FtsZ GTPase Activity Quantification

GTPase activity of FtsZ was measured by quantifying the inorganic phosphate with a colorimetric phosphate quantification assay (BIOMOL GREEN kit, ENZO life sciences, Lörrach, Germany) for 140 s^{48 (link)}. Purified FtsZ-wt or FtsZ-G55-Venus-Q56 were used at 3 µM in Reaction buffer, and polymerization was triggered by 1 mM GTP. 13 µL fractions were added to a 96-Well Flat-Bottom Microplate (UV-Star, Greiner Bio-One GmbH) every 20 s after addition of GTP and mixed with 37 µL of Reaction buffer and 100 µL of BIOMOL GREEN reagent, stopping the reaction. After ~10 min of incubation at RT, the absorbance at 620 nm was measured in a TECAN plate reader (Tecan Group Ltd., Mannedorf, Switzerland) at room temperature. Phosphate concentrations were calculated from a Na₂HPO₄ standard curve in Reaction buffer, and the GTPase activity reaction rate (V, mol Pi/mol FtsZ/min) was determined from the slope of the linear part of phosphate accumulation curves.