In vitro assay of BcFtsZ GTPase activity.

GTPase activity was assayed at 30°C, using a pyruvate kinase–l-lactic dehydrogenase (PK/LDH) spectrophotometric coupled assay (68). The reaction mixture was first set up to contain 50 mM HEPES (pH 7.5), 5 mM MgCl₂, 5 mM KCl, 10 U PK/LDH, 0.25 mM NADH, 0.25 mM phosphoenolpyruvate, and 5.25 µM BcFtsZ. The assay was initiated by the addition of 0.5 mM GTP. Steady-state kinetic parameters were determined by monitoring the absorbance at 340 nm at various concentrations of GTP. The experiments were performed in triplicate, and the kinetic constants were determined by fitting the data to the Michaelis-Menten equation using Origin 8. C109 was added in concentrations ranging from 0.5 µM to 100 µM, and the inhibitory concentration that reduced the activity by half (IC₅₀) was determined using Origin 8. A_[I] is the enzyme activity at inhibitor concentration [I], and A_[0] is the enzyme activity without inhibitor.

The activities of PK and LDH individually were assayed with C109, but no effect was detected, thus excluding the possibility that the molecule can exert an effect on them.