Elastase, coded by the lasB gene, is an important virulence determinant of Pseudomonas aeruginosa. Development of a model of lasB induction is essential to understanding the global induction of virulence factors which contributes to morbidity and mortality from acute and chronic P. aeruginosa infections. P. aeruginosa uses a bacterial cell density-dependent ("quorum sensing") system of gene activation to induce three protease structural genes, including lasB. This activation is mediated by the complex of a regulatory protein (LasR) and an N-acylhomoserine lactone, or autoinducer (PAI-1). Two operators are present in the regulatory region of lasB, one of which (OP1) is responsive to the LasR/PAI-1 complex. Preliminary data suggest that the two operators function synergistically to activate lasB, and that the second operator (OP2) is a minor OP l-like operator. Gel shift and DNase I footprinting assays will be used to elucidate the roles of LasR/PAI-1, OP1, and OP2 in lasB activation. The experiments outlined will determine whether lasB is a class II promoter, and whether the function of OP2 is to facilitate cooperative protein-protein binding. Specific aims are: 1) To determine the function of OP1 in lasB activation: a) Does LasR bind OP1?, b) Does LasR interact with RNAP alphaCTD in a class II-fashion?, and, c) Does LasR directly facilitate isomerization? 2) To determine the function of OP2 in lasB activation: a) Does LasR bind OP2?, b) Do LasR at OP2 and RNAP bind cooperatively?, and, c) Does LasR bind OP1 and OP2 cooperatively?