Figure 10.

Application of DSB-3–based fluorescence PSD assay to cell extracts containing temperature-sensitive bacterial PSD. Wildtype (HB101) and temperature-sensitive psd mutant (EH150) strains of E. coli were grown at 30 °C to early log phase and then either kept at 30 °C or shifted to 42 °C and grown for four generations. Cells were harvested and processed to produce crude extracts. Enzyme assays were performed with crude cell-free extracts (600 ng of protein/μl), 0.5 mm PS, and 3.1 mm Triton X-100 at 30 °C for 30 min. DSB-3 fluorescence detection reactions with the PSD reaction products were conducted as described in the legend to Fig. 8. A, net fluorescence intensities of each PSD assay at 30 min are shown after background correction. Background fluorescence is the value obtained from the PSD assay at zero time with heat-inactivated cell extracts. B, PSD activity of the E. coli cell-free extracts was calculated by applying the standard fluorescence data obtained as described in the legend to Fig. 8C. Standard fluorescence curves with the PE/PS mixed micelles for the three cell types were generated in the presence of corresponding heat-inactivated E. coli extracts. The data are from four independent experiments and are means ± S.E. (error bars).