1. The apparent transmembrane bulk-phase electrical potential ( A $ ) of Clostridium pasteurianum was determined from the distribution ratio of the membrane-permeable cation butyltriphenylphosphonium (BuPh3P+). In glycolysing cells the highest value of d $, calculated on the assumption that there was no energy-dependent binding of BuPh3P+ to the organisms, was recorded in media containing only 2-3 mM K + ions and, even so, was only 100-110mV.2. Efrapeptin, a BF,-directed inhibitor of the membrane H +-ATPase of C1. pustcuriunum, abolished the membrane potential (A$) 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 energisation in this anaerobe. 4. Whilst the addition of valinomycin to cells of C1. 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 nonglycolysing cells of Cl. pasteuvianum. Valinomycin stimulated, but butyricin 7423 inhibited, the uptake of 86Rb+ ions by glycolysing cells of C1. pasteurianum. 5.A mutant strain of Cl. pasteurianum (viz. strain DC3) which possessed a H+-ATPase with diminished sensitivity both to NJV"'dicyclohexy1carbodiimide and to butyricin 7423, exhibited a negligible decrease in A $ 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 C1. pasteurianum is not adequately explained by its ability in vitro to inhibit the membrane H+-ATPase of this organism.6. Bactericidal concentrations of butyricin 7423 neither provoked efflux of Na' ions from Cl. pasteurianum nor exhibited any protonophorous activity. However, at artificially high concentration, butyricin 7423 catalysed the passage of Na' ions as well as of K + ions through multilayer lipid membranes.7. 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.

The bacteriocin butyricin 7423 inhibited the activity of the membrane H+ -ATPase (BFoF1) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF1 component. In vitro studies with the H+-ATPases of mutant strains selected for diminished sensitivity sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BFo component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H+-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular 86Rb+ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H+-ATPase. High extracellular concentrations of K+ ions enabled Cl. pasteurianum to survive exposure to low concentration of this membrane active bacteriocin.