Temperature-dependent regulation of the lpxT gene in Escherichia coli and Pseudomonas aeruginosa

The LpxT protein modifies the outer membrane of Gram negative bacteria by transferring a phosphate group from undecaprenyl-pyrophosphate to the lipid A moiety of the lipopolysaccharide. Recently, we found that the expression of the lpxT gene of Pseudomonas aeruginosa (Pa) is post-transcriptionally regulated by an RNA thermometer (RNAT). An RNAT is a thermo-labile secondary structure that entraps the mRNA Translation Initiation Region (TIR) at low temperature, thus inhibiting ribosome binding. As temperature increases, the RNAT unfolds allowing mRNA translation. The bioinformatic analysis of the Escherichia coli (Ec) lpxT 5’-untranslated region (5’-UTR) shows that the TIR may be sequestered in a stem-loop structure with features typically found in some RNATs, thus suggesting a conserved regulatory strategy for Ec and Pa lpxT. On the other hand, Ec lpxT mRNA was claimed to be a direct target of the sRNA MicA, which was proposed to pair ca. 90 nt upstream of the lpxT AUG and negatively regulate lpxT expression. To assess whether Ec lpxT is regulated by temperature and/or MicA, we have assayed the expression of different lpxT-GFP translational fusions in the BW25113 strain and in its isogenic ∆micA derivative at 28° and 42°C. Moreover, we analysed in the same strains and conditions the transcription profile of the chromosomal lpxT locus. We found that i) the lpxT transcript starts 29 nt upstream of the ORF start codon; ii) the presence of the physiological lpxT 5’-UTR confers thermo-dependent expression to the reporter construct; ii) lpxT-GFP transcripts with a longer 5’-UTR, encompassing the putative MicA interaction site, are poorly expressed at any temperature; iii) MicA does not affect the expression of the reporter constructs. On the whole our results suggest that Ec lpxT may be regulated by a new RNAT. Experiments are in progress in our lab to characterize the Ec lpxT putative RNAT structure and function.