TEMPERATURE-DEPENDENT REGULATION OF THE LPXT GENE IN ESCHERICHIA COLI AND PSEUDOMONAS AERUGINOSA

Bacteria respond to temperature variation through sophisticated regulatory networks that involve different macromolecules and molecular thermosensors. In particular, RNA thermometers (RNATs) are thermolabile secondary structures located within the 5’-UTR of some mRNAs that permit fast response to temperature changes. Typically, the RNATs at low temperature entrap the Translational Initiation Region (TIR) of the cognate mRNA thus inhibiting ribosome binding. When the temperature increases, the RNAT secondary structure becomes unstable and gradually shifts to an open conformation, thus allowing translation. Interestingly, in pathogenic bacteria RNATs have been found to respond to the mammal host temperature (37°C) and control the expression of virulence genes. Therefore, the identification of RNATs in pathogens could provide new information about invasion and pathogenicity strategies. RNATs are characterized by poor sequence conservation and structural variability that make the bioinformatic analysis hardly applicable to RNATs identification. We developed a new genetic tool, namely the Tet-Trap, aimed to the identification of post-transcriptionally regulated genes. We applied the Tet-Trap to find out new genes regulated by temperature in the opportunistic pathogen Pseudomonas aeruginosa. Using this system, we identified four new putative RNATs. Two of them, namely ptxS and lpxT, were validated with both in vitro and in vivo approaches. On the whole, our results strongly suggest the presence of new RNATs in the 5’-UTR of both ptxS and lpxT. The LpxT protein modifies the outer membrane of Gram negative bacteria by transferring a phosphate group from undecaprenyl-pyrophosphate to the position 1 of the Lipid A moiety of the lipopolysaccharide (LPS), generating a Lipid A diphosphate specie. One-third of the lipid A found in the E. coli outer membrane contains an unsubstituted diphosphate unit at position 1. The high proportion of this modification in Lipid A and its wide distribution among Gram-negative bacteria make relevant the clarification of its regulation and biological role. We observed that in E. coli, the lpxT gene 5’-UTR is predicted to fold into an unstable stem-loop entrapping the TIR, thus suggesting a conserved lpxT regulatory strategy in E. coli and P. aeruginosa. This observation prompted us to analyze the expression of the E. coli lpxT gene at different temperatures. We found that the E. coli lpxT gene expression is temperature-responsive. Moreover, toeprinting and reporter translational fusion experiments indicated that thermoregulation was achieved through translation modulation and that the determinants of thermoregulation were located in the lpxT 5’-UTR. Point mutations in the lpxT 5’-UTR predicted to change the stability of the stem-loop involving the TIR or enhancing the complementarity of the SD with the 16S rRNA affected thermoregulation, showing that both these elements cooperate in lpxT regulation. Overall, our results strongly suggest that in E. coli, lpxT translation may be modulated in response to temperature variations through a peculiar mechanism based on the combined action of sub-optimal elements.