Project Summary/Abstract Staphylococcus aureus is a problematic human bacterial pathogen, which is broadly resistant to b-lactam antibiotics. This resistance is inducible and is conferred by a set of genes that encode a b-lactam antibiotic sensor/signal transducer protein, a gene repressor and two resistance determinants (the BlaZ b-lactamase and a unique penicillin-binding protein designated as PBP2a). A key feature of these processes is the recognition of the antibiotic by the b-lactam sensor/signal transduce BlaR, an integral membrane protein. Recognition of the antibiotic by the sensor domain of BlaR unleashes conformational changes through the membrane, which lead to the activation of the protease domain on the cytoplasmic side. This process culminates in expression of the genes for the antibiotic-resistance determinants. In Specific Aim 1, we describe the use of a fluorescent tool in live S. aureus for discovery of agents that shut down the BlaR recognition of the b-lactam antibiotics, which would reverse the resistance phenotype. A discovery funnel is outlined for analysis of structure-activity relationship for these compounds. In Specific Aim 2 we communicate a discovery that the BlaZ b-lactamase is incorporated to the surface of the cytoplasmic membrane in a lipidation- and phosphorylation-dependent manner. We outline a method for purification of the membrane-anchored BlaZ for the purpose of the identification of the sites of phosphorylation. The protein will be used in kinetic studies to compare to the non- membrane-anchored BlaZ, which is not phosphorylated. Plans are detailed for X-ray crystallography for characterization of the structural issues. The generality of the lipidation- and phosphorylation-dependent anchoring of proteins to the membrane surface in S. aureus will be explored for 10 distinct proteins. In each case, plans are outlined to identify the sites of phosphorylation to elucidate rules for phosphorylation and membrane sequestration of these proteins. These studies will shed definitive light on the complex machinery that S. aureus strains have evolved for resistance to b-lactam antibiotics.