Affiliation:
1. Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland,1 and
2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 021152
Abstract
ABSTRACT
The biosynthetic genes
pchDCBA
and
pchEF
, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the
pchR
regulatory gene on the chromosome of
Pseudomonas aeruginosa
. The 4.6-kb region located downstream of the
pchEF
genes was found to contain three additional, contiguous genes,
pchG
,
pchH
, and
pchI
, probably forming a
pchEFGHI
operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the
Yersinia pestis
and
Y. enterocolitica
proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in
pchG
abolished pyochelin formation, whereas mutations in
pchH
and
pchI
did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a
P. aeruginosa
mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed
pchDCBA
and
pchEFG
genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host
Escherichia coli
expressing
pchDCBA
and
pchEFG
together with the
E. coli entD
gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate,
l
-cysteine, ATP, NADPH, and
S
-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in
P. aeruginosa
.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
108 articles.
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