POST-TRANSCRIPTIONAL REGULATION IN GRAM NEGATIVE BACTERIA

Post-transcriptional regulation plays a pivotal role in gene expression control as it ensures a fast response to environmental changes. This is particularly important in unicellular organisms that are exposed to environmental changes. During my PhD, I studied different aspects of this type of regulation in two Gram-negative bacteria: Escherichia coli and the opportunistic pathogen Pseudomonas aeruginosa. In E. coli I investigated the role of the ribosomal protein S1 in the interplay between translation and mRNA decay. In particular we showed that S1 not associated to 30S can sequester and protect mRNA from RNase E, the main endonuclease in E. coli. Another aspect that I considered in my research has been regulation by RNA molecules in P. aeruginosa. In the last decade, it has become clear how RNA-based regulatory mechanisms are important in controlling bacterial gene expression. However, little is known about it in this relevant human pathogen. By RNA deep sequencing we identified more than 150 novel candidate sRNAs in the P. aeruginosa strains PAO1 and PA14, which differ in virulence degree. We confirmed by Northern blotting the expression of 52 new sRNAs, substantially increasing the number of known sRNAs expressed by this bacterium. In this context we developed a genetic screen for the identification of genes post-transcriptionally regulated by RNA determinants and applied this system to the search of RNA thermometers (RNATs), i.e. mRNA determinants that couple translation with temperature changes. We identified four putative RNATs and validated two of them in E. coli. Interestingly, the two are located upstream of genes previously implicated in P. aeruginosa pathogenesis, namely dsbA and ptxS. ptxS RNAT was validated also in P. aeruginosa and represents the first RNAT ever described in this bacterium.