Protein-Protein Interaction and Structure-Function Studies of XcpR, a Protein Involved in Extracellular Protein Secretion of Pseudomonas Aeruginosa
"Pseudomonas aeruginosa is an opportunistic human and animal pathogen whose virulence is dependent on its ability to secrete a number of hydrolytic enzymes and toxins. The extracellular secretion of these proteins requires at least eleven xcp gene products (XcpP-Z) and the bifunctional peptidase/methylase, PilD. Although a number of macromolecular translocation mechanisms have been found in a wide range of bacterial species that involve xcp-related gene products, functions of the Xcp proteins and the process of their assembly into a translocation apparatus have not been determined. Assembly and function of the protein secretion apparatus must involve interactions among its components, and thus, it is essential to understand the structure and function of the individual components in order to investigate their protein-protein interactions. In this thesis I describe experiments involving the generation of temperature-sensitive (ts) mutations in XcpR, a component of the secretion apparatus which contains a consensus ATP-binding site and is thought to energize the secretion process. These mutations result in a deficiency of exoprotein secretion at a non-permissible temperature, and therefore, these conditional xcpR mutations were used to isolate suppressor mutations using a novel direct selection method. In these experiments, I have isolated eight independent ts-mutations in the C-terminal half of xcpR. All of the ts-mutations have been mapped, and their nucleotide substitutions have been identified. Intragenic suppressor mutations have also been isolated for seven of the eight ts-mutants, and they restore exoprotein secretion to various degrees. These suppressors fall into two broad classes. One class is characterized as ""local"" suppressors which map to amino acid residues nearby each of the original ts-mutations, while the second class is characterized as ""global"" suppressors because they can suppress multiple ts-mutations, usually distal in the primary structure of XcpR. The implications of the results obtained from genetic suppressor analyses and further biochemical characterization of XcpR are discussed."
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign