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Abstract
Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules. Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa 3, but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,NȲ,NȲ-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate-and NADH-dependent respiration. NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc 1 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome bc 1 complex. One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c 1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc 1 complex. Haem staining revealed that haem proteins of M r 31000 and M r 23000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M r 23000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome c 1 and, in its absence, the protein of M r 23000 does not remain associated with the membrane. Formation of this M r 23000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM. Although the cytochrome bc 1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.
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