A structural framework for a near-minimal form of life: mass and compositional analysis of the helical mollicute Spiroplasma melliferum BC3.

Spiroplasma melliferum is a wall-less bacterium with dynamic helical geometry. This organism is geometrically well defined and internally well ordered, and has an exceedingly small genome. Individual cells are chemotactic, polar, and swim actively. Their dynamic helicity can be traced at the molecular level to a highly ordered linear motor (composed essentially of the proteins fib and MreB) that is positioned on a defined helical line along the internal face of the cell's membrane. Using an array of complementary, informationally overlapping approaches, we have taken advantage of this uniquely simple, near-minimal life-form and its helical geometry to analyze the copy numbers of Spiroplasma's essential parts, as well as to elucidate how these components are spatially organized to subserve the whole living cell. Scanning transmission electron microscopy (STEM) was used to measure the mass-per-length and mass-per-area of whole cells, membrane fractions, intact cytoskeletons and cytoskeletal components. These local data were fit into whole-cell geometric parameters determined by a variety of light microscopy modalities. Hydrodynamic data obtained by analytical ultracentrifugation allowed computation of the hydration state of whole living cells, for which the relative amounts of protein, lipid, carbohydrate, DNA, and RNA were also estimated analytically. Finally, ribosome and RNA content, genome size and gene expression were also estimated (using stereology, spectroscopy and 2D-gel analysis, respectively). Taken together, the results provide a general framework for a minimal inventory and arrangement of the major cellular components needed to support life.

Unperturbing a non-helically perturbed bacterial flagellar filament: Salmonella typhimurium SJW23.

Salmonella typhimurium SJW23 has a right-handed, non-helically perturbed filament of serotype gt with a unique surface pattern. Non-helical perturbations involve symmetry reduction along the five-start helical lines resulting in layer lines of fractional Bessel orders and a consequent seam. The flagellin gene, fliC(23), which we sequenced, differs from the sequence of the canonic, plain SJW1655 flagellin, fliC(1655). We modified discrete components of fliC(23) in order to localize, in the expressed filament, the submolecular site responsible for the non-helical perturbation. These modifications include (i) deleting the outermost domain D3(23), (ii) replacing D3(23) with D3(1655), (iii) substituting a hydrophilic α-helix at the interface between the neighboring domains D1 and D2 with a hydrophobic one from fliC(1655), and (iv) substituting a serine/glycine pair in the loop connecting the modified α-helix to its neighbor; these modifications were made in the presence and absence of D3(23). We used S. typhimurium SJW1655 both as a reference and as a source for 'spare parts'. The symmetry of the constructs was assessed from the power spectra through changes in the layer lines at a height of 1/105 and 1/35 Å(-1), unique to the non-helical perturbation. Deleting D3(23), either alone or in combination with various substitutions, or replacing it with D3(1655) transforms the non-helically perturbed filament into a plain one as judged by the disappearance of the typical layer lines from the power spectra. We conclude that the non-helical perturbation is a product of unique interactions in the D3(23) density shell. Whereas other minor structural changes may occur at the filaments interior, they are all helically symmetric.

The monomeric, tetrameric, and fibrillar organization of Fib: the dynamic building block of the bacterial linear motor of Spiroplasma melliferum BC3.

Spiroplasmas belong to the class Mollicutes, representing the minimal, free-living, and self-replicating forms of life. Spiroplasmas are helical wall-less bacteria and the only ones known to swim by means of a linear motor (rather than the near-universal rotary bacterial motor). The linear motor follows the shortest path along the cell's helical membranal tube. The motor is composed of a flat monolayered ribbon of seven parallel fibrils and is believed to function in controlling cell helicity and motility through dynamic, coordinated, differential length changes in the fibrils. The latter cause local perturbations of helical symmetry, which are essential for net directional displacement in environments with a low Reynolds number. The underlying fibrils' core building block is a circular tetramer of the 59-kDa protein Fib. The fibrils' differential length changes are believed to be driven by molecular switching of Fib, leading consequently to axial ratio and length changes in tetrameric rings. Using cryo electron microscopy, diffractometry, single-particle analysis of isolated ribbons, and sequence analyses of Fib, we determined the overall molecular organization of the Fib monomer, tetramer, fibril, and linear motor of Spiroplasma melliferum BC3 that underlies cell geometry and motility. Fib appears to be a bidomained molecule, of which the N-terminal half is apparently a globular phosphorylase. By a combination of reversible rotation and diagonal shift of Fib monomers, the tetramer adopts either a cross-like nonhanded conformation or a ring-like handed conformation. The sense of Fib rotation may determine the handedness of the linear motor and, eventually, of the cell. A further change in the axial ratio of the ring-like tetramers controls fibril lengths and the consequent helical geometry. Analysis of tetramer quadrants from adjacent fibrils clearly demonstrates local differential fibril lengths.

