [PubMed]
  • García-Horsman JA, Berry E, Shapleigh JP, Alben JO, Gennis RB. A novel cytochrome c oxidase from Rhodobacter sphaeroides that lacks CuA. Biochemistry. 1994 Mar 15;33(10):3113–3119. [PubMed]
  • Gray KA, Grooms M, Myllykallio H, Moomaw C, Slaughter C, Daldal F. Rhodobacter capsulatus contains a novel cb-type cytochrome c oxidase without a CuA center. Biochemistry. 1994 Mar 15;33(10):3120–3127. [PubMed]
  • Hill S, Viollet S, Smith AT, Anthony C. Roles for enteric d-type cytochrome oxidase in N2 fixation and microaerobiosis. J Bacteriol. 1990 Apr;172(4):2071–2078. [PMC free article] [PubMed]
  • Kelly MJ, Poole RK, Yates MG, Kennedy C. Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air. J Bacteriol. 1990 Oct;172(10):6010–6019. [PMC free article] [PubMed]
  • Kitts CL, Ludwig RA. Azorhizobium caulinodans respires with at least four terminal oxidases. J Bacteriol. 1994 Feb;176(3):886–895. [PMC free article] [PubMed]
  • Mandon K, Kaminski PA, Elmerich C. Functional analysis of the fixNOQP region of Azorhizobium caulinodans. J Bacteriol. 1994 May;176(9):2560–2568. [PMC free article] [PubMed]
  • Moshiri F, Chawla A, Maier RJ. Cloning, characterization, and expression in Escherichia coli of the genes encoding the cytochrome d oxidase complex from Azotobacter vinelandii. J Bacteriol. 1991 Oct;173(19):6230–6241. [PMC free article] [PubMed]
  • Preisig O, Anthamatten D, Hennecke H. Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen-fixing endosymbiosis. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3309–3313. [PMC free article] [PubMed]
  • Preisig O, Zufferey R, Thöny-Meyer L, Appleby CA, Hennecke H. A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum. J Bacteriol. 1996 Mar;178(6):1532–1538. [PMC free article] [PubMed]
  • Smith A, Hill S, Anthony C. The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation. J Gen Microbiol. 1990 Jan;136(1):171–180. [PubMed]
  • Thöny-Meyer L, Stax D, Hennecke H. An unusual gene cluster for the cytochrome bc1 complex in Bradyrhizobium japonicum and its requirement for effective root nodule symbiosis. Cell. 1989 May 19;57(4):683–697. [PubMed]
  • Woese CR. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. [PMC free article] [PubMed]
  • Yang T, O'Keefe D, Chance B. The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii. Biochem J. 1979 Sep 1;181(3):763–766. [PMC free article] [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...