Azetidomonamide and Diazetidomonapyridone Metabolites Control Biofilm Formation and Pigment Synthesis in Pseudomonas aeruginosa.

Synthesis of azetidine-derived natural products by the opportunistic pathogen Pseudomonas aeruginosa 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 P. aeruginosa for the titratable expression of the biosynthetic aze 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 aze 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 P. aeruginosa.

PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa.

Alkyl hydroxyquinoline N-oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen Pseudomonas aeruginosa They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). Although the enzymatic synthesis of HHQ and PQS had been elucidated, the route by which AQNOs are synthesized remained elusive. Here, we report on PqsL, the key enzyme for AQNO production, which structurally resembles class A flavoprotein monooxygenases such as p-hydroxybenzoate 3-hydroxylase (pHBH) and 3-hydroxybenzoate 6-hydroxylase. However, we found that unlike related enzymes, PqsL hydroxylates a primary aromatic amine group, and it does not use NAD(P)H as cosubstrate, but unexpectedly required reduced flavin as electron donor. We also observed that PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which was preferred over 2-ABA as substrate of the downstream enzyme PqsBC. In vitro reconstitution of the PqsL/PqsBC reaction was feasible by using the FAD reductase HpaC, and we noted that the AQ:AQNO ratio is increased in an hpaC-deletion mutant of P. aeruginosa PAO1 compared with the ratio in the WT strain. A structural comparison with pHBH, the model enzyme of class A flavoprotein monooxygenases, revealed that structural features associated with NAD(P)H binding are missing in PqsL. Our study completes the AQNO biosynthetic pathway in P. aeruginosa, indicating that PqsL produces the unstable product 2-hydroxylaminobenzoylacetate from 2-ABA and depends on free reduced flavin as electron donor instead of NAD(P)H.

PqsBC, a Condensing Enzyme in the Biosynthesis of the Pseudomonas aeruginosa Quinolone Signal: CRYSTAL STRUCTURE, INHIBITION, AND REACTION MECHANISM.

Pseudomonas aeruginosaproduces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the ß-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used forin vitrobinding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.

A PQS-Cleaving Quorum Quenching Enzyme Targets Extracellular Membrane Vesicles of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQDS.b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQDS.b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQDS.b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQDS.b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme.

PqsE of Pseudomonas aeruginosa Acts as Pathway-Specific Thioesterase in the Biosynthesis of Alkylquinolone Signaling Molecules.

Pseudomonas aeruginosa uses the alkylquinolones PQS (2-heptyl-3-hydroxy-4(1H)-quinolone) and HHQ (2-heptyl-4(1H)-quinolone) as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. The biosynthesis of HHQ is generally accepted to require the pqsABCD gene products. We now reconstitute the biosynthetic pathway in vitro, and demonstrate that in addition to PqsABCD, PqsE has a role in HHQ synthesis. PqsE acts as thioesterase, hydrolyzing the biosynthetic intermediate 2-aminobenzoylacetyl-coenzyme A to form 2-aminobenzoylacetate, the precursor of HHQ and 2-aminoacetophenone. The role of PqsE can be taken over to some extent by the broad-specificity thioesterase TesB, explaining why the pqsE deletion mutant of P. aeruginosa still synthesizes HHQ. Interestingly, the pqsE mutant produces increased levels of 2,4-dihydroxyquinoline, resulting from intramolecular cyclization of 2-aminobenzoylacetyl-coenzyme A. Overall, our data suggest that PqsE promotes the efficiency of alkylquinolone signal molecule biosynthesis in P. aeruginosa and balances the levels of secondary metabolites deriving from the alkylquinolone biosynthetic pathway.