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 ...

SummaryIntegron are genetic elements able to carry, capture and shuffle the genes embedded in gene cassettes. The attC recombination sites adopt a stable secondary structure when single-stranded that is necessary for their recombination. In this study, we evaluated the impact of the structure of the attC site on expression of the 3Ј gene in class 1 integrons. This was analysed by substituting the attC of the blaIMP-8 gene cassette with various mutated attC sites spanning a wide range of sizes and secondary structures, and measuring the integron-dependent translation of the 3Ј aac(6Ј)-Ib7 gene. In the resulting constructs, the 5Ј-attC site differentially affected the expression of the aac(6Ј)-Ib7 gene. Contrary to what was expected from their proposed role as Rho-independent transcription terminators, the transcription of the aac(6Ј)-Ib7 gene was not affected by the various attC sites. Mutations of natural sites revealed that destabilization of the potential stem-loop structure of the attC site in the transcript could enhance the expression of the 3Ј gene. In particular, the presence of a translated open reading frame was shown to increase translation of the 3Ј gene. These findings might be explained by the capacity of the stem-loop structures to impede ribosome progression.

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