Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558.
Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His-113 excludes this residue as an axial heme ligand. A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B. subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000032:analysis; D005786:Gene Expression Regulation; D005796:Genes; D007118:Immunoassay; D008969:Molecular Sequence Data; D009154:Mutation; D019255:NADPH Oxidases; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D013385:Succinate Dehydrogenase / Q000378:metabolism
D003573:Cytochrome b Group; D012333:RNA, Messenger; D004247:DNA; C036888:cytochrome b558; D013385:Succinate Dehydrogenase; D019255:NADPH Oxidases