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Journal of Bacteriology, February 2001, p. 813-820, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.813-820.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Essential PchG-Dependent Reduction in Pyochelin Biosynthesis of Pseudomonas aeruginosa

Cornelia Reimmann,1,* Hiten M. Patel,2 Laura Serino,1,dagger Mario Barone,1,Dagger Christopher T. Walsh,2 and Dieter Haas1

Laboratoire de Biologie Microbienne, Université de Lausanne, CH-1015 Lausanne, Switzerland,1 and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 021152

Received 12 July 2000/Accepted 2 November 2000

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.

* Corresponding author. Mailing address: Laboratoire de Biologie Microbienne, Université de Lausanne, Bâtiment de Biologie, CH-1015 Lausanne, Switzerland. Phone: 41 21 692 56 32. Fax: 41 21 692 56 35. E-mail: Cornelia.Reimmann{at}lbm.unil.ch.

dagger Present address: Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, United Kingdom.

Dagger Present address: Institut für Mikrobiologie, ETH Zürich, CH-8092 Zürich, Switzerland.

Journal of Bacteriology, February 2001, p. 813-820, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.813-820.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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