Mutations in Cytochrome Assembly and Periplasmic Redox Pathways inBordetella pertussis

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Abstract
Transposon mutagenesis ofBordetella pertussiswas used to discover mutations in the cytochromecbiogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochromecoxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesizec-type cytochromes and possessed insertions in the genes for cytochromecoxidase subunits (ctaC, -D, and -E), hemeabiosynthesis (ctaB), assembly of cytochromecoxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize allc-type cytochromes. Seven of these had transposons indipZ(dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochromecassembly by exogenous dithiothreitol, which was consistent with the cytochromeccysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in theccsBAoperon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent inccsAmutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbAordsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochromecbiogenesis inB. pertussis, a targeted knockout was made indsbB. The DsbB−mutant makes holocytochromesclike the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. AdipZmutant is not corrected by adsbBmutation. Alternative mechanisms to oxidize disulfides inB. pertussisare analyzed and discussed.

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