Summary Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organized quorum‐sensing systems activated by two types of small acyl‐homoserine lactone signal molecules. Recently, a third type of bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the quorum‐sensing systems. However, the molecular mechanism underlying PQS signalling has remained poorly understood. In this study the global transcriptional profile of P. aeruginosa in response to PQS revealed a marked upregulation of genes belonging to the tightly interdependent functional groups of the iron acquisition and the oxidative stress response. Remarkably, most of the differentially regulated genes, as well as the induction of rhlR, could be traced back to an iron‐chelating effect of PQS. Our results amount to the elucidation of how PQS affects P. aeruginosa and have important implications for the understanding of the complex regulatory circuits involved in P. aeruginosa gene regulation.

The administration of macrolides such as azithromycin for chronic pulmonary infection of cystic fibrosis patients has been reported to be of benefit. Although the mechanisms of action remain obscure, anti-inflammatory effects as well as interference of the macrolide with Pseudomonas aeruginosa virulence factor production have been suggested to contribute to an improved clinical outcome. In this study we used a systematic approach and analyzed the impact of azithromycin on the global transcriptional pattern and the protein expression profile of P. aeruginosa PAO1 cultures versus those in untreated controls. The most remarkable result of this study is the finding that azithromycin exhibited extensive quorum-sensing antagonistic activities. In accordance with the inhibition of the quorum-sensing systems, virulence factor production was diminished and the oxidative stress response was impaired, whereas the type III secretion system was strongly induced. Moreover, P. aeruginosa motility was reduced, which probably accounts for the previously observed impaired biofilm formation capabilities of azithromycin-treated cultures. The interference of azithromycin with quorum-sensing-dependent virulence factor production, biofilm formation, and oxidative stress resistance in P. aeruginosa holds great promise for macrolide therapy in cystic fibrosis. Clearly quorum-sensing antagonist macrolides should be paid more attention in the management of chronic P. aeruginosa infections, and as quorum-sensing antagonists, macrolides might gain vital importance for more general application against chronic infections.

The expression of virulence determinants in Pseudomonas aeruginosa is coordinately regulated in response to both the social environment-commonly referred to as quorum sensing-and to environmental cues. In this study we have dissected the various independent regulation levels for pyocyanin production, which is influenced by the homoserine lactone-and Pseudomonas quinolone signal (PQS)-mediated quorum-sensing systems as well as by iron and phosphate availability. We demonstrate that the phosphate regulon is involved in the transcriptional activation of rhlR and the augmentation of PQS and pyocyanin production under phosphate limitation. However, we also observed an enhancement of rhlR transcription under low-iron medium conditions and after the addition of PQS that was independent of the phosphate regulon. These results highlight the complexity of secondary metabolite production in P. aeruginosa via environmental cues and the quorumsensing system.Bacterial organisms that elaborate traits tailored to their surroundings have better chances of surviving the pressures of unfavorable environmental conditions and host defenses. The outstanding capability of Pseudomonas aeruginosa for adaptation is reflected by the large number of putative transcriptional regulators (53), as bacterial differentiation is often controlled by transcription factors whose activity is influenced by local cues. Moreover, it has been recognized that environmental signals (33) as well as the social surrounding control bacterial virulence factor production. Cell-density-dependent gene regulation is commonly referred to as quorum sensing (QS) (20). QS is based on the release of soluble communicator molecules that trigger the transcription of QS-dependent genes when the bacterial population has reached a certain cell density. Many of these genes are involved in bacterial pathogenicity (9,19,39,40,50). Three chemically distinct signal molecules have been identified so far in P. aeruginosa. Two of these are acyl-homoserine lactones (AHL): a butyryl-homoserine lactone and a 3-oxo-dodecanoyl homoserine lactone, which together with their corresponding transcriptional activator proteins (R proteins) comprise the two hierarchically organized QS systems las and rhl (8,27,38,41,44) and control the expression of over 200 genes (23,48,57). The third signal molecule is 2-heptyl-3-hydroxy-4-quinolone (43). This Pseudomonas quinolone signal (PQS) interacts with the las and the rhl systems. While the las system seems to induce the production of PQS, exogenous PQS up-regulates the expression of the rhl system (15, 16, 31). In P. aeruginosa the impact of the rhl QS on the biosynthesis of the secondary metabolites pyocyanin and rhamnolipids is well documented (42). However, the production of these secondary metabolites also seems to be dependent on environmental cues (3,62). A link between QS and iron homeostasis was suggested previously (5,11,21,24,25,52,60). Moreover, PQS was shown to exhibit an iron-chelating activity, and PQS-dependent rhlR induction seems to be ...

CitationPseudomonas aeruginosa cupA-encoded fimbriae expression is regulated by a GGDEF and EAL Cyclic-diguanylate (c-di-GMP) is a widespread bacterial signal molecule that plays a major role in the modulation of cellular surface components, such as exopolysaccharides and fimbriae, and in the establishment of a sessile life style. Here, we report that intracellular c-di-GMP levels influence cupA encoded fimbriae expression in Pseudomonas aeruginosa. In an autoaggregative P. aeruginosa small colony variant (SCV) CupA fimbriae and the intracellular c-di-GMP concentration were found to be enhanced as compared to the clonal wild-type. The expression of CupA fimbriae were dependent on a functional PA1120 and morA gene both encoding a GGDEF domain. Overexpression of the GGDEF domain protein PA1120 complemented the PA1120 and the morA mutant with respect to CupA fimbriae expression. In agreement with these findings, overexpression of the EAL domain containing phenotypic variance regulator (PvrR) in the SCV resulted in a switch to wild-type colony morphology, a decreased intracellular level of c-di-GMP and reduced cupA fimbriae expression. Although a regulation of cupA encoded fimbriae expression via c-di-GMP was clearly demonstrated, cupA expression is also under transcriptional control that is independent of the global intracellular c-di-GMP levels.

The role of intercellular communication in the regulation of bacterial multicellular behavior has received widespread attention, and a variety of signal molecules involved in bacterial communication have been discovered. In addition to the N-acyl-homoserine lactones, 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal, have been shown to function as signal molecules in Pseudomonas aeruginosa. In this study we unraveled the biosynthetic pathway of HAQs using feeding experiments with isotope-labeled precursors and analysis of extracted HAQs by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Our results show that the biosynthesis of various HAQ metabolites is directed via a common metabolic pathway involving a "head-to-head" condensation of anthranilic acid and ␤-keto fatty acids. Moreover, we provide evidence that the ␤-keto-(do)decanoic acids, crucial for the biosynthesis of the heptyl and nonyl derivatives of the 4-hydroxyquinolines in P. aeruginosa, are at least in part derived from a common pool of ␤-hydroxy(do)decanoic acids involved in rhamnolipid biosynthesis.