ACS Chemical Biology Images were collected using an Applied Precision assembled DeltaVi- sion EpiFluorescence microscope containing an Olympus Plan Apo 100x oil objective with 1.42 and a working distance of 0.15 mm. Images were processed and Pearson’s coefficient colocalization analysis of 50 spores per condition was performed using SoftWoRX Explorer Suite. Acquisition of epi-fluorescence images utilized FITC (Ex: 490/20; Em: 528/28), rhodamine (Ex: 555/28; Em: 617/73), and Cy5 (Ex: 640/ 20; Em: 685/40) to visualize fluorescein, rhodamine, and BODIPY-633 fluorescence, respectively. Functional Competition Assay. Competitive binding of nisin and vancomycin was evaluated utilizing a functional assay in which vancomycin prevented nisin-mediated loss of membrane potential via lipid II binding.26 Spores were incubated in BHI medium (germinating conditions), in the presence of 10 mM L-Ala and inosine (germinating conditions), or in 0.1 M MOPS pH 6.8 (nongerminating conditions) with 3,30-diethyloxacarbocyanine iodide (DIOC2) for 60 min at 37 C followed by incubation with 0 or 100 μM vancomycin for 2 min. Nisin was added to cultures and the effect on membrane potential disruption was immediately assayed with flow cytometry through 10,000 counts to observe the population of spores that exhibited reduced membrane potential in the presence of nisin. The MIC of vancomycin against B. anthracis is 0.5 3.5 μM,27 29 but the antimicrobial effects of vancomy- cin under the conditions used here are not manifested until spores have been germinated and incubated in the presence of vancomycin for 90 min, which is well after the time frame of interest in this investigation. Experiments investigating spore hydration, oxidative metabolism, mem- brane potential, and membrane integrity were performed as previously described.9 Procedures used for competition binding experiments are described in the Supporting Information. Site-Directed Mutagenesis. Mutagenesis of nisA was performed using the QuikChange mutagenesis kit from Stratagene. Reactions with complementary mutagenic primers (Supplementary Table S1) were performed as described in the QuikChange protocol with pRSFDuet- 1nisAB as the plasmid template 30 for the generation of NisA S5A and M21P mutants. After cycling of the reaction mixture 18 times in a thermal cycler (MJ Research), the resulting mixture was digested with DpnI (New England Biolabs), and the resulting DNA was used to transform supercompetent E. coli DH5R cells. The resulting mutant plasmids were isolated, and the entire gene was sequenced to ensure that only the appropriate mutations were introduced. For double mutations, a second round of site-directed mutagenesis was performed utilizing pRSFDuet-1nisAB-M21P as the plasmid template for the generation of NisA N20P/M21P and M21P/K22P mutants. Heterologous Production of Nisin and Nisin Mutants. Electro-competent E. coli BL21(DE3) cells were cotransformed with pRSFDuet-1 containing nisA variants and the nisB gene and pACYC- Duet-1 containing the nisC gene30 (Supplementary Table S2). Over- night culture grown from a single colony transformant was used as inoculum to grow 2 L of terrific broth medium (0.12% Pancreatic Digest of Casein, 0.24% Yeast Extract, BD Biosciences; 0.094% K2HPO4, 0.022% KH2PO4, Fisher Chemical) containing 50 mg/L kanamycin and 25 mg/L chloramphenicol at 37 C until the OD600 nm reached about 0.6. The incubation temperature was then changed to 18 C, and the culture was induced with 0.5 mM IPTG. The induced cells were shaken continually at 18 C for an additional 18 h. The cells were harvested by centrifugation (10,000 g for 10 min, Beckman JA-10 rotor). Purification of the modified precursor peptides is described in the Supporting Information. Cleavage of Modified Prenisin with Trypsin and Purifica- tion of Nisin Variants. Modified prenisin (500 μM) and trypsin (30 μM, Worthington Biochemicals) were incubated at RT for 3 h with 150 rpm