ACS Chemical Biology Figure 1. Structures of nisin, vancomycin, and lipid II. (A) The structure of nisin A. Dehydroalanine (Dha) and dehydrobutyrine (Dhb) are indicated in green and magenta, respectively. Lanthionine and methyllanthionine cross-links are indicated in red and blue, respec- tively. The numbers indicate the locations of the amino acids that were mutated. The N-terminal fragment resulting from chymotrypsin clea- vage (c-nisin) is highlighted in maroon. (B) The structure of lipid II in B. anthracis. The vancomycin binding site is indicated in red, and the nisin binding site is indicated in blue. m-DAP: diaminopimelic acid. (C) Transmission electron microscopy image of a B. anthracis Sterne 7702 endospore. (D) The structure of vancomycin. outgrowth.8,10,20 To better understand the mechanism of inhibi- tion of spore outgrowth, the eﬀects of nisin, vancomycin, and nisin analogues (Figure 1A,D) on germination and outgrowth of B. anthracis Sterne 7702 spores were compared; vancomycin and the nisin analogues bind to lipid II but do not form pores. These studies show that nisin does bind lipid II on spores but that lipid II binding is not suﬃcient for outgrowth inhibition, whereas ARTICLES membrane disruption is essential. In contrast, lipid II binding is suﬃcient for antimicrobial activity against vegetative cells.6 Using ﬂuorescently labeled nisin analogues, no evidence was found for a covalent mechanism of inhibition. ’ METHODS Spore Preparations. Spores were prepared from B. anthracis Sterne 7702 or B. anthracis 34F2, as described previously.21 Both strains do not contain the plasmid pX02 that encodes the genes for capsule production. Enumeration of spores was performed using a Petroff- Hauser hemocytometer under a light microscope at 400x magnification (Nikon Alphaphot YS). A typical spore preparation yielded 10 mL of spores at a concentration of 2.0 109 spores/mL. Labeling of Nisin and Vancomycin. Nisin was purified and characterized as described previously.9 Nisin was reacted with NHS- fluorescein (Pierce) or with NHS-BODIPY-633 (Invitrogen) to gen- erate analogues with a single fluorescein group (f-nisin) or a single BODIPY group (b-nisin) after purification. The reactions were carried out according to the manufacturer’s protocols except that a 0.75:1 molar ratio of NHS-fluorophore and nisin was used. Labeling reactions were stopped with the addition of 100-fold molar excess of Tris after a 3 h reaction time at 25 C. Reactions were analyzed via matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS) (Applied Biosystems), which indicated that all nisin was consumed. Proteolytic digest of the fluores- cently labeled compounds with trypsin and chymotrypsin and subse- quent analysis by LC MS showed that the label was located predominantly (>85%) at the N-terminus of b-nisin and at the C-term- inal Lys of f-nisin. Vancomycin was labeled with NHS-rhodamine (Pierce) to obtain r-vancomycin, with NHS-fluorescein to generate f-vancomycin, or with NHS-BODIPY-633 to produce b-vancomycin as previously described.22 Vancomycin labeling occurs on the amino group of the vancosamine sugar (Figure 1D).22 All labeled compounds were purified by reverse phase-high performance liquid chromatography (RP- HPLC) utilizing a C4 semipreparative column (Waters) with a linear gradient of 0 100% acetonitrile with 0.1% trifluoroacetic acid over 40 min. Acetonitrile, TFA, and water were removed from fractions containing nisin or vancomycin by rotary evaporation followed by lyophilization. Prior to use, lyophilized nisin and vancomycin were dissolved in 0.1 M MOPS pH 6.8 to yield the desired concentration. Truncation of Nisin with Chymotrypsin. Proteolysis of nisin with chymotrypsin was performed as previously described.23 Reactions were analyzed by MALDI-TOF MS. The N-terminal chymotryptic segment (c-nisin, Figure 1A) was purified from full length nisin and the C-terminal fragment by RP-HPLC utilizing a C4 semipreparative column (Waters) with a linear gradient of 0 100% acetonitrile over 40 min. Acetonitrile, TFA, and water were removed from fractions containing c-nisin by rotary evaporation followed by lyophilization. Culturing B. anthracis Spores. B. anthracis 34F2 mutants were graciously provided by Dr. David Popham (Virginia Tech). B. anthracis Sterne 7702, B. anthracis 34F2, B. anthracis 34F2 ΔsleB,24 B. anthracis 34F2 ΔcwlJ1,24 or B. anthracis 34F2 ΔsleBΔcwlJ1 25 spores at a concentration of 4.0 106 spores/mL were incubated in brain heart infusion medium (BHI; BD Bioscience) supplemented with nisin (at 1 or 10 μM), c-nisin (10 μM), nisin variants (S5A, N20P/M21P, or M21P/K22P at 10 μM), vancomycin (at 0.1, 1, 10, or 100 μM), or with 0.1 M 3-(N-morpholino)propanesulfonic acid (MOPS; Sigma) at pH 6.8 as a control. For nongerminating conditions, 0.1 M MOPS was substituted for BHI medium. All incubations were performed at 37 C under aeration at 180 rpm on a rotary shaker and under ambient CO2. Differential Interference Contrast (DIC) and Epi-fluores- cence Microscopy. Live epi-fluorescence microscopy was performed by mounting samples on glass slides in 0.5% agarose under coverslips. 745 dx.doi.org/10.1021/cb1004178 |ACS Chem. Biol. 2011, 6, 744–752