Experimental and Theoretical Investigation of Signaling in Quorum Sensing of Pseudomonas Aeruginosa

Quorum sensing (QS) is an intercellular communication system by which some bacterial cells are capable of indirectly monitoring their own population density through exchange of signal molecules. The expression of virulence factors is kept low until the population density (signal molecule concentration) reaches a threshold value, after which the host system is surprised by a stealth attack. The focus of this study is on the Quorum Sensing regulatory system of Pseudomonas aeruginosa called the Las system.
In this thesis, two distinct methods to obtain information about the system are considered. First, Surface Enhanced Raman Spectroscopy (SERS) is applied to the signal molecule known as N -dodecanoyl-L-homoserine lactone. To the best of our knowledge we here for the first time demonstrate SERS to detect a QS signal molecule below 1 nM concentration in both ultrapure water and under physiological conditions. Based on our results, SERS shows promise as a highly suitable tool for in situ measurements of low Acyl-Homoserine Lactone (AHL) concentrations in biofilms containing QS bacteria. Secondly, a conventional fluorescent monitor system is used to follow the response to signal molecules. A kinetic model is applied in order to extract knowledge about the interaction between signal molecule and regulator. In the model, the regulator monomer is decaying rapidly due to proteases while all dimerized regulators are protected against proteases. Moreover, the LasR regulator can fold into its stable dimer conformation in the absence of its cognate signal. In the presence of signal the dimer is activated through cooperative binding of the two signal molecules.