Iron and peroxide regulation of the PrrF sRNAs and a conserved Dps-like protein in Pseudomonas aeruginosa and Pseudomonas fluorescens

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron to cause infection. Iron can also be toxic due to its participation in Fenton chemistry, resulting in the production of reactive oxygen species (ROS). Thus, P. aeruginosa regulates the uptake, use, and storage of iron to mitigate the effects of ROS. P. aeruginosa uses several mechanisms to manage oxidative stress, including superoxide dismutases to detoxify the superoxide radical, catalases to break down H2O2, and members of the ferritin superfamily to store iron. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are predicted to pair with and destabilize mRNA transcripts for several oxidative stress response proteins, including sodB, katA, and brnD, encoding a novel bacterioferritin-like Dps protein. In this study, we developed brnD reporter constructs that are responsive to PrrF-mediated iron regulation. We demonstrated that PrrF-mediated regulation of brnD occurs in the 5 untranslated region (UTR) of its mRNA, likely via a conserved region of complementarity with the PrrF sRNAs. We further demonstrated that brnD mRNA levels are increased upon hydrogen peroxide treatment. Surprisingly, peroxide treatment also resulted in elevated levels of the PrrF sRNAs, and this induction occurred specifically at the prrF2 promoter. Lastly, we investigated these regulatory effects in Pseudomonas fluorescens, revealing similar iron regulation of PrrF sRNAs and the brnD ortholog, as well as peroxide-induced expression of the PrrF sRNAs. Combined, these data highlight a distinct class of iron-responsive Dps-like proteins with potential functional conservation across the pseudomonads, and they reveal a novel aspect of the oxidative stress response involving the PrrF sRNAs. IMPORTANCEIron is a crucial micronutrient for Pseudomonas aeruginosa survival and virulence, yet it can also be toxic due to production of reactive oxygen species. The PrrF sRNAs are produced in iron limiting conditions and block the expression of several mRNAs involved in P. aeruginosas oxidative stress response. Here, we make the surprising discovery that oxidative stress induces expression of the PrrF sRNAs. We also provide evidence that PrrF regulation of two mRNA targets is hindered upon oxidative stress. Oxidative stress similarly induced the PrrF sRNAs in Pseudomonas fluorescens, indicating conservation of this novel regulatory event. This study therefore highlights a novel and conserved regulatory link between iron homeostasis and oxidative stress protection in the pseudomonads.