A genetic approach to thermosensors identification in Pseudomonas aeruginosa

Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis riboswitches is widespread in Bacteria and controls relevant phenotypic traits. Unfortunately, the identification on a genomic scale of genes post-transcriptionally regulated is not an easy task, as modulation of translation efficiency is not always reflected by changes in the mRNA amount. We devised a reporter genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermosensors, a class of riboswitches that modulate gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of ecological niches, genes differentially expressed at 37 °C and at lower temperatures may be implicated in infection and survival in the human host. We prepared in a plasmid vector a translational fusion library of P. aeruginosa DNA fragments (PaDNA) inserted upstream of Tip2, a short peptide able to inactivate the TetR repressor upon expression1. The library was assayed in streptomycin resistant merodiploid rpsL+/rpsL-31 E. coli strain in which the dominant rpsL+ allele (which confers streptomycin sensitivity) was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e. putatively expressing PaDNA-Tip2 fusions at 37 °C and not at 28°C) were sequenced. We identified six new putative thermosensors. Interestingly, two of them are located upstream of genes implicated in P.aeruginosa pathogenesis. Experiments are in progress to validate the results of our survey. 1Goeke, D., et al. (2012) J. Mol. Biol. 416:33-45