The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis

Abstract

Pathogenic bacteria, such asYersinia pseudotuberculosisencounter 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. ThetrxAgene 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 ofsodB,sodCandkatA. However, they barely conferred translational repression inY.pseudotuberculosisat 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region ofkatYwe 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 H2O2scavenger. Consistent with the upregulation ofkatY, we observed an improved protection ofY.pseudotuberculosisat 37°C. Our findings suggest a multilayered regulation of the oxidative stress response inYersiniaand an important role of RNAT-controlledkatYexpression at host body temperature.