Mode of action and structure–activity relationship studies of geobacillin I

Recently, we characterized two lanthipeptides, geobacillin I and geobacillin II, from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2.¹⁸ Geobacillin I contains seven thioether bridges, one dehydroalanine (Dha) and one dehydrobutyrine (Figure 1c). The N-terminal A and B rings of geobacillin I are very similar to the corresponding rings of nisin; however, the C-terminal structures are very different (Figure 1c). The nisin A and B rings are involved in lipid II binding,¹³ and hence we anticipated that geobacillin I might also bind lipid II. The three amino-acid linker peptide between the C and D rings of nisin has been shown to be indispensable for pore formation activity.^{17, 19, 20, 21} For instance, the ΔN20ΔM21 and N20P/M21P mutants of nisin lost pore formation ability against Gram-positive bacteria.^{17, 19} Geobacillin I has only a single amino acid between the C and D rings, similar to the ΔN20ΔM21 mutant of nisin. Thus, based on the available data on nisin, we anticipated that geobacillin I would bind to lipid II but not form pores in the membrane of Gram-positive bacteria. In this work, we tested these expectations experimentally.

We also investigated the efficiency of pore formation by geobacillin I using propidium iodide (PI)—a membrane impermeable fluorescent dye. Upon pore formation or membrane disruption, PI can enter the cell, resulting in an increase in fluorescence intensity because of the interaction of PI with nucleic acids. PI uptake was monitored at nine different concentrations with each experiment conducted in triplicate (Figure 2b and Supplementary Figure S3). The data showed only twofold lower efficiency in PI uptake for geobacillin I, with IC₅₀ values for nisin at 0.3 μM compared with 0.6 μM for geobacillin I (Figure 2b and Supplementary Figure S3).

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