TR Purple Quorum Sensing

Background  Quorum-sensing is a method of decentralized cell-cell communication commonly used by bacteria to initiate multiple-cell activities such as forming bacterial colonies (otherwise known as biofilms), cell growth and cellular attack (infections). Quorum-sensing has also been implicated in the environmental sensing mechanisms of bacteria.  Quorum-sensing is influenced by chemical signals called autoinducers (related to cytokines) that have been found to be unique to each bacteria species. Evidence shows that each bacterium can differentiate between its own released autoinducers and those of other bacteria. The two purposes of quorum-sensing have hence been proposed: as a cell-cell communication mechanism (the Miller-Bassler hypothesis) and an environmental sensor (the Redfield hypothesis).   Problem  The activities mediated by quorum-sensing via a chemical pathway make it a good candidate for a new generation of antibiotics, either by inhibiting the binding of the autoinducer signals or by changing the pathway itself. Quorum-sensing can even be used to “rewire” bacteria in order to create new biological circuits, better biofilms, or more effective drug vectors. We will investigate the inhibition or promotion of autoinducer binding to bacteria receptors through protein and genetic engineering.   Proposed Methods  We will be using a widely studied quorum-sensing model bacteria V. harveyi to investigate quorum-sensing as the signaling pathway has been elucidated - using the autoinducer-2 and the LuxN protein. Our goal will be to induce mutagenesis in the suspected binding motifs of receptor proteins involved with binding autoinducer-2 (Module 1). We will compare signals to the following controls: (1) a sample that has had the autoinducer-2 producing gene silenced (Module 2), (2) a sample that has a competitive-inhibiting chemical added, (3) a mock, (4-optional) a sample with a known mutation in the quorum-sensing pathway. Assays used will be a gas chromatography, mass spectrometry compared analysis to detect autoinducers, gene sequencing and gel analysis (to make sure our mutagenesis is doing what it’s supposed to do), protein assays (i.e. Western; to make sure our receptors are being produced properly), and a biofilm-based model using optical tweezers (to keep cells in a specific, reproducible environmental condition).   References  1.	Swem LR, Swem DL, Wingreen NS, Bassler BL. Deducing receptor signaling parameters from in vivo analysis: LuxN/AI-1 quorum sensing in Vibrio harveyi. Cell. 2008 Aug 8;134(3):390-1.  2.	Long T, Tu KC, Wang Y, Mehta P, Ong NP, Bassler BL, Wingreen NS. Quantifying the integration of quorum-sensing signals with single-cell resolution. PLoS Biol. 2009 Mar 24;7(3):e68.  3.	Williams JW, Cui X, Levchenko A, Stevens AM. Robust and sensitive control of a quorum-sensing circuit by interlocking feedback loops. Mol Syst Biol. 2008;4:234.  4.	Timp W, Mirsaidov U, Matsudaira P, Timp G. Jamming prokaryotic cell-to-cell communication in a model biofilm. Lab Chip. 2009 Apr 7;9(7):925-34.