Cloning and expression in Escherichia coli of sdhA, the structural gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex

Abstract

Bacillus subtilis cytochrome b558 is a transmembrane protein which anchors succinate dehydrogenase (SDH) to the cytoplasmic membrane and is reduced by succinate. The structural gene for this cytochrome was cloned and expressed in Escherichia coli. Random BamHI or BglII fragments of B. subtilis 168 DNA were cloned in the BamHI site of plasmid pHV32. The derived plasmids were used to transform B. subtilis SDH mutants to chloramphenicol resistance by integration of the plasmid via DNA homology. Of some 3,000 transformants tested, 6 were SDH positive and had pHV32 integrated close to the sdh operon. Two plasmids, pKIM2 and pKIM4, with an insert of B. subtilis DNA of 5.7 and 3.4 kilobases, respectively, were generated by transforming E. coli with DNA from the SDH-positive transformants after cleavage with EcoRI or BglII and ligation. In E. coli carrying either of the two plasmids, about 4% of total membrane protein was B. subtilis cytochrome b558. E. coli (pKIM2) also contained antigen which reacted with antibodies specific for the flavoprotein and the iron-sulfur protein subunit of B. subtilis SDH. Enzymatically active, membrane-bound B. subtilis SDH could not be demonstrated in E. coli (pKIM2). The B. subtilis DNA insert in pKIM2 could transform B. subtilis sdhA (cytochrome b558), sdhB (flavoprotein), and sdhC (iron-sulfur protein) mutants to the wild type. The results suggest that pKIM2 carries the whole B. subtilis sdh operon. The data confirm the gene order and the proposed direction of transcription of the B. subtilis sdh operon. Most likely the sdh genes in E. coli(pKIM2) are controlled by their natural promoter.