Structure of the cytoskeleton of Spiroplasma melliferum BC3 and its interactions with the cell membrane.

Spiroplasma melliferum is a wall-less bacterium with dynamic helical symmetry. Taking advantage of the simplicity of this primitive lifeform, we have used structural (electron tomography and freeze fracture of whole cells; cryoelectron tomography and diffraction analysis of isolated cytoskeletons) and proteomic approaches to elucidate the basic organizing principles of its minimal yet functional cytoskeleton. From among approximately 30 Spiroplasma proteins present in a highly purified cytoskeletal fraction, we identify three major putative structural proteins: Fib, MreB, and elongation factor Tu. Fib assembles into a single flattened ribbon that follows the shortest helical line just under the plasma membrane and acts as a linear motor, whereas MreB is present as a matching array of membrane-associated fibrils parallel and associated with the motor. We also identify a prominent previously unknown filamentous network that occupies much of the cytoplasm and appears to cross-link the ribosomes. The abundant potentially filament-forming protein elongation factor Tu may be a component of this network, but the tomography data are most consistent with DNA as the core component. The results provide new information on the minimal organization necessary to support the scaffolding and motile functions of a minimal cytoskeleton.

The flagellar filament structure of the extreme acidothermophile Sulfolobus shibatae B12 suggests that archaeabacterial flagella have a unique and common symmetry and design.

Archaea, constituting a third domain of life between Eubacteria and Eukarya, characteristically inhabit extreme environments. They swim by rotating flagellar filaments that are phenomenologically and functionally similar to those of eubacteria. However, biochemical, genetic and structural evidence has pointed to significant differences but even greater similarity to eubacterial type IV pili. Here we determined the three-dimensional symmetry and structure of the flagellar filament of the acidothermophilic archaeabacterium Sulfolobus shibatae B12 using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Processing of the cryo-negatively stained filaments included analysis of their helical symmetry and subsequent single particle reconstruction. Two filament subunit packing arrangements were identified: one has helical symmetry, similar to that of the extreme halophile Halobacterium salinarum, with ten subunits per 5.3 nm repeat and the other has helically arranged stacked disks with C(3) symmetry and 12 subunits per repeat. The two structures are related by a slight twist. The S. shibatae filament has a larger diameter compared to that of H. salinarum, at the opposite end of the archaeabacterial phylogenetic spectrum, but the basic three-start symmetry and the size and arrangement of the core domain are conserved and the filament lacks a central channel. This similarity suggests a unique and common underlying symmetry for archaeabacterial flagellar filaments.

The archaeabacterial flagellar filament: a bacterial propeller with a pilus-like structure.

