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The overall goal of this application is to determine the molecular mechanisms of quorum sensing and the role of autoinducer-2 (AI-2) in modulating Campylobacter adaptation to various environments. <P> Aim 1: Define the effect of the G92D amino acid substitution on the enzymatic function of LuxS. <P> Aim 2: Characterize a two-component sensor system and its regulation by the presence of AI-2 in the growth media of Campylobacter. <P> Aim 3: Examine the effects of the luxS null mutation on the adaptation of Campylobacter to various environments using in vitro and in vivo experiments.
APPROACH: Specific aim 1 will take advantage of the AI-2 deficient 81116 culture to evaluate the effect of the G92D substitution on the enzymatic activity of LuxS. This naturally occurring AI-2 deficient strain demonstrates a phenotypic change in motility upon mutagenesis of the luxS gene similar to that previously associated with the loss of luxS in other AI-2 producing strains of Campylobacter. These findings provide a strong rationale for additional work to understand the role of the amino acid G92 in the function of the LuxS enzyme. Site-directed mutagenesis and the AI-2 reporter assay will be used to demonstrate the specific role of G92 in AI-2 production. Additionally, in order to address both the metabolic and AI-2 synthase function of the mutant LuxS, the S-ribosylhomocysteinase activity of the recombinant mutant enzyme will be compared to that of the recombinant wild-type enzyme.<P> In specific aim 2 we will focus on confirming the regulation of Cj0889c-Cj0890c by AI-2 and characterizing the role of the two-component sensor system in gene regulation and environmental adaptation. <P>Finally, specific aim 3 will provide a comprehensive evaluation of in vitro and in vivo phenotypic changes associated with the mutation of luxS in Campylobacter. Conventional and germ-free animals will be utilized in this specific aim in order to control for AI-2 interference by normal enteric flora. When completed, the findings will significantly improve our understanding of the quorum sensing mechanism in Campylobacter. In all experimental designs, the activity of AI-2 will be monitored using the Vibrio harveyi AI-2 reporter assay. Appropriate controls and statistical evaluation of the data will be utilized when necessary.