title: Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model.
abstract: Unlike other cytochromes, c-type cytochromes have two covalent bonds formed between the two vinyl groups of haem and two cysteines of the protein. This haem ligation requires specific assembly proteins in prokaryotes or eukaryotic mitochondria and chloroplasts. Here, it is shown that Bordetella pertussis is an excellent bacterial model for the widespread system II cytochrome c synthesis pathway. Mutations in four different genes (ccsA, ccsB, ccsX and dipZ) result in B. pertussis strains unable to synthesize any of at least seven c-type cytochromes. Using a cytochrome c4:alkaline phosphatase fusion protein as a bifunctional reporter, it was demonstrated that the B. pertussis wild-type and mutant strains secrete an active alkaline phosphatase fusion protein. However, unlike the wild type, all four mutants are unable to attach haem covalently, resulting in a degraded N-terminal apocytochrome c4 component. Thus, apocytochrome c secretion is normal in each of the four mutants, but all are defective in a periplasmic assembly step (or export of haem). CcsX is related to thioredoxins, which possess a conserved CysXxxXxxCys motif. Using phoA gene fusions as reporters, CcsX was proven to be a periplasmic thioredoxin-like protein. Both the B. pertussis dipZ (i. e. dsbD) and ccsX mutants are corrected for their assembly defects by the thiol-reducing compounds, dithiothreitol and 2-mercaptoethanesulphonic acid. These results indicate that DipZ and CcsX are required for the periplasmic reduction of the cysteines of apocytochromes c before ligation. In contrast, the ccsA and ccsB mutants are not corrected by exogenous reducing agents, suggesting that CcsA and CcsB are required for the haem ligation step itself in the periplasm (or export of haem to the periplasm). Related to this suggestion, the topology of CcsB was determined experimentally, demonstrating that CcsB has four transmembrane domains and a large 435-amino-acid periplasmic region.
authors.affiliation: Departments of Biology, and Molecular Microbiology, Campus Box 1137, Washington University, One Brookings Drive, St Louis, MO 63130, USA.
authors.forename: C S
authors.initials: CS
authors.lastname: Beckett
authors.forename: J A
authors.initials: JA
authors.lastname: Loughman
authors.forename: K A
authors.initials: KA
authors.lastname: Karberg
authors.forename: G M
authors.initials: GM
authors.lastname: Donato
authors.forename: W E
authors.initials: WE
authors.lastname: Goldman
authors.forename: R G
authors.initials: RG
authors.lastname: Kranz
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D001886:Bordetella pertussis / Q000201:enzymology* / Q000235:genetics*; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D010088:Oxidoreductases; C094848:DsbD electron transport protein, E coli; D000469:Alkaline Phosphatase