Fig. 4

Frequency of spontaneous rifampicin mutation in DNA glycosylase-deficient (CC101 mutM mutY ksgA) E. coli. Cells were introduced with the empty vector (pYP73), ksgA gene, or ksgA (1-213) gene. (a) Procedure for measuring spontaneous mutation frequencies. Each strain of E.coli was grown overnight at 37 °C in LB medium containing 0.1 mM IPTG and the appropriate antibiotics. The undiluted or diluted culture were plated on LB plate or that containing rifampicin and incubated 37 °C for 24 h. We counted the colonies and calculated the number of the viable cells and the rifampicin-resistant cells and determined the spontaneous mutant frequency from these numbers. (b) The result of spontaneous mutant frequency measurement. Mean + S.E. values of six independent experiments are shown (n = 6). An asterisk (*) indicates p values < 0.05. (c) Comparison of expression levels by western blotting. Crude extracts were obtained from E.coli and quantified by BCA method. Crude extracts and purified proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane. Lanes 1-3, crude extract from E.coli introduced with ksgA gene (88, 44, and 18 μg, respectively); lanes 4-6, that with ksgA (1-213) gene (88, 44, and 18 μg, respectively); lane 7, purified His-KsgA (0.75 μg); lane 8, purified His-KsgA (1-213) (0.75 μg)