Figure 3.

Functional characterization of wt and F40W and F135W mutants of EcFtsZ. (A) GTP hydrolysis activity was initiated by the addition of 1 mM GTP and stopped by adding 10% perchloric acid. Each protein (25 μM) sample was incubated in buffer containing 50 mM Mes (pH 6.5), 10 mM MgCl₂, and 50 mM KCl for 5 min at 30°C. Percentages of activity are with respect to wt EcFtsZ. The errors were obtained by linear regression analysis. (B) Polymerization of F40W and F135W mutants and wt EcFtsZ (25 μM) in a solution containing 50 mM Mes (pH 6.5), 50 mM KCl, and 10 mM MgCl₂, followed by turbidity changes at 30°C. The addition of GTP is indicated by the arrow. (C) In vivo complementation was estimated from the number of colonies obtained after titration at the restrictive temperature (42°C) compared with the number of colonies obtained at the permissive temperature (30°C). C+ corresponds to the transformation of E. coli VIP-2(DE3) strain with wt EcFtsZ, and C− with the vector alone (see Materials and Methods).