Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

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Abstract
B. subtilis succinate dehydrogenase (SDH) is composed of 2 unequal subunits designated Fp (MW, 65,000) and Ip (MW, 28,000). The enzyme is structurally and functionally complexed to cytochrome b558 (MW, 19,000) in the membrane. A total of 21 B. subtilis SDH-negative mutants were isolated. The mutants fall into 5 phenotypic classes with respect to the presence and localization of the subunits of the SDH-cytochrome b558 complex. One class contains mutants with an inactive membrane-bound complex. Membrane-bound enzymatically active SDH could be reconstituted in fused protoplasts of selected pairs of SDH-negative mutants. Most likely reconstitution is due to the assembly of preformed subunits in the fused cells. On the basis of the reconstitution data, the mutants tested could be divided into 3 complementation groups. The combined data of the present and previous work indicate that the complementation groups correspond to the structural genes for the 3 subunits of the membrane-bound SDH-cytochrome b558 complex. A total of 31 SDH-negative mutants of B. subtilis were now characterized. The respective mutations all map in the citF locus at 255.degree. on the B. subtilis chromosomal map. In the present paper, the nomenclature for the genetics of SDH in B. subtilis was revised. All mutations which give an SDH-negative phenotype will be called sdh followed by an isolation number. The designation citF will be omitted, and the citF locus will be divided into 3 genes: sdhA, sdhB and sdhC. Mutations in sdhA affect cytochrome b558, mutations in sdhB affect Fp and mutations in sdhC affect Ip.