mixing on a platform shaker. The resulting mixture was checked by MALDI-TOF MS, and the desired proteolytic fragment corresponding to mature nisin or its variants were observed-calculated ARTICLES for heterologously expressed nisin (h-nisin): 3352.5152 m/z (M þ H), observed 3352.5005 m/z; calculated for h-nisin S5A: 3354.5608 m/z (M þ H), observed 3354.4946 m/z; calculated for h-nisin N20P/M21P: 3301.5673 m/z (M þ H), observed 3301.7642 m/z; and calculated for h-nisin M21P/K22P 3287.9826 m/z (M þ H), observed: 3287.8738 m/z. Nisin and variants were purified from the cleavage reaction via RP- HPLC utilizing a C4 semipreparative column with a linear gradient of 0 100% acetonitrile with 0.1% TFA over 40 min. The fractions containing nisin variants were lyophilized and analyzed by MALDI- TOF and ESI MS. ’ RESULTS AND DISCUSSION Fluorescently Labeled Nisin Analogues Behave Similarly to Wild-type Nisin. To further evaluate the mechanism by which nisin inhibits the outgrowth of germinated B. anthracis spores, bodipy-labeled nisin (b-nisin) and fluorescein-labeled nisin (f-nisin) were synthesized to probe nisin binding to spores using fluorescence microscopy. The fluorescein label, which was located predominantly at the C-terminal lysine residue of nisin, or the bodipy label, which was located predominantly at the N-terminus, did not have major deleterious effects on nisin activity, as fluorescently labeled nisin inhibited B. anthracis growth with IC90 values of 5.4 and 6.5 μM for b-nisin and f-nisin, respectively, which correspond to a 6- and 7-fold loss in activity, respectively, compared to unmodified nisin.9 These findings are consistent with observations in previous reports using nisin labeled with 5-(aminoacetamido)fluorescein at the C-terminal carboxylate, which also resulted in an active analogue.5 Addi- tional experiments that confirm very similar activities of fluores- cently labeled nisin analogues and wild-type nisin are presented in later sections. Importantly, control studies revealed that these labeled nisin analogues neither induced nor inhibited germina- tion initiation, as monitored by the loss of optical density associated with spore hydration during germination (not shown), analogous to unmodified nisin.9,10 The binding of b-nisin was compared with a known lipid II binding probe, ﬂuorescein-vancomycin, (f-vancomycin).22 Van- comycin interacts with the D-Ala-D-Ala part of lipid II,2,31,32 which is distinct from the binding site of nisin that features the pyrophosphate group (Figure 1B).33 Fluorescently labeled van- comycin has been used previously to investigate lipid II localiza- tion on vegetative bacilli.22,34 Like nisin, vancomycin (at concen- trations up to 100 μM) neither induced nor inhibited spore germination (Supplementary Figure S1). To verify that ﬂuores- cently labeled nisin can be used to detect the localization of lipid II, vegetative B. anthracis cells were incubated with f-nisin or b-nisin. Previous studies have demonstrated that nisin relocalizes lipid II to patches in bacilli.5 In our experiments, f-nisin and b-nisin also localized in patches on B. anthracis (Supplementary Figure S2). Collectively, these experiments provide support that the ﬂuorescently labeled nisin analogues bind to lipid II. Nisin Binds to Lipid II of Germinated Spores. In the presence of both b-nisin and f-vancomycin (both at 1 μM), the outgrowth of germinated spores was inhibited, and b-nisin and f-vancomycin were associated with spores (Figure 2A), although with distinct binding patterns. Vancomycin was localized diffu- sely over the entire surface of germinated spores, whereas nisin was localized in a more punctate fashion and predominantly near the poles of the germinated spores, where colocalization with vancomycin was observed (Figure 2A). Co-localization analysis utilizing Pearson’s coefficient confirmed that vancomycin was 746 dx.doi.org/10.1021/cb1004178 |ACS Chem. Biol. 2011, 6, 744–752