, 2008b). The potential-sensitive

fluorescent cyanine dye diSC3(5) was used for assessing RAD001 price the sakacin A-induced dissipation of ΔΨ. By adding glucose to Listeria cells, a negative-inside ΔΨ was generated, resulting in the quenching of the probe fluorescence as a consequence of probe accumulation within the cells. As shown in Fig. 2, Listeria cells were able to maintain ΔΨ in the presence of nigericin (arrow 4) that dissipates transmembrane ΔpH. When sakacin A was added to glucose-energized and nigericin-treated cells, the fluorescence of the probe increased, as a result of its release from the cell interior (arrow 5). This indicates a depolarization of the cytoplasmic membrane consequent to the addition of sakacin A. Figure 4 also makes it evident that the decrease in fluorescence induced by the addition of glucose has an amplitude very similar to the fluorescence increase

ensuring from the addition of sakacin A. The ionophore valinomycin was used at the end of these experiments (arrow 6) to completely dissipate ΔΨ (McAuliffe et al., 1998). The pH-sensitive fluorescent probe cFDASE was used to assess the transmembrane ΔpH in Listeria cells. As also shown in Fig. 2, the fluorescence of the probe rapidly increased upon addition of lactose to cells (arrow 1), consequent to increased FK866 mw internal pH. When sakacin A was added (arrow 2), a rapid decrease in the signal was observed. No further signal increase was observed when nigericin was added (arrow 3), indicating 3-mercaptopyruvate sulfurtransferase that sakacin A completely dissipated the transmembrane ΔpH of Listeria cells. The effects of sakacin A on isolated cell walls were studied by measuring the time course of turbidity decrease

in cell wall suspensions at sakacin A concentration close to the MIC. As shown in Table 1, turbidity decreased by c. 20% within 30 min of sakacin A addition. After 24 h, the sample treated with sakacin A gave a turbidity decrease (38–40%) not significantly different (P > 0.05) from that obtained with lysozyme. Isolated Listeria cell walls were exposed to various antimicrobials, and the solubilized material was analyzed by MALDI-TOF MS. The differences in the MS spectra in Fig. 3 indicate that individual antimicrobials had specific mechanisms of action and suggest that Listeria cell walls were broken down by sakacin A into fragments in the 1000–2500 Da range. In separate set of experiments, isolated Listeria cell walls were treated for 24 h at 30 °C with increasing amounts of sakacin A, and the released fragments (Fig. 4) were sequenced by MS/MS. No fragments were released in the absence of sakacin A or with sakacin A concentrations lower than 0.1 mg mL−1. As summarized in Table 2, products containing fragments from both the polysaccharide and the peptide components of the peptoglycan were evident at sakacin A concentrations of 0.