On the Mode of Action of the Bacteriocin Butyricin 7423. Effects on Membrane Potential and Potassium-Ion Accumulation in Clostridium pasteurianum

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Abstract
The apparent transmembrane bulk-phase electrical potential (.DELTA..psi.) of C. pasteurianum was determined from the distribution ratio of the membrane-permeable cation butyltriphenylphosphonium (BuPh3P+). In glycolyzing cells the highest value of .DELTA..psi., calculated on the assumption that there was no energy-dependent binding of BuPh3P+ to the organisms, was recorded in media containing only 2-3 mM K+ ions and, even so, was only 100-110 mV. Efrapeptin, a BF1-directed inhibitor of the membrane H+-ATPase [EC 3.6.1.3] of C. pasteurianum, abolished the membrane potential (.DELTA..psi.) and caused complete efflux of actively-transported K+ ions. Thus protonmotive hydrolysis of ATP generated by substrate level phosphorylation is the sole means of membrane energization in this anaerobe. At low (sublethal) concentrations, butyricin 7423 stimulated K+ efflux from C. pasteurianum without measurably affecting its membrane potential. At lethal and supralethal concentrations of this bacteriocin, both .DELTA..psi. and active K+ uptake were abolished. While the addition of valinomycin to cells of C. pasteurianum suspended in media of low K+ concentration generated a diffusion potential to which BuPh3P+ would respond, addition of butyricin 7423 in place of valinomycin caused no such effect. Also, unlike valinomycin, butyricin 7423 did not increase the rate of K+ efflux from non-glycolyzing cells of C. pasteurianum. Valinomycin stimulated, but butyricin 7423 inhibited, the uptake of 86Rb+ ions by glycolyzing cells of C. pasteurianum. C. pasteurianum mutant strain DC 3, which possessed a H+-ATPase with diminished sensitivity both to N,N''-dicyclohexylcarbodiimide and to butyricin 7423, exhibited a negligible decrease in .DELTA..psi. and in K+ accumulation ratio in response to concentrations of butyricin 7423 that were bactericidal to the wild-type, parent organism. Even so, the bactericidal action of butyricin 7423 on C. pasteurianum is not adequately explained by its ability in vitro to inhibit the membrane H+-ATPase of this organism. Bactericidal concentrations of butyricin 7423 neither provoked efflux of Na+ ions from C. pasteurianum nor exhibited any protonophorous activity. At artificially high concentration, butyricin 7423 catalyzed the passage of Na+ ions as well as of K+ ions through multilayer lipid membranes. As a non-protonophorous uncoupler, butyricin 7423 appears to act in a similar manner to that of the membrane-active colicins. Yet no evidence was obtained that butyricin 7423 at its minimum lethal concentration might form a gated ion channel in the cytoplasmic membrane of the target cell, or act as a classic ionophore.