Common prokaryotic motility modes are swimming by means of rotating internal or external flagellar filaments or gliding by means of retracting pili. The archaeabacterial flagellar filament differs significantly from the eubacterial flagellum: (1) Its diameter is 10-14 nm, compared to 18-24 nm for eubacterial flagellar filaments. (2) It has 3.3 subunits/turn of a 1.9 nm pitch left-handed helix compared to 5.5 subunits/turn of a 2.6 nm pitch right-handed helix for plain eubacterial flagellar filaments. (3) The archaeabacterial filament is glycosylated, which is uncommon in eubacterial flagella and is believed to be one of the key elements for stabilizing proteins under extreme conditions. (4) The amino acid composition of archaeabacterial flagellin, although highly conserved within the group, seems unrelated to the highly conserved eubacterial flagellins. On the other hand, the archaeabacterial flagellar filament shares some fundamental properties with type IV pili: (1) The hydrophobic N termini are largely homologous with the oligomerization domain of pilin. (2) The flagellin monomers follow a different mode of transport and assembly. They are synthesized as pre-flagellin and have a cleavable signal peptide, like pre-pilin and unlike eubacterial flagellin. (3) The archaeabacterial flagellin, like pilin, is glycosylated. (4) The filament lacks a central channel, consistent with polymerization occurring at the cell-proximal end. (5) The diameter of type IV pili, 6-9 nm, is closer to that of the archaeabacterial filament, 10-14 nm. A large body of data on the biochemistry and molecular biology of archaeabacterial flagella has accumulated in recent years. However, their structure and symmetry is only beginning to unfold. Here, we review the structure of the archaeabacterial flagellar filament in reference to the structures of type IV pili and eubacterial flagellar filaments, with which it shares structural and functional similarities, correspondingly.

The cytoskeleton of spiroplasma: a complex linear motor.

Spiroplasma are wall-less, helical bacteria from the class Mollicutes. The Mollicutes (Mycoplasma, Acholeplasma, Spiroplasma) evolved by regressive evolution to generate one of the simplest and minimal free-living and self-replicating forms of life. The spiroplasmas are the more advanced members in the class and are the closest to their clostridial ancestors. Spiroplasmas were discovered and identified as such only in 1972 and the finding of a unique and well-defined internal cytoskeleton, believed to be uncommon in bacteria, followed in 1973. Structural analysis suggests that the core of the spiroplasmal cytoskeleton is a flat, monolayered ribbon comprised of the 59-kDa fib gene product. The ribbon follows the shortest helical line of the polar cell from end to end. The structural building blocks of the cytoskeletal ribbon are fibrils assembling into a structure with approximately 10-nm axial and lateral repeats. Differential length changes of the fibrils may generate a wide dynamic spectrum of helical and non-helical geometries allowing for directional motility in low Reynolds number environments. The presence of other cytoskeletal elements (FtsZ, FtsA, EF-TU, MreB) has been demonstrated only recently in Spiroplasma cells. The cellular and molecular structure and dynamics of spiroplasmas and their cytoskeletal elements are reviewed.

Refining the structure of the Halobacterium salinarum flagellar filament using the iterative helical real space reconstruction method: insights into polymorphism.

The eubacterial flagellar filament is an external, self-assembling, helical polymer approximately 220 A in diameter constructed from a highly conserved monomer, flagellin, which polymerizes externally at the distal end. The archaeal filament is only approximately 100 A in diameter, assembles at the proximal end and is constructed from different, glycosylated flagellins. Although the phenomenology of swimming is similar to that of eubacteria, the symmetry of the archebacterial filament is entirely different. Here, we extend our previous study on the flagellar coiled filament structure of strain R1M1 of Halobacterium salinarum. We use strain M175 of H.salinarum, which forms poly-flagellar bundles at high yield which, under conditions of relatively low ionic-strength (0.8 M versus 5 M) and low pH ( approximately 2.5 versus approximately 6.8), form straight filaments. We demonstrated previously that a single-particle approach to helical reconstruction has many advantages over conventional Fourier-Bessel methods when dealing with variable helical symmetry and heterogeneity. We show here that when this method is applied to the ordered helical structure of the archebacterial uncoiled flagellar filament, significant extensions in resolution can be obtained readily when compared to applying traditional helical techniques. The filament population can be separated into classes of different morphologies, which may represent polymorphic states. Using cryo-negatively stained images, a resolution of approximately 10-15 A has been achieved. Single alpha-helices can be fit into the reconstruction, supporting the proposed similarity of the structure to that of type IV bacterial pili.

Shaping and moving a spiroplasma.

