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

Abstract

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.

Growth inhibition of putrefactive anaerobe 3679 caused by stringent-type response induced by protonophoric activity of sorbic acid

“…Dimethyloxazolidinedione used for pHi determinations penetrated the cells within 1 min and remained at a constant level for at least 10 min. For pH, calculations, the internal water volume determined from the weight difference between wet and dry cells (59) was 0.026 Iul per 107 cells, being identical with that reported for C. pasteurianum (8). (Table 4), an amino acid that is specifically required by PA 3679 strain L (5).…”

Uncoupling by Acetic Acid Limits Growth of and Acetogenesis by Clostridium thermoaceticum

“…Also, the ApH of C. thermoaceticirm was relatively resistant to dissipation in the presence of oxygen, even though these cells are obligate anaerobes. It is probably for this reason that in C. pasteurianium a proton electrochemical gradient could be demonstrated, even though the cells were briefly exposed to air during preparation or sampling (2,5,29).…”

Proline reduction byClostridium sporogenesis coupled to vectorial proton ejection

“…For this reason it is premature to enquire into the source of the alkalinisation preceding the prolineinduced acidification, although it could be explained by a proline-H ÷ symport or by the combined activity of a cation-proline symport with a cation-H + antiport. Amongst the clostridia, it is known that Clostridium pasteurianum possesses an active K ÷ uniporter [19], which would serve to explain why the addition of an ionophore such as valinomycin or a 'permeant' ion such as thiocyanate was not required to observe proton motive activity. Indeed, neither treatment increased the net observable stoichiometry of proline-induced proton translocation (data not shown).…”

“…These were from previously disclosed sources [19,20]. Tetrachloro salicylanilide (TCS) was a generous gift from Professor W.A.…”