The cationic peptide antibiotics Pep 5, nisin and subtilin depolarize bacterial and artificial membranes by formation of voltage-dependent multi-state pores. Studies with non-energized liposomes indicated that the peptides do not span the membrane in the absence of a membrane potential. The effects of Pep 5 and nisin on neutral membranes, as studied by membrane fluidity, phase transition points and carboxyfluorescein e&x, were small compared to melittin. Acidic liposomes were affected more strongly, indicative of primarily electrostatic interactions with phospholipid head groups. Subtilin may slightly enter the hydrophobic core as suggested by tryptophan fluorescence quenching and liposome fusion experiments.

The peptide antibiotic subtilin was shown to induce a rapid efflux of amino acids from intact bacterial cells and cytoplasmic membrane vesicles, and to prevent amino acid uptake by cells preincubated with the peptide. Upon addition of subtilin the trans-membrane potential (dy) was greatly reduced. Starved bacterial cells were less sensitive to subtilin than energized cells. Depolarization of cells by carbonyl cyanide rn-chlorophenylhydrazone prevented subtilin action, but its activity could be restored by a valinomycin-induced potassium diffusion potential. Using this technique, we deduced a threshold potential of about -90 to -100 mV to be essential for subtilin action on intact cells. A similar value was obtained in macroscopic conductance measurements with black lipid membranes. The current-voltage characteristic was symmetric, i.e. subtilin induced membrane currents with trunsnegative and trans-positive voltages. Single-channel experiments revealed short-lived multi-state pores of the alamethicin type. The pores had lifetimes of several hundred milliseconds and pore diameters of up to approximately 2 nm.Subtilin is a cationic peptide antibiotic which is produced by Bacillus subtilis ATCC 6633 and which belongs to a group of antibiotics recently named lantibiotics [I]. Other members of this group are nisin 121, Pep 5 [3], and epidermin [l]. The prominent feature of lantibiotics is the content of the unusual amino acids lanthionin, 3-methyl lanthionine, dehydrobutyrine and dehydroalanine. The biosynthesis of lantibiotics was shown to proceed by enzymic processing of ribosomal precursors [l, 4, 51. The structure of subtilin was elucidated by GroB et al. (Fig. 1) [6]; however, there have been no reports yet on its mode of action.We have recently investigated the action of the lantibiotics Pep 5 and nisin. Both depolarize energized bacterial membranes [7, 81 and form voltage-dependent multi-state pores in black lipid bilayers 19, 101. Pore formation requires the existence of a transmembrane potential in both intact cells and artificial bilayers; the potential must have the proper orientation (trans-negative) and a sufficient magnitude (-80 to -100 mV at neutral pH). The largest pores observed in black lipid bilayers have pore diameters of up to 1 nm and are stable for milliseconds, whereas smaller pores have lifetimes of several hundred milliseconds [9,10]. Through these pores vital gradients equilibrate with the outside (e.g. H', K', amino acids, ATP); loss of metabolites and salts and the concomitant breakdown of the membrane potential are considered to be the primary cause of cell death [ l l , 121. A secondary effect, which may contribute to the overall bactericidal action of Pep 5 and nisin, is a rapid induction of autolysis [13, 141.Correspondence to H.-G. Sahl, Institut fur Medizinische Mikrobiologie und Immunologie der Universitat Bonn, D-5300 Bonn 1-Vcnusberg, Federal Republic of Germany Abbreviations. CCCP, carbonyl cyanide rn-chlorophenylhydrazone; Phy2GroPCho, diphytanoylglycerophosphocholine; Ph,P+, ...

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