Dissecting communication in microbial communities

The aim of our work is to identify novel molecules that interact with quorum sensing systems mediated by N-acyl homoserine laetone (AHL) signal molecules and to study the role of quorum sensing in the environments where these signals are produced. Initially, we surveyed libraries of synthetic compounds and metagenomic libraries for inducers and inhibitors of quorum sensing. I developed a high throughput screen to identify small molecules that activate or inhibit AHL-mediated cell-cell communication. In a library of 16,000 structurally diverse compounds, I identified nine antagonists and fourteen agonists of LasR-mediated quorum sensing. I also devised an intracellular screen to identify metagenomic clones that induce or inhibit a quorum sensing biosensor that a traditional overlay screen would not have detected. This screen identified a novel signal mimic from the uncultivated microbiota residing in the gypsy moth midgut, novel LuxIR homologues, and a functional LuxIR regulon with extremely low similarity to known LuxIR homologues from microorganisms in soil. This work expands our understanding of quorum sensing to include the uncultivated bacteria, which represent the majority of bacteria in most environments.; In order to gain a better understanding of the role of communication in a community of bacteria, I studied the effects of disrupting signaling in the bacterial community residing in the cabbage white butterfly (CWB) larval midgut. As an initial step to determine whether AHL signals are exchanged in the highly alkaline environment of the midgut, I constructed AHL-sensing bioluminescent reporter strains in the wild type Pantoea and an AHL-deficient mutant of this strain. The biosensors detected AHLs produced and exchanged by Pantoea in the midgut of CWB larvae. I also examined the role of signals in pathogenesis produced by Pseudomonas aeruginosa PA01. Mortality induced by P. aeruginosa PA01 was significantly reduced by interfering with signaling either by introducing a P. aeruginosa signaling mutant or administering a potent inhibitor of quorum sensing to the insect. These results are the first report of the functional exchange of AHLs among bacteria in the alkaline environment of the CWB larval midgut.