| Quorum sensing is a chemical communication process bacteria use to control collective behaviors. Quorum-sensing bacteria produce, secrete, and detect extracellular molecules called autoinducers to monitor cell density. In the bioluminescent marine bacterium Vibrio harveyi, five small regulatory RNAs, called Qrr1-5 (for quorum regulatory RNAs), lie at the center of the quorum-sensing pathway and control quorum-sensing transitions. The Qrr sRNAs post-transcriptionally regulate multiple mRNA targets: They activate the low-cell-density master transcriptional regulator aphA and repress the high-cell-density master transcriptional regulator luxR. They repress the autoinducer synthase luxM, the receptor luxN, and the transcriptional activator luxO..;The aim of this work is to discover whether the Qrr sRNAs have roles beyond controlling quorum-sensing components and if so, to define the mechanisms used by the five Qrr sRNAs to control their different targets, and to understand the function each portion of the Qrr sRNAs plays in regulation. This work identifies sixteen novel target mRNAs of the Qrr sRNAs and shows that genes that are directly controlled by the Qrr sRNAs are the most rapid to respond to quorum-sensing autoinducers. This work also shows that the Qrr sRNAs use four distinct mechanisms to control their particular targets: the Qrr sRNAs repress luxR through catalytic degradation, repress luxM through coupled degradation, repress luxO through sequestration, and activate aphA by revealing the ribosome-binding site while the sRNAs themselves are degraded. The particular regulatory mechanism is determined by the different base-pairing interactions between the Qrr sRNAs and each mRNA target. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response. The Qrr sRNAs each possess four predicted stem-loops. This work shows that each stem-loop plays a different role in target regulation and Qrr sRNA stability: the first two stem-loops play major roles in base-pairing with target mRNAs and the first stem-loop is also crucial for protecting the Qrr sRNAs from RNase E-mediated degradation. The third stem-loop plays an accessory role in base-pairing and stability. The fourth stem-loop functions as a rho-independent terminator. |
