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

RIOS COLOMBO, N.S.; CHALON M.C.; BELLOMÍO A

San Miguel de Tucuman

Congreso; Reunion Anual de la Sociedad Argentina de Biofisica; 2016

Sociedad Argentina de Biofisica

EnterocinCRL35 and Pediocin PA-1 are pediocin-like bacteriocins that act on Gram-positive bacteria membrane. In order to study the mechanism by which they induce loss of membrane integrity, in previous work we constructed "suicide probes"1: fused genes of bacteriocins with EtpM, a bitopic membrane protein. We demonstrated that these bacteriocins are able to exert a bactericidal effect on Gram-negative bacteria when anchored in the membrane, regardless of their specific receptor(Man-PTSsystem).The aim of this study was to evaluate the effect of these bacteriocins in cell membrane potential and permeability, when expressed as suicide probes in Gram-negative bacteria such as E. coli.On the one hand, we employed the potentiometric indicator DiSC3(5),a fluorophore that exhibits changes in fluorescence intensity2. These changes are dependent on transmembrane potential Δψ, and they are feasibly to be measured overtime. The results showed that the expression of fusions EtpM-bacteriocins generates Δψ dissipation by membrane depolarization. In contrast, neither the control strain that expressed only EtpM (membrane anchor), nor the strain that co-expresses the suicide probe with Enterocin CRL35immunity protein (MunC) showed a significant change in membrane potential.On the other hand, we used LIVE / DEAD BacLigh tkit, which includes two fluorophores that penetrate the membrane according to its integrity. This allows to discriminate between live and dead cells3. The images obtained by fluorescence microscopy clearly evinced that suicide probes expression disturbs membrane permeability, so bacteria emitted red fluorescence (dead cells), while control and co-expressing EtpM-bacteriocin/MunC strains conserve membrane integrity, so they emitted green fluorescence (live cells).