ACS Chemical Biology ARTICLES Figure 4. Lipid II binding is not suﬃcient to inhibit membrane potential establishment via membrane disruption. (A) Functional competition assay using nisin-induced dissipation of the membrane potential as read-out. The data is plotted as the population of spores exhibiting a native membrane potential per 10,000 spores as observed by ﬂow cytometry using the DiOC2 dye. Preincubation with vancomycin blocks nisin-mediated membrane potential dissipation. * indicates a p-value <0.05 between nisin (1 μM) treated spores and spores pre- treated with vancomycin (100 μM) prior to nisin addition. ** indicates a p-value <0.05 between nontreated spores and spores treated with vancomycin (100 μM) only. (B, C) At time 0 (prior to the addition of 10 μM nisin, c-nisin, or vancomycin) and 30 min aliquots were removed from the cultures and evaluated for membrane potential by measuring the DiOC2-associated B. anthracis ﬂuorescence by ﬂow cytometry. Data are rendered as the fold change in membrane potential relative to spores in the presence of 0.1 M MOPS pH 6.8 at the indicated time point. reduction of spore trans-membrane potential (Figure 4C). Be- cause these studies were carried out under conditions where vancomycin or c-nisin reduced the binding of b-nisin to spores (Figures 3A C), these results suggest that nisin-dependent reduction of the trans-membrane potential requires binding to lipid II in newly germinated spores. Lipid II Binding Is Associated with Nisin-Dependent Al- terations in Membrane Integrity. Nisin disrupts the membrane integrity of germinated spores,9 but the data thus far do not determine whether nisin-lipid II interactions are required for this Figure 5. C-Terminal region of nisin is essential for membrane disrup- tion. At time 0 (prior to the addition of BHI as germinant and 10 μM nisin, c-nisin, or vancomycin) and 30 min, aliquots were removed from the cultures and evaluated for the following: (A, C) Membrane disrup- tion by measuring PI uptake by the entire population of B. anthracis spores using ﬂow cytometry. (B) Population of spores that exhibited an increase in ﬂuorescence associated with PI uptake. The data are plotted as the percent of spores that took up PI. Data in panels A and C are rendered as the fold change in PI uptake relative to spores in the presence of 0.1 M MOPS pH 6.8 at the indicated time point. activity. Therefore, experiments were conducted to examine the effect on membrane integrity monitored by the uptake of the membrane-impermeable dye propidium iodide (PI) upon nisin- induced membrane disruption utilizing flow cytometry.9 Nisin induced significantly more PI uptake than c-nisin in over 92% of the population of spores (Figure 5A,B). Vancomycin was not able to induce an increase in PI uptake (Figure 5C). In addition, only nisin was able to render the germinated spore metabolically inactive prior to outgrowth (Supplementary Figure S8). Collec- tively, these results suggest that an association between nisin and lipid II is important for the disruption of membrane integrity. 749 dx.doi.org/10.1021/cb1004178 |ACS Chem. Biol. 2011, 6, 744–752