Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa

Abstract

The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation. The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD. The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate. One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P. aeruginosa cells. The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine. Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element ⍀Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites. Furthermore, the pchD::⍀Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit. A full-length pchDCBA transcript of ca. 4.4 kb could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron-mediated repression of the pchDCBA operon.

Isochorismate Synthase (PchA), the First and Rate-limiting Enzyme in Salicylate Biosynthesis of Pseudomonas aeruginosa

“…A similar regulatory mechanism has been observed for another siderophore of P. aeruginosa, pyoverdin (19). When cells have accumulated excess iron the Fur repressor is activated, which switches off the expression of the pyochelin and pyoverdin biosynthetic genes (15,16,20).…”

“…Sodium isochorismate, used as a reference, was a kind gift from E. W. Leistner (University of Cologne) or was prepared and purified as described previously (21). Racemic dihydroaeruginoate (Dha) and chorismate were prepared by the methods of Serino et al (15) and Grisostomi et al (26), respectively.…”

“…This enzyme catalyzes the conversion of chorismate to isochorismate and is the product of pchA, the last gene of the pchDCBA operon in P. aeruginosa (14,15). The subsequent reaction is catalyzed by the pchB product, isochorismate pyruvate-lyase (21), which produces salicylate (Fig.…”

“…1), which are produced and excreted by P. aeruginosa during iron limitation (12). Pyochelin synthesis starts from chorismate (13)(14)(15), a branch point intermediate in aromatic biosynthesis, and uses two molecules of L-cysteine ( Fig. 1).…”

Salicylic Acid Produced by the Rhizobacterium Pseudomonas aeruginosa 7NSK2 Induces Resistance to Leaf Infection by Botrytis cinerea on Bean

“…Plasmid pME3388 carries the pchR...pchD promoter region of the pchDCBA operon (33) fused to the reporter gene lacZ (C. Reimmann and D. Haas, personal communication). The pchDCBA operon is required for pyochelin and SA production in P. aeruginosa, and its transcription is highly iron-regulated (33). Reporter gene activity in P. aeruginosa 7NSK2 (pME3388) was checked in vitro in casamino acids broth amended with 0 to 20 µM FeCl 3 .…”