Summary: Following Tn5 mutagenesis of biovar 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 -type cytochromes and cytochromes , but increased levels of cytochrome Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor -tetramethyl--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 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome complex. One of the mutants contained Tn in a gene homologous to that encoding cytochrome , and in the other the Tn was in DNA homologous to that encoding the cytochrome component of the cytochrome complex. Haem staining revealed that haem proteins of 31000 and 23000 were absent from membranes from the mutants whereas an additional soluble -type cytochrome protein of 23000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome and, in its absence, the protein of 23000 does not remain associated with the membrane. Formation of this 23000 component was specifically blocked in a third respiratory-defective mutant which contained Tn in a region of DNA showing homology to a gene previously shown to encode the membrane-bound -type cytochrome CycM. Although the cytochrome complex is essential for symbiotic nitrogen fixation, the other membrane-bound -type cytochrome (CycM) is not.


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