Social Evolution and Regulatory Architecture of Pseudomonas aeruginosa Quorum Sensing

Graduation date: 2017Cell-cell communication in bacteria is understood to facilitate the coordination of population-wide cooperative behavior in the form of concerted gene expression. The opportunistic pathogen Pseudomonas aeruginosa uses such a communication mechanism to regulate a large group of genes important to virulence strategies in this bacterium. This general mechanism of communication is termed quorum sensing (QS) and restricts activation of target genes to high cell density when cooperation is beneficial. QS in P. aeruginosa, like many Gram-negative Proteobacteria, is mediated through the synthesis of diffusible N-acyl-homoserine lactone (AHL) signals by LuxI-type synthases, and recognition by LuxR-type receptors that function as transcriptional regulators. P. aeruginosa harbors two complete AHL QS synthase/receptor pairs termed LasI/R and RhlI/R. Here we use P. aeruginosa QS as a model system to investigate mechanisms that help maintain cooperative, QS-dependent secretion in the face of non-cooperating cheater mutants, and that define the cell density threshold that triggers the activation of QS target gene expression.\ud We begin with analysis of an in vitro evolution system in which P. aeruginosa must express QS-controlled extracellular proteases in order to grow. In this system, QS-deficient cheater mutants evolve over time. They take advantage of protease production by the QS-proficient wild-type. Curiously, QS-deficient cheaters only\ud reach a frequency of about 25% during the duration of the experiment. They do not enrich to levels that would cause a collapse of the population, generally referred to as a “tragedy of the commons”. Genomic sequence analysis revealed a previously unknown mutation in this system in the transcriptional regulator PsdR. Mutations in the gene coding for PsdR derepress growth rate limiting nutrient uptake and metabolism, a non-social adaptation. Combining mutational analysis with phenotypic assays and measurements of relative fitness, we show that rapid fixation of PsdR mutation in evolving populations serves to preserve cooperation and prevent a tragedy of the commons.\ud Next, we focus on the mechanisms that determine the threshold of QS induction in P. aeruginosa. We constructed a set of isogenic mutant strains deficient in one, two, or three anti-activator proteins that serve to delay QS activation: QteE, QscR, and QslA. While these anti-activator proteins are understood to bind LasR and RhlR QS receptors, it is yet unclear why multiple anti-activators are needed, and how they work in concert to achieve the QS threshold. Using phenotypic assays, QS gene activation kinetics, and transcriptomic profiling, we found additive effects in the deletion of multiple anti-activator genes with largely overlapping sets of anti-activator-affected genes. Progressive deletion of anti-activators advances the induction threshold and increases expression levels. Our results suggest some anti-activators may even co-associate with R-proteins in exerting their effect.\ud Together, these studies contribute new mechanistic understanding of how P. aeruginosa uses QS to coordinate cooperative behaviors to specific conditions, and how this cooperative communication system may be safeguarded against social exploitation