ACS Chemical Biology Figure 2. Nisin and vancomycin binding to spores. At time 30, 60, and 120 min, samples were removed and visualized by epi-ﬂuorescence microscopy. (A) Incubation of germinated spores with 1 μM bodipy- nisin (b-nisin, red) and ﬂuorescein-vancomycin (f-vancomycin, green). Co-localization is indicated by yellow in merged images. (B) Incubation of germinated spores with 1 μM b-nisin (red). (C) Incubation of germinated spores with 1 μM f-vancomycin (green). For each panel, a single spore is shown for clarity, but the image is representative of all B. anthracis spores within that sample. localized at sites where nisin was found at all time points (n = 50; Supplementary Figure S3). It is possible that the clustering of b-nisin near the poles reflects the localization of lipid II that is required for the biosynthesis of new cell wall during the out- growth process. However, we cannot rule out that the observed strong localization at the poles may be the result of nisin-induced relocalization of lipid II. The distinct binding patterns of b-nisin and f-vancomycin were not caused by intrinsic diﬀerences between bodipy and ﬂuorescein, because switching the labels (e.g., using ﬂuorescein- labeled nisin and bodipy-labeled vancomycin) resulted in similar ARTICLES binding patterns (Supplementary Figure S4A C). Furthermore, the patterns of nisin and vancomycin binding were independent of the order in which the compounds were introduced (Supplementary Figure S5A). Finally, the incubation of spores with b-nisin under germinating conditions in the absence of vancomycin also resulted in punctate localization at the pole of the spore with some diﬀuse ﬂuorescence associated with the spore membrane (Figure 2B). Complete colocalization of nisin and vancomycin was not expected since vancomycin binds the D-Ala-D-Ala structure present in both the pentapeptide of the non-cross-linked cell wall and cortex in addition to the pentapeptide of lipid II,26 whereas nisin will bind to the pyrophosphate that is only present in lipid II (Figure 1B).33 Reﬂective of these diﬀerences in binding speciﬁcities, nisin did not detectably associate with dormant spores (Supplementary Figure S5B), indicating that lipid II was not accessible prior to germination initiation. In contrast, vanco- mycin was associated with dormant spores (Supplementary Figure S5B), which may be due to the accessibility of non- cross-linked peptidoglycan within the spore cortex. Incubation of spores with f-vancomycin (1 μM) in the absence of nisin showed relatively uniform labeling at 30 min, with more punctuate labeling at 60 min (Figure 2C). Outgrowth of spores into bacilli altered the localization of vancomycin to bands that cross the long axis of the bacilli at 120 min (Figure 2C). This localization of vancomycin in outgrown spores is similar to lipid II localization observed previously in vegetative bacilli of B. subtilis and reﬂects the helical localization of lipid II along the long axis of the rod-shaped cell.22,34 To provide support that b-nisin binds to lipid II associated with germinated spores, competition binding experiments were performed. These studies revealed that a 100-fold molar excess of unlabeled vancomycin signiﬁcantly reduced binding of b-nisin to the surface of germinated spores (Figure 3A), presumably by preventing nisin interaction with lipid II as a consequence of steric hindrance. Likewise, a 100-fold molar excess of unmodiﬁed nisin signiﬁcantly inhibited the binding of f-vancomycin to the surface of spores under germinating conditions (Figure 3B). As mentioned above, complete competition is not expected since these two compounds, in addition to sharing lipid II as target, also have nonoverlapping binding sites (non-cross-linked cell wall for vancomycin and the membrane for nisin). Importantly, 100-fold molar excess of unmodiﬁed nisin also signiﬁcantly reduced the binding of b-nisin to germinated spores (Figure 3C), providing additional evidence that b-nisin and wild-type nisin bind to the same target. Alternative Approaches with Fluorescently Labeled Nisin Analogues. In an effort to determine whether the time- dependent labeling patterns observed with fluorescently labeled nisin and vancomycin reflect degradation and processing of the cortex and cell wall, a series of cortex hydrolase mutants were evaluated for their ability to alter nisin and vancomycin localization. Previous studies demonstrated that single knockouts of sleB or cwlJ1 slowed the rate of cortex hydrolysis while the double mutant, ΔsleBΔcwlJ1, eliminated cortex hydrolysis in a B. anthracis 34F2 background.24,25 In the presence of both b-nisin and f-vancomycin (both at 1 μM), the outgrowth of germinated spores of these mutants were inhibited, and b-nisin and f-vanco- mycin were associated with spores (Supplementary Figure S6A), with a similar binding pattern observed for the spore variants as shown above for B. anthracis 7702 (Figure 2). Thus, although the structure of the cell wall is changing during cortex hydrolysis, the 747 dx.doi.org/10.1021/cb1004178 |ACS Chem. Biol. 2011, 6, 744–752