NATURE | VOL 413 | 6 SEPTEMBER 2001 | www.nature.com 39 articles Prokaryotic origin of the actin cytoskeleton Fusinita van den Ent, Linda A. Amos & Jan Lo Èwe MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK ............................................................................................................................................................................................................................................................................ It was thought until recently that bacteria lack the actin or tubulin ®lament networks that organize eukaryotic cytoplasm. However, we show here that the bacterial MreB protein assembles into ®laments with a subunit repeat similar to that of F-actinÐthe physiological polymer of eukaryotic actin. By elucidating the MreB crystal structure we demonstrate that MreB and actin are very similar in three dimensions. Moreover, the crystals contain proto®laments, allowing visualization of actin-like strands at atomic resolution. The structure of the MreB proto®lament is in remarkably good agreement with the model for F-actin, showing that the proteins assemble in identical orientations. The actin-like properties of MreB explain the ®nding that MreB forms large ®brous spirals under the cell membrane of rod-shaped cells, where they are involved in cell-shape determination. Thus, prokaryotes are now known to possess homologues both of tubulin, namely FtsZ, and of actin. A central component of the eukaryotic cytoskeleton is ®lamentous actin. Actin is the most abundant protein in many eukaryotic cells, and is conserved from yeast to humans 1 . In 1942, Straub isolated monomeric actin (G-actin) and discovered that raising the salt concentration causes G-actin to polymerize into ®lamentous actin (F-actin) 2 . Electron microscopy and X-ray ®bre diffraction have shown that F-actin consists of two proto®laments that are twisted gently around one another to form a right-handed double helix. The subunits in each actin proto®lament have an approximately 55 A Ê spacing 3 , and the helical pitch is variable owing to torsional ¯exibility of the proto®laments 4 . Under appropriate conditions actin will polymerize into a variety of polymers 5 . When actin is treated with gadolinium it assembles into sheets and cylinders of straight proto®laments 6 . The ®lamentous polymers of actin deter- mine the shape of many eukaryotic cells, besides having a vast range of other functions. The three-dimensional structure of G-actinÐ which has a relative molecular mass of 43,000 (M r 43K)Ðhas been determined in complexes with various actin-binding proteins that prevent actin polymerization 7±10 . Actin is a member of a larger superfamily of proteins 11,12 , which includes Hsp70 (ref. 13), cell-division protein FtsA 14 , and sugar kinases 15,16 . Crystal structures have revealed that each member of the actin superfamily has the characteristic core of actin, and is distinguished by additional insertions or deletions that are neces- sary for the speci®c function of each family member. A sequence database search revealed that the bacterial proteins MreB and StbA have sequence patterns in common with the actin superfamily 11 . MreB, among all proteins of the superfamily, is most closely related to actin in overall size 11 . The mreB gene is located in the gene cluster mre (murein cluster e). It is, together with mrd, the principal operon involved in determination of cell shape in bacteria 17±19 . Recent evidence shows that MreB assembles into a cytoskeleton-like structure in Bacillus subtilis 20 . MreB and the closely related protein Mbl are important in regulating the cell shape of B. subtilis; immuno¯uorescence reveals that elongated polymers of MreB and Mbl encircle the cell as large spirals under the cell membrane. The MreB proteins are widely distributed among rod-shaped, ®lamentous and helical bacteria 20 , suggesting that an MreB cytoskeleton is important to generate a non-spherical shape. To investigate whether MreB can self assemble into actin-like ®laments, we cloned and puri®ed MreB from Thermotoga maritima. Biochemical and electron microscopic analyses show that the protein forms ®laments with a longitudinal repeat similar to that of eukaryotic actin. Elucidation of the crystal structure of MreB shows that it is indeed clearly related to actin. Furthermore, the crystal packing reveals the ®lamentous structure of an actin-like protein at atomic resolution. Here we provide biochemical and structural evidence for MreB as the bacterial actin homologue. Polymerization assays The gene encoding MreB1 from T. maritima was ampli®ed by polymerase chain reaction (PCR) and cloned for overexpression in Escherichia coli strain C41 (see Methods). Thermotoga maritima has two mreB genes. We also cloned mreB2, but the protein was mostly insoluble (data not shown). A BLAST search with both proteins revealed that MreB1 is more closely related to MreB from B. subtilis, with 56% overall identity. MreB forms polymers under various conditions, as was initially investigated in a pelleting assay (Fig. 1). To polymerize, MreB requires ATP (Fig. 1a, lane 1) or GTP (lane 5). It can form ®laments in the absence of magnesium (lane 4). Polymers are formed over a wide pH range, the optimum being pH 6±7 (Fig. 1b). In contrast to actin polymerization, which requires physiological salt concentra- tions, T. maritima MreB is able to form ®laments over a wide range of salt concentrations, as high as 4 M NaCl. The fact that thermo- philic organisms usually possess relatively high intracellular salt concentrations could explain the ability of T. maritima MreB to assemble in high salt. Electron microscopy The nature of MreB polymers, found in the pelleting assay, was investigated by electron microscopy of negatively stained samples. A variety of polymers formed under different conditions (see Fig. 2a, e). The simplest polymers are thin ®laments that appear to consist of two proto®laments (each composed of a string of monomers), but such individual thin ®laments are rare. More common are pairs of thin ®laments, which are often curved (Fig. 2a) depending on the conditions used. Those shown in Fig. 2a are much more highly curved than would be required to produce the curved ®laments observed in vivo 20 . From our images it is unclear how the curvature is accommodated into the structure. The ®ltered image (Fig. 2d) of the polymer in Fig. 2b (diameter of about 160 A Ê ) apparently shows two thin ®laments, with an approxi- mately 51 A Ê longitudinal spacing (Fig. 2c). The thin ®laments appear to lack the distinct twist of the two-stranded helices of F- actin, although they often appear to have a slight twist. At low NaCl concentrations (25 mM NaCl), MreB forms two-dimensional © 2001 Macmillan Magazines Ltd