In a number of bacterial pathogens, the production of virulence factors is induced at 37°C; this effect is often regulated by mRNA structures formed in the 5′ untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37°C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37°C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5′ UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect.RNA thermometers | bacterial virulence | gene regulation | polarity | quorum sensing A free-living γ-proteobacterium, Pseudomonas aeruginosa is one of the most clinically important opportunistic pathogens. It is responsible for acute infections in immune-compromised individuals and is the primary cause of death in patients with cystic fibrosis (1). The wide pathogenic capacity of this bacterium depends on its ability to produce and secrete multiple virulence factors that are regulated at the transcriptional level by the quorum-sensing (QS) response (2). Several mechanistic details of this intricate regulatory network remain to be elucidated at the molecular level.The QS response is mediated by the bacterial production of acyl-homoserine lactones (autoinducers) that act as signal molecules interacting with transcriptional regulators of the LuxR family. In P. aeruginosa, Las and Rhl QS regulation is arranged in a hierarchical cascade. LasR interacts with 3-oxo-dodecanoylhomoserine lactone (3O-C12-HSL) produced by the LasI enzyme, and activates the transcription of several genes encoding virulence factors and also of rhlR, lasI, and rhlI; rhlR encodes the second QS transcriptional regulator, whereas the product of the rhlI gene is the enzyme that produces butanoyl-homoserine lactone (C...

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