Suprastructures and dynamic properties of Mycobacterium tuberculosis FtsZ

Abstract

Tuberculosis causes the most death in humans by any bacterium. Drug targeting of bacterial cytoskeletal proteins requires detailed knowledge of the various filamentous suprastructures and dynamic properties. Here, we have investigated by high resolution electron microscopy the assembly of cell division protein and microtubule homolog FtsZ from Mycobacterium tuberculosis (MtbFtsZ) in vitro in the presence of various monovalent salts, crowding agents and polycations. Supramolecular structures, including two-dimensional rings, three-dimensional toroids, and multistranded helices formed in the presence of molecular crowding, were similar to those observed by fluorescence microscopy in bacteria in vivo. Dynamic properties of MtbFtsZ filaments were visualized by light scattering and real time total internal reflection fluorescence microscopy. Interestingly, MtbFtsZ revealed a form of dynamic instability at steady state. Cation-induced condensation phenomena of bacterial cytomotive polymers have not been investigated in any detail, although it is known that many bacteria can contain high amounts of polycations, which may modulate the prokaryotic cytoskeleton. We find that above a threshold concentration of polycations which varied with the valence of the cation, ionic strength, and pH, MtbFtsZ mainly formed sheets. The general features of these cation-induced condensation phenomena could be explained in the framework of the Manning condensation theory. Chirality and packing defects limited the dimensions of sheets and toroids at steady state as predicted by theoretical models. In first approximation simple physical principles seem to govern the formation of MtbFtsZ suprastructures.

FIGURE 1.

Electron micrographs of suprastructures of MtbFtsZ induced by molecular crowding under various conditions. A, 300 mm potassium acetate, MC, pH 6.6; scale bar, 500 nm. B, 300 mm potassium acetate, MC, pH 7.7; scale bar, 200 nm. C, 300 mm rubidium chloride, pH 6.6, MC; scale bar, 500 nm. D, 300 mm rubidium chloride, pH 7.7, MC; scale bar, 200 nm. E, 300 mm NaCl, MC, pH 6.6; scale bar, 200 nm. F, 300 mm NaCl MC, pH 7.7; scale bar, 200 nm.

FIGURE 2.

A closer view of toroids formed in 300 mm rubidium MC, pH 6.6. A, two dimensional rings. B, three-dimensional toroids showing packing defects (arrow). Scale bars, 200 nm.

FIGURE 3.

Typical electron micrographs of MtbFtsZ filaments formed in aqueous solutions under various conditions. A, 300 mm potassium acetate, pH 6.6. B, 300 mm potassium acetate, pH 7.7. C, 300 mm rubidium chloride, pH 6.6. D, 300 mm rubidium chloride, pH 7.7. E, 300 mm NaCl, pH 6.6. F, 300 mm NaCl, pH 7.7. Scale bars, 200 nm.

FIGURE 4.

Polymerization and steady-state kinetics. A, light-scattering time courses of MtbFtsZ polymerization after adding GTP at 24 °C: blue, 300 mm KCl, pH 6.6; green, 300 mm rubidium chloride, pH 6.6; red, 300 mm NaCl, pH 6.6; black, 300 mm NaCl, pH 7.7; pink, 300 mm KCl, pH 7.7; light blue, 300 mm rubidium chloride, pH 7.7. B, critical concentration of MtbFtsZ: red, 300 mm NaCl, pH 6.6; green, 300 mm NaCl, pH 7.7; blue, 300 mm KCl, pH 6.6; black, 300 mm rubidium chloride, pH 6.6. C, TIRF microscopy image of polymerizing MtbFtsZ bundle 50 s after adding GTP. D, same bundle as in C about 300 s later. E, time courses of individual polymerizing MtbFtsZ bundles. F, behavior of individual bundles at steady state.

FIGURE 5.

Light-scattering signal of MtbFtsZ as a function of concentration of various cations. Each sample contained initially 0.5 mg/ml MtbFtsZ followed by sequential addition of concentrated cations: red, hexamine cobalt; orange, polylysine; blue, spermidine; light blue, spermine; yellow, barium chloride; pink, manganese chloride; green, MgCl₂; black, CaCl₂. B, effect of ionic strength on the bundle formation of barium chloride at four different NaCl concentrations: red, 75 mm; blue, 150 mm; green, 300 mm; black, 500 mm. C, variation of the bundling onset of hexamine cobalt (blue), spermine (green), and barium chloride (red) versus the NaCl concentration. Data points represent the average over three individual experiments. D, millimolar concentrations of nucleotides reverse the formation of MtbFtsZ bundles. Light scattering increases after adding spermine up to 10 mm (red). Then additional nucleotide was added (blue), and light scattering started to decrease after adding ∼8 mm additional GTP.

FIGURE 6.

Electron micrographs of MtbFtsZ bundles formed by hexamine cobalt. A, polymerized MtbFtsZ in 150 mm NaCl, pH 7.0. B, after adding 2 mm hexamine cobalt. C, after adding 6 mm hexamine cobalt. D, after adding 16 mm hexamine cobalt. E, in the presence of zinc chloride, sheets formed at pH 7.0, whereas F, toroids were predominant at pH 7.7. G, closer view of the substructure of a cation-induced sheet and H, its Fourier transform, which shows two reflections at about 42 Å (arrow 1) and a layer line at about 40 Å (arrow 2).

FIGURE 7.

Several examples of cation-induced MtbFtsZ sheets about 12 h after formation. A, 16 mm hexamine cobalt. B, 100 mm MgCl₂; the arrow marks an area where sheets have assembled side by side. C, closer view at hexamine cobalt-induced sheets shown in A. Sheets show a strong tendency to twist. More examples of chirality of sheets are shown in D, spermine-induced sheets, and E, polylysine-induced sheets. The arrows mark areas where the sheets are twisted or tend to roll up. Arrow 1 marks a straight area. Scale bars, 200 nm.