Topological study of the beta-lactam-sensor proteins from Methicillin resistant Staphylococcus aureus (MRSA)

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

Villa Carlos Paz

Congreso; XLII Reunion Anual de la Sociedad Argentina de Biofisica; 2013

Sociedad Argentina de Biofisica

The membrane proteins BlaR1 and MecR1 play key roles in resistance to beta-lactam antibiotics in MRSA strains. It is proposed that they sense and transduce the signal to unleash the transcription of the resistance genes.1 However, the topology of these proteins and the molecular events that are involved in the transmission of the signal remain controversial. Here we report a topology study of the proteins expressed in E. coli (where we have previously shown that BlaR1 is active) based in a strategy that involves the construction of C-terminal fusions to EGFP.2 This reporter does not fold correctly when translocated to the periplasmic space through the Sec System and, therefore, it does not fluoresce when fused to a periplasmic loop. In order to determine the number of membrane-spanning domains and the localization of the different domains, we characterized the different fusion proteins by fluorescence spectroscopy in whole cells and by fluorescence microscopy, and we normalized the expression of the different constructions in E.coli by Western blot analysis. Acknowledgements The PEW Charitable Trusts, ANPCyT, CONICET   References Llarrull, L.I., Fisher, J.F., Mobashery, S. Antimicrob. Agents and Chemother. 2009. 4051. McKenzie, N.L., Nodwell, J.R. Microbiology. 2009. 1812.