Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa

The biosynthetic genes pchDCBA and pchEF, which are known to be required fo r the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene o n the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downs tream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon, The deduce d 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 I: enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG ab olished pyochelin formation, whereas mutations in pchH and pchI did not aff ect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin b iosynthetic genes were expressed from a vector promoter, uncoupling them fr om Fur-mediated repression by iron and PchR-dependent induction by pyocheli n. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes mere sufficient for salicy late, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG tog ether with the E. coli entD gene, which provides a phosphopantetheinyl tran sferase necessary for PchE and PchF activation. The PchG protein was purifi ed and used in combination with PchD and phosphopantetheinylated PchE and P chF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was att ributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P, aeruginosa.