Modifications in membrane potential and permeability by expression of suicide probes EtpM-bacteriocin and its immunity protein on E. coli

RÍOS COLOMBO NS; CHALÓN MC; BELLOMIO A

San Miguel de Tucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) ? IX IberoAmerican Congress of Biophysics ? XLV Reunion Anual SAB; 2016

SAB

p { margin-bottom: 0.25cm; line-height: 120%; }a:link { }Enterocin CRL35 andPediocin PA-1 are pediocin-like bacteriocins that act onGram-positive bacteria membrane. In order to study the mechanism bywhich they induce loss of membrane integrity, in previous work weconstructed "suicide probes": fused genes of bacteriocinswith EtpM, a bitopic membrane protein. We demonstrated that thesebacteriocins are able to exert a bactericidal effect on Gram-negativebacteria when anchored in the membrane, regardless of their specificreceptor (Man-PTS system). The aim of this study was to evaluate theeffect of these bacteriocins on cell membrane potential andpermeability, when expressed as suicide probes in Gram-negativebacteria such as E. coli. On the one hand, we employed thepotentiometric indicator DiSC3(5), a fluorophore that exhibitschanges in fluorescence intensity. These changes are dependent ontransmembrane potential, and they are feasibly to be measured overtime. The results showed that the expression of fusionsEtpM-bacteriocins generates potential dissipation by membranedepolarization. In contrast, neither the control strain thatexpressed only EtpM (membrane anchor), nor the strain thatco-expresses the suicide probe with Enterocin CRL35 immunity protein(MunC) showed a significant change in membrane potencial. On theother hand, we used LIVE/DEAD BacLight kit, which includes twofluorophores that penetrate the membrane according to its integrity.This allows to discriminate between live and dead cells. The imagesobtained by fluorescence microscopy clearly evinced that suicideprobes expression disturbs membrane permeability, so bacteria emittedred fluorescence (dead cells), while control and co-expressingEtpMbacteriocin / MunC strains conserve membrane integrity, so theyemitted green fluorescence (live cells).