STRUCTURAL GENES FOR SALICYLATE BIOSYNTHESIS FROM CHORISMATE IN PSEUDOMONAS-AERUGINOSA

Salicylate is a precursor of pyochelin in Pseudomonas aeruginosa and b oth compounds display siderophore activity. To elucidate the salicylat e biosynthetic pathway, we have cloned and sequenced a chromosomal reg ion of P. aeruginosa PAO1 containing two adjacent genes, designated pc hB and pchA, which are necessary for salicylate formation. The pchA ge ne encodes a protein of 52 kDa with extensive similarity to the choris mate-utilizing enzymes isochorismate synthase, anthranilate synthase ( component I) and p-aminobenzoate synthase (component I), whereas the 1 1 kDa protein encoded by pchB does not show significant similarity wit h other proteins. The pchB stop codon overlaps the presumed pchA start codon. Expression of the pchA gene in P. aeruginosa appears to depend on the transcription and translation of the upstream pchB gene. The p chBA genes are the first salicylate biosynthetic genes to be reported. Salicylate formation was demonstrated in an Escherichia coli entC mut ant lacking isochorismate synthase when this strain expressed both the pchBA genes, but not when it expressed pchB alone. By contrast, an en tB mutant of E. coli blocked in the conversion of isochorismate to 2,3 -dihydro-2,3-dihydroxybenzoate formed salicylate when transformed with a pchB expression construct. Salicylate formation could also be demon strated in vitro when chorismate was incubated with a crude extract of P. aeruginosa containing overproduced PchA and PchB proteins; salicyl ate and pyruvate were formed in equimolar amounts. Furthermore, salicy late-forming activity could be detected in extracts from a P. aerugino sa pyoverdin-negative mutant when grown under iron limitation, but not with iron excess. Our results are consistent with a pathway leading f rom chorismate to isochorismate and then to salicylate plus pyruvate, catalyzed consecutively by the iron-repressible PchA and PchB proteins in P. aeruginosa.