Slow polymerization of Mycobacterium tuberculosis FtsZ

Abstract

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

FIG. 1

Polymerization of M. tuberculosis FtsZ. M. tuberculosis FtsZ (10 μM) was assayed under the same conditions as those used for E. coli FtsZ (22). The protein was incubated in 50 mM MES-NaOH (pH 6.5)–10 mM MgCl₂–25 mM KCl in a fluorometer cuvette at 30°C. The 90° angle light scattering was monitored to obtain a baseline. After 6 min, 1 mM GTP was added (●). The dynamic nature of the assay is shown in the other curve (○), in which the addition of 1 mM GTP was followed by 20 mM EDTA, 25 mM MgCl₂, 1 mM GTP, and finally 20 mM EDTA.

FIG. 2

GTP dependence of FtsZ polymerization. FtsZ (10 μM) polymerization was initiated by the addition of various amounts of GTP (arrow) or the nonhydrolyzable GTP analogue 5′-guanylylimidodiphosphate (GMP-PNP).

FIG. 3

Effects of salt on FtsZ polymerization. FtsZ polymerization was initiated by the addition of 0.05 mM GTP (arrow). (A) Effect of various amounts of KCl. (B) Effect of various amounts of NaCl.

FIG. 4

M. tuberculosis FtsZ polymerization requires Mg²⁺. FtsZ polymerization was initiated by the addition of 0.04 mM GTP (arrow) in the presence of various amounts of MgCl₂.

FIG. 5

M. tuberculosis FtsZ polymerizes above a critical concentration. Polymerization of FtsZ at different concentrations was initiated by the addition of 0.05 mM GTP (arrow). (Inset) The net maximum change in light scattering is plotted against the FtsZ concentration. The intercept on the abscissa is the critical concentration of FtsZ required for polymerization to proceed.

FIG. 6

Effect of pH on FtsZ polymerization. FtsZ (13 μM) was polymerized at three pH values in a constant-ionic-strength buffer (50 mM HEPES, 50 mM MES, 100 mM ethanolamine) containing 100 mM KCl and 5 mM MgCl₂. Polymerization was initiated by the addition of 0.04 mM GTP (arrow).

FIG. 7

Inhibition of M. tuberculosis FtsZ polymerization by SRI 7614 (ethyl 6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate). FtsZ (13 μM) was incubated with different concentrations of SRI 7614 in polymerization buffer. Polymerization was initiated by the addition of 0.04 mM GTP (arrow). (Inset) Determination of IC₅₀ value. The net maximum change in light scattering as a percentage of the control is plotted versus the log of the inhibitor concentration. The line is the best fit to the data from three experiments.

FIG. 8

Time course for GTP hydrolysis of M. tuberculosis FtsZ. At various times, 25-μl aliquots were withdrawn and assayed for release of ³²P. The kinetics of GTP hydrolysis with 100 mM KCl and 5 mM MgCl₂ were determined at a concentration slightly above the minimum concentration required for polymerization (5 μM) and at a concentration that gave both polymerization and depolymerization within 60 min (13 μM; Fig. 7). Inhibition by SRI 7614 (10 mM, six times the IC₅₀ value) was determined with 13 μM FtsZ. (Inset) Expansion of the time course for 5 μM FtsZ.

FIG. 9

Electron microscopic analysis of FtsZ polymers. Polymerization was initiated at different MgCl₂ concentrations. (a) No MgCl₂; (b) ordered aggregates of parallel protofilaments formed at 5 mM MgCl₂; (c) 10 mM MgCl₂ (insets, blowups showing bending [top] and fraying [bottom]); (d) 10 mM SRI 7614 and 5 mM MgCl₂. Magnifications, ×40,000 (a to c) and ×50,000 (d).