BIOSYNTHESIS OF PYOCHELIN AND DIHYDROAERUGINOIC ACID REQUIRES THE IRON-REGULATED PCHDCBA OPERON IN PSEUDOMONAS-AERUGINOSA

The high-affinity siderophore salicylate is an intermediate in the bio synthetic pathway of pyochelin, another siderophore and chelator of tr ansition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upst ream of the salicylate biosynthetic genes pchBA was sequenced and foun d to contain two additional, contiguous genes, pchD and pchC, having t he same orientation, The deduced amino acid sequence of the 60-kBa Pch D protein was similar to those of the EntE protein (2,3-dihydroxybenzo ate-AMP ligase) of Escherichia coli and other adenylate-forming enzyme s, suggesting that salicylate might be adenylated at the carboxyl grou p by PchD, The 28-kDa PchC protein showed similarities to thioesterase s of prokaryotic and eukaryotic origin and might participate in the re lease of the product(s) formed from activated salicylate, One potentia l product, dihydroaeruginoate (Dha), was identified in culture superna tants of iron-limited P. aeruginosa cells, The antifungal antibiotic D ha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine, Inactivation of the chromosomal p chD gene by insertion of the transcription and translation stop elemen t Omega 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::Omega Sm /Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA gene s constitute a transcriptional unit, A full-length pchDCBA transcript of ca, 4,4 lib could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promot ers, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pch R gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron-mediated repression of the pchDCBA opero n.