Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes.
The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.
D000595:Amino Acid Sequence; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001421:Bacteria, Anaerobic / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D005796:Genes; D005798:Genes, Bacterial; D005838:Genotype; D009097:Multienzyme Complexes / Q000235:genetics*; D013385:Succinate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic
D000906:Antibodies; D000936:Antigen-Antibody Complex; D009097:Multienzyme Complexes; C033727:fumarate reductase complex; D013385:Succinate Dehydrogenase