Assessing the Importance of Stem-Loop One in Stability of and Target Regulation by Qrr4 in the Quorum Sensing Pathway

Abstract

Quorum sensing is a cell-to-cell communication process performed by bacteria such as Vibrio harveyi to transition between individual and group activities. The pathway is initiated by changes in concentrations of extracellular molecules called autoinducers that induce a signal transduction cascade. Five Qrr small RNAs lie at the heart of the signaling cascade. They function to establish gene expression patterns by controlling the translation of the mRNA encoding the transcription factors, LuxR and AphA, the two master regulators central in the quorum sensing circuit. The Qrr1-5 sRNAs are each predicted to contain four stem-loops, and they regulate targets through four distinct mechanisms. Previous work developed and used a method called RSort-Seq to determine the importance of nucleotides within the Qrr4 sequence for specific target regulation. Surprisingly, this method revealed that individual mutations in stem-loop one (SL1) do not perturb Qrr4 function, even though SL1 has been proven to be critical for Qrr stability. To further understand the contribution of SL1 to Qrr4 function, the individual nucleotide analysis was extended to understand the role of particular nucleotides in SL1 in stability and base pairing. Using GFP screens and rifampicin stability assays, six nucleotides were studied in depth to show that RSort-Seq alone could not reveal individual SL1 nucleotides important for stability. Additionally, two bases were found to be responsible for differentiating between different sRNA regulatory mechanisms of repression.