Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers

In a number of bacterial pathogens, the production of virulence factors is induced at 37 degrees 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 degrees 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 degrees 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.