Project description:In this study, we used transcriptional profiling to define the adaptive response of C. violaceum to oxidative stress induced by cumene hydroperoxide (CHP) and to identify the OhrR regulon. DNA microarray and northern blot analysis revealed that in CHP-treated cells occur strong upregulation of genes involved in many pathways for stress protection, including antioxidant enzymes (catalase and peroxidases), thioredoxin, glutaredoxin and lipoyl-dependent reducing systems, DNA repair enzymes, heat shock response (σ32 regulon), iron limitation (Fur regulon) and nitrogen starvation. Genes encoding glyoxalases, glutathione S-transferases and oxygenases were also induced, suggesting further catabolism of the aromatic compound CHP. Pathways downregulated by CHP stress include electron transport chain, nucleotide biosynthesis and unsaturation of fatty acids. Further, we identified two upregulated genes (OhrA and a protein with GGDEF domain for c-di-GMP synthesis) and three downregulated genes (hemolysin, chitinase and collagenase) in the ohrR mutant. Using a mouse infection model, we demonstrate that the ohrR mutant, but not the ohrA mutant, is attenuated for virulence and showed a decreased bacterial burden in the liver. Therefore, we have defined the CHP stimulon and determined that C. violaceum uses the organic hydroperoxide sensor OhrR for regulate expression of genes required to antioxidant defense and to modulate virulence in its interaction with the host. Overall design: Two experimental procedures comparing WT control vs. WT CHP or WT control vs. ohrR mutant. Each comparison was performed using three biological replicates.