Investigation of temperature-regulated mechanisms in Yersinia pseudotuberculosis

As a pathogenic bacterium, Yersinia pseudotuberculosis is confronted with constantly changing conditions during infection of warm-blooded hosts and after excretion into the environment. In order to adapt quickly to these changing stresses, the bacterial cell uses sophisticated regulatory mechanisms at the DNA, RNA and protein level. Among the various environmental influences, temperature fluctuations play a prominent role. Bacteria are able to monitor the ambient temperature by cis-acting RNA thermometers (RNATs), which are localized in the 5'-UTR and form a complex RNA secondary structure. These RNA structures gradually melt as the temperature rises, liberating the ribosome binding site and thus regulating the expression of the downstream open reading frame. In contrast to such gradually melting RNATs, "cold thermosensors" undergo a complete rearrangement of the RNA structure and expose the ribosome binding site (RBS) at low temperatures. In Y. pseudotuberculosis, we have described several RNATs that regulate virulence genes coding for LcrF, the master regulator of virulence, or for components of type 3 secretion system and and an exotoxin.

Parallel analysis of RNA structures (PARS) has identified an ok RNAT candidate upstream of yopH, coding for an essential effector protein of the T3SS. Translational control by this RNAT was demonstrated by reporter gene studies. Point mutations were introduced to create open and closed RNAT versions and tested for functionality. In addition, the structure and RNAT-like melting of the RBS was shown by in vitro structure probing.

In addition, we search for cold thermosensors that regulate gene expression once the bacterium leaves the host and re-enters the environment. Several candidates have emerged from our global RNA structuromic studies and will be subject to structure-function analyses.