Membrane permeabilization of Escherichia coli by Class IIa bacteriocins of Gram (+) bacteria

CHALON, MC.; BARRAZA DANIELA E.; ACUÑA, LEONARDO; SESMA F.; MORERO RD; MINAHK CJ; BELLOMIO AUGUSTO

San Miguel de Tucuman

Congreso; XLI reunión anual de la Sociedad Argentina de Biofísica; 2012

INSIBIO-CONICET-UNT

Membrane permeabilization of Escherichia coli by Class IIa bacteriocins of Gram (+) bacteria Chalón MC, Barraza D, Acuña L, Sesma F1, Morero RD, Minahk CJ, Bellomio A. INSIBIO (CONICET-UNT). Inst. de Química Biológica. Fac. de Bioqca. Qca. y Fcia UNT. 1 CERELA (CONICET). San Miguel de Tucumán. E-mail: chalon@fbqf.unt.edu.ar Class IIa bacteriocins secreted by Gram (+) bacteria act on the cell membrane of phylogenetically related strains to the producer strain. They bind to a membrane protein which plays as a specific receptor and cause membrane depolarization and loss of small organic molecules of the target cell. It is not known exactly what the function of the receptor is, and it is speculated that it could act as an anchor allowing the approximation of the bacteriocin to the membrane. A subsequent insertion of an amphipathic helix into the hydrophobic core of the membrane takes place. Bacteriocin producing cells synthesize an immunity protein that protects them somehow preventing the action of the bacteriocin. It was demonstrated that when the bacteriocin is added from the extracellular medium forms a ternary complex, bacteriocin-receptor- immunity protein. Gram (-) bacteria lack the receptor and are therefore insensitive to class IIa bacteriocin. To better understand the mechanism of insertion of the bacteriocins into the membrane and the role of the receptor, we fused class IIa bacteriocins to the C-terminal domain of EtpM, an integral monotopic membrane protein of E. coli, under the control of the pBAD promoter. Therefore, EtpM is translocated to the periplasm with the bacteriocin and this fusion protein remains anchored by the N-terminal in the membrane upon induction with arabinose. EtpM-bacteriocin induces a decrease in the cell viability of the cells expressing the fusion. Interestingly, the co-expression of the immunity protein protects from this effect. Furthermore, we investigated the membrane potential before and after inducing the expression of the fusion ETPM-bacteriocin. It was observed that the expression of the fusion polypeptide leads to the loss of the membrane potential. Once again, the coexpression of the immunity protein prevents the electrochemical gradient dissipation. The results obtained indicate that the anchorage of the bacteriocin to the membrane is sufficient to insert a class IIa bacteriocin into the cell membrane. Furthermore, the immunity protein acts efficiently avoiding the dissipation of the membrane potential even in the absence of a specific receptor, indicating that the bacteriocin-protein complex could be formed even in the absence of the receptor.