Azetidomonamide and Diazetidomonapyridone Metabolites Control Biofilm Formation and Pigment Synthesis in <i>Pseudomonas aeruginosa</i>

Abstract

Synthesis of azetidine-derived natural products by the opportunistic pathogen <i>Pseudomonas aeruginosa</i> is controlled by quorum sensing, a process involving the production and sensing of diffusible signal molecules that is decisive for virulence regulation. In this study, we engineered <i>P. aeruginosa</i> for the titratable expression of the biosynthetic <i>aze</i> gene cluster, which allowed the purification and identification of two new products, azetidomonamide C and diazetidomonapyridone. Diazetidomonapyridone was shown to have a highly unusual structure with two azetidine rings and an open-chain diimide moiety. Expression of <i>aze</i> genes strongly increased biofilm formation and production of phenazine and alkyl quinolone virulence factors. Further physiological studies revealed that all effects were mainly mediated by azetidomonamide A and diazetidomonapyridone, whereas azetidomonamides B and C had little or no phenotypic impact. The P450 monooxygenase AzeF which catalyzes a challenging, stereoselective hydroxylation of the azetidine ring converting azetidomonamide C into azetidomonamide A is therefore crucial for biological activity. Based on our findings, we propose this group of metabolites to constitute a new class of diffusible regulatory molecules with community-related effects in <i>P. aeruginosa</i>.