Affiliation:
1. Washington University, Department of Biology, Campus Box 1137, 1 Brookings Drive, St. Louis, Missouri 63130
Abstract
ABSTRACT
Transposon mutagenesis of
Bordetella pertussis
was used to discover mutations in the cytochrome
c
biogenesis pathway called system II. Using a tetramethyl-
p
-phenylenediamine cytochrome
c
oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize
c
-type cytochromes and possessed insertions in the genes for cytochrome
c
oxidase subunits (
ctaC
, -
D
, and -
E
), heme
a
biosynthesis (
ctaB
), assembly of cytochrome
c
oxidase (
sco2
), or ferrochelatase (
hemZ
). Eighteen mutants were unable to synthesize all
c
-type cytochromes. Seven of these had transposons in
dipZ
(
dsbD
), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome
c
assembly by exogenous dithiothreitol, which was consistent with the cytochrome
c
cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the
ccsBA
operon, 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 in
ccsA
mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (
dsbA
or
dsbB
). To examine whether the periplasmic disulfide bond pathway is required for cytochrome
c
biogenesis in
B. pertussis
, a targeted knockout was made in
dsbB
. The DsbB
−
mutant makes holocytochromes
c
like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A
dipZ
mutant is not corrected by a
dsbB
mutation. Alternative mechanisms to oxidize disulfides in
B. pertussis
are analyzed and discussed.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
32 articles.
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