FIGURE 7.

In vivo analysis of the Walker motif mutants of Hda. A, MK86 [Δhda ΔoriC rnhA] cells harboring pACYC177 (Vector), pACYC/ntHda (WT), pACYC/ntHdaA1B1 (A1B1), pACYC/ntHdaB2 (B2), or pACYC/ntHdaA1B2 (A1B2) were grown at 37 °C in the presence of [³²P]orthophosphate. Nucleotide-bound DnaA protein was immunochemically isolated from cell lysates, and the recovered nucleotides were analyzed as described under “Experimental Procedures.” Proportions (%) of ATP-DnaA included in the total amount of ATP/ADP-DnaA molecules are shown (ATP/(ADP+ATP)). The origin and the migration positions of ATP and ADP are indicated. The asterisk indicates a nonspecific signal, which was seen in the samples prepared using pre-immune serum. B, experiments shown in A were independently repeated three times, and the results with standard deviations are shown. C, expression levels of cells used in this experiment. Western blotting was performed using the indicated cells (A₆₀₀ of 0.15, 67μl). The amount of ntHda was quantified and deduced using a quantitative standard of purified Hda-cHis (Deduced amount). ntHda is the nontagged form of Hda. The relative level of wild-type ntHda was 180-fold increased compared with that of MG1655 (25).