Fig. 7

Effect of cFP exposure on the membrane permeability of B. vulgatus. A Estimation of the EC₅₀ of Triton X-100. Various concentrations of Triton X-100, ranging from 0.0003% to 0.1%, were exposed to B. vulgatus MGM001 cells, which were incorporated with a membrane-associating fluorescent dye, DiSC3(5), as described in “Methods.” Released fluorescence from the N% Triton X-100-treated cells (termed Δ_Ν%) was obtained by subtracting the values of basal RFU (before treatment of N% Triton X-100) from the maximal RFU (after treatment of N% Triton X-100). These values were normalized with the estimates of DiSC3(5) released from the cells treated with 0.1% Triton X-100 (Δ_0.1%), at which almost all B. vulgatus cells were permeabilized. The ratios ([Δ_Ν%/Δ_0.1%] × 100) were plotted against the concentrations of Triton X-100. The arrow indicates the effective concentration (EC₅₀) of Triton X-100 to release 50% of Δ_0.1%. B Release of DiSC3(5) from cFP-treated B. vulgatus. A fluorescent dye, DiSC3(5), was used to examine the change in permeability of bacterial membrane. B. vulgatus cells, which had been exposed to 4 mM cFP for 6 h, were mixed with 0.4 μΜ DiSC3(5) for 1 h. After unincorporated dyes were washed out, RFUs from DiSC3(5) associated with cells were measured. At 30 min (as indicated with a black vertical arrow), cells were treated with 0.002% Triton X-100, at which approximately 50% of the B. vulgatus cells were permeabilized (as shown in A). The released fluorescence from the Triton X-100-treated cells was obtained by subtracting the values of basal RFUs (averaged RFUs for 10 min before treatment of 0.002% Triton X-100) from the maximal RFUs (averaged RFUs for 10 min after treatment of 0.002% Triton X-100). As a control, the cells untreated with cFP were followed by the same procedure and then Δ_{0 mM cFP} was compared with Δ_{4 mM cFP}. Each line was derived from the average values from the total six experiments