SummaryUsing Bacillus subtilis as a model organism, we investigated thermotolerance development by analysing cell survival and in vivo protein aggregate formation in severely heat-shocked cells primed by a mild heat shock. We observed an increased survival during severe heat stress, accompanied by a strong reduction of heat-induced cellular protein aggregates in cells lacking the ClpXP protease. We could demonstrate that the transcription factor Spx, a regulatory substrate of ClpXP, is critical for the prevention of protein aggregate formation because its regulon encodes redox chaperones, such as thioredoxin, required for protection against thiol-specific oxidative stress. Consequently B. subtilis cells grown in the absence of oxygen were more protected against severe heat shock and much less protein aggregates were detected compared to aerobically grown cells. The presented results indicate that in B. subtilis Spx and its regulon plays not only an important role for oxidative but also for heat stress response and thermotolerance development. In addition, our experiments suggest that the protection of misfolded proteins from thiol oxidation during heat shock can be critical for the prevention of cellular protein aggregation in vivo.

Spx is a Bacillus subtilis transcription factor that interacts with the alpha subunits of RNA polymerase. It can activate the thiol stress response regulon and interfere with the activation of many developmental processes. Here, we show that Spx is a central player orchestrating the heat shock response by up-regulating relevant stress response genes as revealed by comparative transcriptomic experiments. Moreover, these experiments revealed the potential of Spx to inhibit transcription of translation-related genes. By in vivo and in vitro experiments, we confirmed that Spx can inhibit transcription from rRNA. This inhibition depended mostly on UP elements and the alpha subunits of RNA polymerase. However, the concurrent up-regulation activity of stress genes by Spx, but not the inhibition of translation related genes, was essential for mediating stress response and antibiotic tolerance under the applied stress conditions. The observed inhibitory activity might be compensated in vivo by additional stress response processes interfering with translation. Nevertheless, the impact of Spx on limiting translation becomes apparent under conditions with high cellular Spx levels. Interestingly, we observed a subpopulation of stationary phase cells that contains raised Spx levels, which may contribute to growth inhibition and a persister-like behaviour of this subpopulation during outgrowth.