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.
Highlights
Bacterial cell division protein FtsZ, a homolog of eukaryotic tubulin, assembles at midcell into a ring called the Z-ring
Suprastructures Induced by Molecular Crowding—First, we studied the suprastructures formed by Mycobacterium tuberculosis FtsZ (MtbFtsZ) in the presence of the crowding agents MC or polyvinyl alcohol by electron microscopy
The average diameter was about 170 nm, whereas fully condensed toroids had an average inner diameter of approximately 125 nm and an outer diameter of approximately 250 nm (Fig. 1, A and B). This indicates that MtbFtsZ toroids grow approximately from a protofilament loop onto which more MtbFtsZ condenses both to the outside and inside, similar to DNA toroid formation [5] but different from E. coli FtsZ toroids, which formed protofilament loops about 200 nm in diameter from which the toroid grew only to the outside [3]
Summary
Chemicals—Methyl cellulose (MC) was from WAKO, polyvinyl alcohol was from Kanto Chemical, and fluorophores were from Invitrogen. The protein concentration was determined as described [4]. Preparation of FtsZ Suprastructures and Electron Microscopy— Monomeric MtbFtsZ was mixed with the appropriate amount of crowding agent at 24 °C (final FtsZ concentration was mostly 12.5 M), and polymerization was induced by addition of nucleotide to a final concentration of 2 mM. Sometimes a GTP regeneration system was added as described [12] MtbFtsZ in appropriate buffer was polymerized by the addition of nucleotide. Cations were added as concentrated salts, and light scattering was monitored until a steady state was reached. MtbFtsZ was diluted to the desired concentration, using NaMg buffer, pH 6.6) in the presence of 10 mM dithiothreitol and crowding agents (1% MC or 8% polyvinyl alcohol). Filament length was analyzed and error estimation determined using the programs Aquacosmos, ImageJ, and KaleidaGraph 3.6 as previously described in more detail [15]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
