Bacteria adapt rapidly and continuously to their ever-changing environment by employing precise regulatory mechanisms at all levels—from transcription to translation, and protein degradation. Temperature as stimulus is particularly important for pathogens, which transition from cool ambient temperatures to an abrupt warm environment and back. We are gradually piecing together the intricate details of how Yersinia pseudotuberculosis responds to temperature changes.

In this study, we explored how trans-regulatory elements, such as the small RNA CyaR, structurally respond to temperature and how it would affect its regulatory efficiency. Our previous structuromic datasets reveal compelling conformational changes at 25 and 37°C in CyaR, where the seed region is hidden by a secondary structure that could impede further interactions with its regulatory target. Our initial comparative transcriptome analysis between Yersinia WT and a ΔCyaR mutant shows ompX is differentially regulated between the two temperatures in exponential phase. Complementation assays validate ompX as the first direct interaction partner of CyaR in pseudotuberculosis.

Moreover, in-line probing experiments provide compelling mechanistic insights into the structural interplay of CyaR::ompX interactions at 25 and 37°C. Our results suggest that intramolecular folding of the ompX 5'-UTR is favored at lower temperatures. As both transcripts breathe open at higher temperatures, intermolecular interactions between ompX and CyaR start to prevail. This in turn affects the ability of the 30S to form translation initiation complexes. Effective competition of CyaR with the ribosomal subunit reduces translation efficiency at 37°C. Two additional regulatory levels, the reduced stability of the ompX transcript and the OmpX protein at elevated temperatures contribute to the net result that OmpX production is higher at 25°C.