Mass Distribution and Spatial Organization of the Linear Bacterial Motor ofSpiroplasma citriR8A2

Abstract

In the simple, helical, wall-less bacterial genus Spiroplasma, chemotaxis and motility are effected by a linear, contractile motor arranged as a flat cytoskeletal ribbon attached to the inner side of the membrane along the shortest helical line. With scanning transmission electron microscopy and diffraction analysis, we determined the hierarchical and spatial organization of the cytoskeleton of Spiroplasma citri R8A2. The structural unit appears to be a fibril, ϳ5 nm wide, composed of dimers of a 59-kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit of the intact ribbon is a pair of aligned fibrils, along which pairs of dimers form tetrameric ring-like repeats. On average, isolated and purified ribbons contain 14 fibrils or seven well-aligned fibril pairs, which are the same structures observed in the intact cell. Scanning transmission electron microscopy mass analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified cytoskeletons indicate that the 59-kDa protein is the only constituent of the ribbons.The mollicutes (Spiroplasma, Mycoplasma, and Acholeplasma spp.) are the smallest and simplest free-living and selfreplicating forms of life. The mollicutes evolved by regressive evolution and genome reduction from the genus Clostridium (for a review, see reference 15). The reduced genome of the mollicutes yields a minimal inventory of cellular components and extreme structural simplicity. Nonetheless, the cells have a well-defined shape, are chemotactic and motile, and demonstrate a variety of cell movements and dynamic morphologies. For example, mycoplasmas and acholeplasmas glide on solid and semisolid surfaces, while the spiroplasmas (2) are free swimmers (for a review, see reference 11).The mollicutes lack cell walls and flagella but have an internal, contractile cytoskeleton that functions as a linear motor (for a review, see reference 20). The spiroplasmas have a particularly unique cellular geometry, such that the Spiroplasma cell can be viewed as a membranous tube to which a flat cytoskeletal ribbon of parallel fibrils is attached along the shortest helical line on the inner surface of the cellular tube. Both tube and cytoskeleton are mutually coiled into a dynamic helix, the latter driving the former. The cytoskeletal ribbon functions as a linear motor by differentially changing the length of its fibril components (21).The genus Spiroplasma was first described by Saglio et al. (16). Williamson (25) and Williamson and Whitcomb (26) reported the isolation of Spiroplasma cytoskeletons by cell lysis and detergent extraction. It has also been shown that cytoskeletons can be released from Spiroplasma cells by repeated freezing and thawing (19). Further electron microscopy studies on Spiroplasma spp. (4,5,17,27) led to a model of a flat, polar, dense cytoskeletal ribbon ϳ94 nm wide attached to the inner surface of the cell membrane and constructed from ϳ4.5-to 5.0-nm-diameter fibrils with an ϳ9-nm repeat. The cytoskeleton accounts for ϳ1% of the total cell...

The Monomeric, Tetrameric, and Fibrillar Organization of Fib: The Dynamic Building Block of the Bacterial Linear Motor of Spiroplasma melliferum BC3

“…The underlying building block of the fibril core is a circular dynamic tetramer of the 59-kDa protein Fib. 13,24 Fib (GenBank ID: M62504), unlike flagellin, is unique to spiroplasmas and bears no similarity to any amino acid or DNA sequences in the currently available databases 32 (however, see the text below regarding specific motif searches). A stable cluster of cytoskeletal and other proteins copurifies with the linear motor ribbon, of which it may be part or with which it may be complexed.…”

“…13,25 The general quantitative organization of the motor, its fibrillar nature, and molecular building blocks were deduced from a concerted analysis of helical cell geometry, 23 sequencing, 32 protein density, diffractometry of isolated whole motors and disrupted components, direct electron microscopy (EM) observation, 13 and linear mass density determined by scanning transmission electron microscopy (STEM). 24 However, the molecular structural details and dynamicsconstituting the core of this article-have not been resolved yet.…”

“…15,21,22 Within the Mollicutes, spiroplasmas are unique in having a dynamic helical cell geometry and a linear cytoskeleton, which functions also as a linear motor. 13,[23][24][25][26] The Mollicutes, although lacking flagella, are motile and chemotactic; spiroplasmas are free swimmers, 14,[27][28][29] while the other members glide. 30,31 The spiroplasmal linear motor is attached to the inner face of the helical membranal tube and follows the shortest path along it.…”

Shaping and Moving a <i>Spiroplasma</i>

“…The cell has 5 helical repeats and is nearly fully stretched. The nearly straight cytoskeletal ribbon is depicted in black following the innermost (shortest) helical line [Trachtenberg and Gilad, 2001;Trachtenberg et al, 2003a]. The ribbon is a monomolecular layer comprised of 7 fibrils and attached to the inner surface of the cell membrane.…”

Mass Analysis of Individual Spiroplasma Cells and their Cytoskeletons

“…The average linearmass-density of a freeze-dried cell is ~3.74 MDa/nm (Fig 1a, 2a) and the total mass of an average cell is ~2.22×10 10 Da/cell, equivalent to ~3.69×10 -14 gram/cell. The average mass density of a double layered, collapsed, cytosol-free, membrane vesicle is ~11.08 kDa/nm 2 (Fig 1b, 2b), while the linear mass density of individual cytoskeletal fibrils (Fig 1c, 2c) is 13.5 kDa/nm [5]. The number of fibrils per ribbon, deduced from the mass per length of straight, uniform ribbons divided by the linear mass density of individual fibrils, is therefore 13.8 (Fig 1c, 2d).…”