Study of the topology of the beta-lactam sensors/transducers BlaR1 and MecR1 from Staphylococcus aureus

BELLUZO, B.S.; PERIS, D.; LLARRULL, L.I.*

Rosario

Congreso; L Reunion Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2014

The PC1 beta-lactamase and the PBP2a transpeptidase represent the main line of defense of Methicillin-Resistant Staphylococcus aureus (MRSA) to beta-lactam antibiotics. The level of expression of these two proteins is regulated by the sensor/transducer membrane proteins BlaR1 and MecR1, respectively. These two proteins are embedded in the plasma membrane and are thought to sense the presence of the antibiotic and to subsequently deliver a signal that triggers both systems, by mechanisms still not fully understood. The topology of BlaR1 and MecR1 is a key to unveiling the signal transduction pathway. We studied the topology of BlaR1 and MecR1 in E. coli by constructing C-terminal fusions to EGFP. When the reporter protein EGFP is translocated across the cytoplasmic membrane unfolded through the Sec system, it does not recover its fluorescence. The characterization of the different fusion proteins by fluorescence spectroscopy in whole cells and by fluorescence microscopy allowed us to determine the topology of the proteins. Using mutants in the Sec and Tat translocation systems we verified that the insertion of these proteins in the membrane depends on the Sec translocon. We also tested the ability of full length proteins to bind the fluorescent penicillin Bocillin-FL in intact cells, and we hence corroborated the periplasmic localization of the C-terminal sensor domain in E. coli.