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Daniel Scheller, Franziska Becker, Andrea Wimbert, Dominik Meggers, Stephan Pienkoß, Christian Twittenhoff, Lisa Knoke, Lars I. Leichert, Franz Narberhaus

• Pathogenic bacteria, such as \(\textit {Yersinia pseudotuberculosis}\) encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5’-untranslated region (5’-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The \(\it trxA\) gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5’-UTRs of \(\it sodB\), \(\it sodC\) and \(\it katA\). However, they barely conferred translational repression in \(\textit {Y. pseudotuberculosis}\) at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary \(H_{2}O_{2}\) scavenger. Consistent with the upregulation of \(\it katY\), we observed an improved protection of \(\textit {Y. pseudotuberculosis}\) at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled \(\it katY\) expression at host body temperature.