RNA-seq analysis of Ralstonia solanacearum nitrosative stress response mutants

Ralstonia solanacerum causes bacterial wilt disease on many important host crops, including tomato and potato. R. solanacearum cells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient and oxygen content of xylem sap, R. solanacearum grows extremely well inside the host, using denitrification to respire in this hypoxic environment. R. solanacearum growth in planta also depends successful mitigation of oxidative stress produced by both the bacterium and the host. We found that proteins encoded by norA and hmpX that are predicted to interact with nitric oxide (NO) and protect against cellular damage caused by NO. Reducing NO toxicity is especially important for Rs that generates NO via denitrifying respiration in planta, and encounters NO produced by plants as a defense and signaling molecule. By analyzing transcriptomes of mutants lacking these genes, we found that iron, sulfur, and nitrogen metabolism genes were consistently upregulated in mutants missing these genes. Our transcriptomic analysis suggests R. solanacearum mutants lacking norA and hmpX suffer oxidative stress from an increase in cellular toxicity caused by NO that leads to damage of iron, sulfur, and nitrogen metabolic proteins. Our results suggest that NorA and HmpX, contribute to oxidative stress mitigation in denitrifying cultures. Overall design: We analyze gene expression of WT R. solanacearum strain GMI1000 and three GMI1000 mutant strains lacking genes involved in the nitrosative stress response. ?norA and ?hmpX missing NO binding protein, in denitrification promoting low-oxygen conditions to induce nitrosative stress in culture. Each treatment or mutant included three biological replicates from separate overnight R. solanacearum cultures.