Thermogenetic tools to monitor temperature-dependent gene expression in bacteria

► Several reporter gene systems can be used to analyze RNA thermometers in vivo. ► Both inducible as well as constitutively regulated systems are applicable. ► GFP and the β-galactosidases LacZ and BgaB (thermostable) are suitable reporters. ► Besides moderate heat shock, LacZ is optimal for analyzing cold shock regulation. ► Novel RNA thermometer candidates were found upstream of the htrA gene from Salmonella and Escherichia coli. Free-living bacteria constantly monitor their ambient temperature. Drastic deviations elicit immediate protective responses known as cold shock or heat shock response. Many mammalian pathogens use temperature surveillance systems to recognize the successful invasion of a host by its body temperature, usually 37°C. Translation of temperature-responsive genes can be modulated by RNA thermometers (RNATs). RNATs form complex structures primarily in the 5′-untranslated region of their transcripts. Most RNATs block the ribosome binding site at low temperatures. Translation is induced at increasing temperature by melting of the RNA structure. The analysis of such temperature-dependent RNA elements calls for adequate test systems that function in the appropriate temperature range. Here, we summarize previously established reporter gene systems based on the classical β-galactosidase LacZ, the heat-stable β-galactosidase BgaB and the green fluorescent protein GFP. We validate these systems by testing known RNATs and describe the construction and application of an optimized bgaB system. Finally, two novel RNA thermometer candidates from Escherichia coli and Salmonella will be presented.