ACS Chemical Biology Figure 3. Competition assays with nisin and vancomycin. (A) Compe- tition assay of spore binding by unlabeled vancomycin and b-nisin. * indicates a p-value <0.05 between b-nisin-only treated spores (1 μM) and spores pretreated with unlabeled vancomycin (100 μM) prior to b-nisin addition. ** indicates a p-value <0.05 between b-nisin-treated (1 μM) spores and spores treated with unlabeled vancomycin (100 μM). (B) Binding competition assay of unlabeled nisin in competition with b-vancomycin. * indicates a p-value <0.05 between b-vancomycin (1 μM) treated spores and spores pretreated with unlabeled nisin (100 μM) prior to b-vancomycin addition. ** indicates a p-value <0.05 between b-vancomycin (1 μM) treated spores and spores treated with unlabeled nisin (100 μM). (C) Binding assay of unlabeled c-nisin in competition with b-nisin. * indicates a p-value <0.05 between b-nisin (1 μM) treated spores and spores pretreated with unlabeled c-nisin (100 μM) prior to b-nisin addition. * indicates a p-value <0.05 between control (0 μM nisin) spores and spores pretreated with unlabeled nisin or unlabeled c-nisin (100 μM) prior to b-vancomycin addition. In A C, the data are plotted as the mean ﬂuorescent intensity (MFI) associated with the binding of the labeled antibiotic. (D) At time 0, 5, and 10 h, samples were removed and visualized by DIC microscopy. For each panel, a single spore is shown for clarity, but the image is representative of all other B. anthracis spores within that sample. The data are representative of those from three independent experiments with 10 μM nisin or c-nisin. accessible binding sites for vancomycin and nisin do not appear to change much. We note that a complementary approach to change the lipid II content of outgrowing spores by using the lipid II biosynthesis inhibitors bacitracin and fosfomycin was not possible because B. anthracis is highly resistant to these compounds.35 37 ARTICLES Although the collective results indicate that nisin binds to lipid II on the spore surface, the microscopy data do not reveal whether lipid II is the only nisin target in germinated spores. Previous studies suggested that nisin may bind covalently to a protein target during outgrowth inhibition through a Michael- type addition of a Cys of a spore protein to Dha5 of nisin.8,10,20 However, extensive eﬀorts to conﬁrm and identify a cognate protein receptor using ﬂuorescently or biotin-labeled nisin as probes were unsuccessful (data not shown), consistent with earlier results emerging from mutagenesis of Dha5.38 Our results also argue against a covalent mechanism involving the other electrophilic sites in nisin. Lipid II Binding Is Not Sufficient to Inhibit Outgrowth. To evaluate whether lipid II binding alone is sufficient for nisin-dependent outgrowth inhibition, we studied the action of a purified amino- terminal chymotryptic fragment of nisin (c-nisin, Figure 1A) that retained the A, B, and C rings of the full-length parent com- pound. c-Nisin contains the necessary N-terminal portion of nisin required for lipid II binding33 but is unable to form pores.38 Competition assays between c-nisin and b-nisin revealed that the binding of b-nisin to germinated spores was significantly reduced in the presence of 100-fold molar excess of c-nisin (Figure 3C), once more providing evidence that b-nisin recognizes and binds lipid II. When incubated with germinated spores, c-nisin did not demonstrate significant inhibition of spore outgrowth (Figure 3D, Supplementary Table S4), although a slight delay in the elongation of vegetative bacilli was observed. These data indicate that while the chymotrypsin-derived amino terminal nisin fragment binds lipid II, this fragment alone does not inhibit the outgrowth of germinated spores. This result is different from a previous study, which demonstrated that nisin fragments missing the D and E rings still inhibited B. subtilis spore outgrowth.38 Possible explanations for the different observations include differences in spore structure between B. subtilis and B. anthracis or differences in the methods to determine spore outgrowth. In the study on B. subtilis, outgrowth was measured by monitoring optical density at 600 nm, whereas the current work utilized microscopy to provide direct visual observation of spore outgrowth. Consistent with the observations that nonpore forming, lipid II-binding nisin analogues do not inhibit spore outgrowth, vancomycin was also incapable of inhibiting the outgrowth of germinated spores (Figure 2). Together, these results support the model that lipid II binding alone is not sufficient to inhibit the outgrowth of germinated spores. Lipid II Binding Is Associated with Loss of Membrane Potential. Nisin prevents the establishment of a membrane potential in germinated spores.9 Although the results presented here support binding of nisin to lipid II, they do not establish whether lipid II is important for nisin-dependent membrane potential dissipation. To evaluate this possibility, the membrane potential was first measured with germinated spores in the presence of vancomycin, monitoring the uptake of the cationic membrane potential dye DiOC2 upon the establishment of a membrane potential utilizing flow cytometry.9 In these experiments, vancomycin did not cause much of a decrease in membrane potential (Figure 4A,B). In contrast, nisin prevented the establishment of a membrane potential in a large fraction of the population (Figure 4A C). Furthermore, a significant decrease of nisin-dependent reduction of the trans-membrane potential of spores was observed in the presence of a 100-fold molar excess of vancomycin (Figure 4A). Moreover, germination of spores in the presence of c-nisin resulted in significantly less 748 dx.doi.org/10.1021/cb1004178 |ACS Chem. Biol. 2011, 6, 744–752