Filament Structure, Organization, and Dynamics in MreB Sheets

Abstract

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several m long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.Despite usually being constrained by a cell wall, bacterial shapes are highly diverse, reflecting the large phylogenetic range. For example Escherichia coli, Bacillus subtilis, and Thermatoga maritime are straight rods, Vibrio cholera is a curved rod, Borrelia burgdorferi forms flat waves, whereas Spiroplasma species are helical. One of the main cytoskeletal proteins involved in determining the shapes of bacteria is thought to be MreB an actin homolog whose atomic structure is very similar to G-actin, despite the low sequence homology (1). It can assemble into polymers both in vitro (1) and in vivo (2). Several studies suggest that MreB plays roles in chromosome segregation (3), polar localization of proteins (4), and maintenance of cell shape and resistance to external mechanical stresses (2). Peptidoglycan cell wall synthesis has been linked to the role of the MreB homolog MbI in B. subtilis (5); however, mechanisms by which MreB may provide mechanical support either directly to the cell or indirectly by affecting peptidoglycan wall integrity remain unclear.In vivo studies of MreB have mainly been limited to visualization under the fluorescence microscope (2, 5). At the low resolution of this technique (ϳ0.2 m), MreB was seen to form cable-like structures, which spiral around the periphery of the cell in B. subtilis, presumably just underneath the cytoplasmic membrane. By electron microscopy (EM), 2 MreB has been observed in vitro to form straight or curved sheets and bundles (1...

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

“…1E). Although protofilaments have not been observed for other eukaryotic actins, they have been observed for the bacterial actin homolog MreB (13,14).…”

Archaeal actin from a hyperthermophile forms a single-stranded filament

“…The remarkable diversity of filaments formed by bacterial actinlike proteins (such as the bacterial cell-shape-determining protein MreB, plasmid partitioning protein ParM, actin-like segregation protein AlfA, and magnetosome cytoskeleton protein MamK) has become clear (9)(10)(11)(12)(13)(27)(28)(29)(30)(31). This seems to reflect the large sequence divergence of the bacterial actin-like proteins (32), in striking contrast to the anomalous sequence conservation of eukaryotic actins.…”

Novel Actin-like Filament Structure from Clostridium tetani

“…The cell shape-determining protein MreB from various bacterial species has been shown to assemble as single-stranded protofilaments (2,3,35). In contrast, all force-generating motors known to be involved in segregating plasmids (ParM-R1, pSK41-ParM, and AlfA) were found to be helical polymers composed of two protofilaments gently winding around each other (2,(7)(8)(9)36).…”

“…First, MreB, which is involved in maintaining cell shape and is found in most prokaryotes (1), forms linear protofilaments arranged in rafts (2,3). Second, ParM-like systems, the polymerizing motors that ensure ordered plasmid segregation prior to cell division, form helical filaments constructed from two protofilaments that resemble F-actin, although they differ in helical parameters and in handedness (4).…”

“…The structures of two classes of bacterial actin-like proteins (Alps) 3 have been determined previously. First, MreB, which is involved in maintaining cell shape and is found in most prokaryotes (1), forms linear protofilaments arranged in rafts (2,3).…”