Temperature-responsive regulation of the lpxT gene in Escherichia coli

RNA thermometers (RNATs) are thermo-labile mRNA secondary structures that modulate translation initiation in response to temperature changes. In a previous work, we found that the expression of Pseudomonas aeruginosa LpxT (Pa LpxT), a membrane protein phosphorylating the lipid A moiety of the lipopolysaccharide, was induced in response to a temperature upshift and we proposed that an RNAT was responsible for such regulation (Delvillani et al. 2014). Here we show that the expression of the Escherichia coli LpxT orthologous protein (Ec LpxT) is also temperature-responsive. Increased Ec LpxT expression at high temperature (i.e. 37-42 vs. 28°C) depends in vivo on post-transcriptional mechanisms. Moreover, in vitro assembly of the 30S ribosomal subunit and fmet-tRNA on the Ec lpxT transcript is less efficient at 28°C than at higher temperature, suggesting that translation initiation of lpxT mRNA is impaired at low temperature. Indeed, the short lpxT 5’-UTR (UnTranslated Region) confers thermo-dependent expression to a reporter translational fusion transcribed from a heterologous promoter. The lpxT 5’-UTR is predicted to form a stem-loop (5’SL) involving the suboptimal Shine-Dalgarno region (SDr); the 5’SL is expected to be quite unstable even at 28°C, thus questioning its ability to prevent ribosome binding at low temperature. However, point mutations changing the stability of the 5’SL secondary structure or ameliorating the complementarity of SDr with the 16S rRNA affect thermoregulation, showing that both elements cooperate in lpxT gene expression regulation. On the whole, our results suggest that Ec lpxT is regulated by an unusual RNA thermometer that exploits the combination of sub-optimal elements to confer temperature-responsive translation to the mRNA. Delvillani, F., Sciandrone, B., Peano, C., Petiti, L., Berens, C., Georgi, C., Ferrara, S., Bertoni, G., Pasini, ME, Dehò, G., Briani, F. (2014) Tet-Trap, a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa. RNA. 20: 1963-1976