ACS Chemical Biology ARTICLES Figure 6. A ﬂexible hinge region, not Dha5, is essential for preventing establishment of a membrane potential, inhibition of spore outgrowth, and membrane disruption. At time 0 and 30 min, aliquots were removed from the cultures and evaluated for the following: (A) Membrane potential by measuring the ﬂuorescence of DiOC2-associated B. anthracis using ﬂow cytometry. (B) Membrane disruption by measuring PI uptake by B. anthracis using ﬂow cytometry. (C) Population of spores exhibiting an increase in ﬂuorescence associated with PI uptake. The data are plotted as the percent of spores that took up PI. (A, B) Data are rendered as the fold change in membrane potential (A) or PI uptake (B) relative to spores in the presence of 0.1 M MOPS pH 6.8 at the indicated time point. (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. All experiments were performed with 10 μM nisin or analogue. Modification of the Nisin Hinge Region Results in Loss of Outgrowth Inhibition. Previous studies have reported conflict- ing results as to whether Dha5 is essential for outgrowth inhibition.8,10,20,38 Therefore, experiments were conducted to determine the necessity of Dha5 for membrane disruption resulting in outgrowth inhibition. Nisin and nisin-Dha5Ala were heterologously produced in E. coli30 (h-nisin and h-nisinS5A, respectively). Both compounds inhibited membrane potential establishment and spore outgrowth and increased PI uptake (Figure 6, Supplementary Table S3) to the same extent. These results show that Dha5 is not essential for spore outgrowth inhibition, as reported previously by Moll and co-workers,38 and that the mutation does not change the mechanism of inhibition. A second region of interest for mutation is the hinge region between the three N-terminal rings and the two C-terminal rings. The double mutants N20P/M21P and M21P/K22P retain lipid II binding aﬃnity but not pore-forming activity.5,6,39 These mutants were prepared in this study by heterologous expression in E. coli (h-nisin N20P/M21P and h-nisin M21P/K22P) and analyzed for their ability to disrupt the spore membrane and inhibit membrane potential establishment and spore outgrowth. As depicted in Figure 6, both mutants lost these activities (see also Supplementary Table S5). Importantly, IC99 experiments against M. ﬂavus indicated that the heterologously expressed nisin variants have the same biological activities as those puriﬁed from L. lactis6 and that these variants have high activity against vegeta- tive cells of B. anthracis Sterne 7702 (Supplementary Table S6) . These data corroborate the ﬁndings with the truncated analogue c-nisin and demonstrate that the ability to form pores in the spore membrane is essential for outgrowth inhibition. In addition, they show that membrane disruption is not essential for inhibiting growth of vegetative cells but is essential for inhibiting spore outgrowth. Conclusions. Previous work implicated nisin-mediated altera- tions in the membrane integrity and prevention of the establish- ment of a membrane potential as the mechanism by which nisin inhibits spore outgrowth.8 10,26 Whether or not interactions with lipid II were important for these activities was not deter- mined. Utilizing fluorescently labeled nisin and vancomycin, competition experiments, and nisin analogues, lipid II was identified in this study as the target for nisin to induce inhibition of spore outgrowth. Lipid II binding alone is not sufficient for 750 dx.doi.org/10.1021/cb1004178 |ACS Chem. Biol. 2011, 6, 744–752