Azetidomonamide and Diazetidomonapyridone Metabolites ControlBiofilm Formation and Pigment Synthesis in Pseudomonas aeruginosa

Synthesis of azetidine-derived natural products bythe opportunistic pathogenPseudomonas aeruginosais controlledby quorum sensing, a process involving the production and sensingof diffusible signal molecules that is decisive for virulenceregulation. In this study, we engineeredP. aeruginosafor thetitratable expression of the biosyntheticazegene cluster, whichallowed the purification and identification of two new products,azetidomonamide C and diazetidomonapyridone. Diazetidomona-pyridone was shown to have a highly unusual structure with twoazetidine rings and an open-chain diimide moiety. Expression ofazegenes strongly increased biofilm formation and production ofphenazine and alkyl quinolone virulence factors. Furtherphysiological studies revealed that all effects were mainly mediatedby azetidomonamide A and diazetidomonapyridone, whereas azetidomonamides B and C had little or no phenotypic impact. TheP450 monooxygenase AzeF which catalyzes a challenging, stereoselective hydroxylation of the azetidine ring convertingazetidomonamide C into azetidomonamide A is therefore crucial for biological activity. Based on ourfindings, we propose this groupof metabolites to constitute a new class of diffusible regulatory molecules with community-related effects inP. aeruginosa.