Bacteria communicate through a process known as quorum sensing, in which small chemical signals are produced and released into the environment. As the population increases, these signals (called autoinducers) accumulate and, when they reach sufficient concentration, are detected by other bacteria. Bacteria modulate gene expression in response to these signals, and thus are able to regulate their behavior in a population-wide manner [72-79]. Many important behaviors are regulated by quorum sensing, including virulence, motility, and biofilm formation [80-83]. While most autoinducers mediate communication within a given bacterial species, a novel signal molecule, termed autoinducer-2 (AI-2), appears to be a universal signal molecule, facilitating signaling between species [25, 78]. Many species of bacteria are known to carry the gene for LuxS, the AI-2 synthase, including pathogens such as Salmonella typhimurium, Bacillus anthracis, Yersinia pestis, and Vibrio cholerae [84]. Thus, AI-2 mediated quorum sensing offers a possible mechanism for controlling bacterial behavior of significant human pathogens [33, 34, 37, 46, 47, 85]. A variety of bacterial species, including Escherichia coli, B. anthracis, and S. typhimurium have the lsr operon, a set of genes responsible for recognizing, internalizing, and processing AI-2 [20, 21]. Once internalized, AI-2 is phosphorylated by LsrK, giving rise to phospho-AI-2 [5]. Two proteins encoded in the lsr operon, LsrF and LsrG, further process phospho-AI-2, but the details of these reactions are not understood at the molecular level [21]. By internalizing and modifying the signal molecule, these bacteria interfere with the AI-2 mediated communication of other species, gaining a competitive advantage. This work will investigate the mechanisms and products of the AI-2 processing reactions catalyzed by E. coli LsrF and LsrG and a putative functional homolog of LsrF from Sinorhizobium meliloti. Cell extracts will be analyzed by mass spectrometry and NMR to identify the products of these reactions. Potential catalytic residues have been identified through structural analysis of these proteins; these residues will be mutated and the resulting proteins and protein/ligand complexes studied by x-ray crystallography. Mutants will be screened for catalytic activity in NMR-based in vivo assays that monitor rates of uptake of 13C-labeled AI-2. The work proposed here seeks to characterize, structurally and functionally, the proteins responsible for degrading P-AI-2 and thus could be of great utility in developing therapies that exploit AI-2 mediated quorum sensing as a means to control bacteria. PUBLIC HEALTH RELEVANCE: Bacteria, including many human pathogens, communicate via small chemical signals, coordinating behaviors such as virulence and biofilm formation in a population-wide manner. This work investigates the structure and function of proteins involved in processing an interspecies signal molecule called autoinducer-2. Understanding the processing of this signal molecule is an important step towards exploiting this communication to control bacterial behavior.