The Pseudomonas aeruginosa PrrF1 and PrrF2 Small Regulatory RNAs Promote 2-Alkyl-4-Quinolone Production through Redundant Regulation of the antR mRNA

is an opportunistic Gram-negative pathogen that requires iron for growth and virulence. Under low-iron conditions, transcribes two highly identical (95%) small regulatory RNAs (sRNAs), PrrF1 and PrrF2, which are required for virulence in acute murine lung infection models. The PrrF sRNAs promote the production of 2-akyl-4(1 )-quinolone metabolites (AQs) that mediate a range of biological activities, including quorum sensing and polymicrobial interactions. Here, we show that the PrrF1 and PrrF2 sRNAs promote AQ production by redundantly inhibiting translation of , which encodes a transcriptional activator of the anthranilate degradation genes. A combination of genetic and biophysical analyses was used to define the sequence requirements for PrrF regulation of , demonstrating that the PrrF sRNAs interact with the 5' untranslated region (UTR) at sequences overlapping the translational start site of this mRNA. The Hfq protein interacted with UA-rich sequences in both PrrF sRNAs ( [dissociation constant] = 50 nM and 70 nM). Hfq bound with lower affinity to the mRNA (0.3 μM), and PrrF was able to bind to mRNA in the absence of Hfq. Nevertheless, Hfq increased the rate of PrrF annealing to the UTR by 10-fold. These studies provide a mechanistic description of how the PrrF1 and PrrF2 sRNAs mediate virulence traits, such as AQ production, in The iron-responsive PrrF sRNAs play a central role in regulating iron homeostasis and pathogenesis, yet the molecular mechanisms by which PrrF regulates gene expression are largely unknown. In this study, we used genetic and biophysical analyses to define the interactions of the PrrF sRNAs with Hfq, an RNA annealer, and the mRNA, which has downstream effects on quorum sensing and virulence factor production. These studies provide a comprehensive mechanistic analysis of how the PrrF sRNAs regulate virulence trait production through a key mRNA target in .