The Mollicutes (Spiroplasma, Mycoplasma and Acholeplasma) are the most minimal cells known to exist, being the smallest and simplest free-living and self-replicating forms of life. Phylogenetically, the Mollicutes are related to gram-positive bacteria and have evolved, by regressive evolution and genome reduction, from Clostridia. The smallest genome in this group (Mycoplasma genitalium - 5.77 x 10(5) bp) is only twice that of a large virus (e.g., Entomopox viruses). The largest Mollicute genome (Spiroplasma LB12 - 2.2 x 10(6) bp) is only about half that of, e.g., Escherichia coli. Structurally, the Mollicutes lack cell walls and flagella, but have internal cytoskeletons and are motile and chemotactic. Only a cholesterol-containing unit membrane envelops the cells. No analogs to the bacterial chemotactic and motility (che, mot, fla) genes, genes for a two-component signal transduction system, genes associated with gliding, or genomic homologs for the eukaryotic cytoskeleton and motor proteins were found in the Mollicutes. The Spiroplasmas are unique amongst the Mollicutes in having a well-defined basic helical cell geometry. In this respect, the Spiroplasma cell can, essentially, be viewed as a helical dynamic membranal tube (diameter approximately 0.2 microm; equivalent to that of one eukaryotic flagellar axoneme or to a bacterial flagellar bundle). A flat cytoskeletal ribbon of parallel fibrils is attached to the inside of the cellular tube. Both tube and cytoskeleton are mutually coiled into a dynamic helix driven by differential length changes of the fibrils, which function as linear motors. The cytoskeletal ribbon follows the shortest (inner) helical line on the inner surface of the cellular tube. Being helical allows for further analytical reduction and consequent structural quantification of Spiroplasma. Of particular importance is the ability to correlate light and electron microscopy data and to calculate the fibril lengths (and corresponding molecular dimensions) in the helical and nonhelical dynamic states. The structural unit of the contractile cytoskeleton is a approximately 50-Angstrom-wide filament comprised of pairs of the 59-kD fib gene product. The monomers are arranged in pairs with opposite polarities allowing for a approximately 100-Angstrom-long axial repeat. The functional unit of the contractile cytoskeletal ribbon is a fibril comprised of an aligned pair of filaments. Neighboring repeats form a tetrameric ring with a lateral repeat of approximately 100 A. The axial length of the rings may shorten by approximately 40%, driving the changes in the fibril lengths and, consequently, helical dynamics. Local length changes result in helical symmetry breaking and nonreciprocating cell movements allowing for net directional displacement. Flexing allows for changes in swimming direction.

The axial alpha-helices and radial spokes in the core of the cryo-negatively stained complex flagellar filament of Pseudomonas rhodos: recovering high-resolution details from a flexible helical assembly.

Of the two known "complex" flagellar filaments, those of Pseudomonas are far more flexible than those of Rhizobium. Their diameter is larger and their outer three-start ridges and grooves are more prominent. Although the symmetry of both complex filaments is similar, the polymer's linear mass density and the flagellin molecular mass of the latter are lower. A recent comparison of a three-dimensional reconstruction of the filament of Pseudomonas rhodos to that of Rhizobium lupini indicates that the outer flagellin domain (D3) is missing in R.lupini. Here, we concentrate on the structure of the inner core of the filament of P.rhodos using field emission cryo-negative staining electron microscopy and a hybrid helical/single particle reconstruction technique. Averaging 158 filaments caused the density band corresponding to the radial spokes to nearly average out due to their variability and inferred flexibility. Treating the Z=0 cross-sections through the aligned individual three-dimensional density maps as images, classifying them by correspondence analysis (using a mask containing the radial spokes domain) and re-averaging the subclasses (using helical reconstruction techniques) allowed a recovery of the radial spokes and resolved the alpha-helices in domain D0 and the triple alpha-helical bundles in domain D1 at a resolution of 1/7A(-1). Although the perturbed components of the helical lattice are present along the entire filament's radius, the interior of the complex filament is similar to that of the plain one, whereas it's exterior is altered. Reconstructions of vitrified and cryo-negatively stained plain, right-handed filaments of Salmonella typhimurium SJW1655 prepared and imaged under conditions identical with those used for P.rhodos confirm the similarity of their inner cores and that the secondary structures in the interior of the flagellar filament can, under critical conditions of image recording and correction, be resolved in negative stain.

Bacterial flagellar microhydrodynamics: Laminar flow over complex flagellar filaments, analog archimedean screws and cylinders, and its perturbations.

The flagellar filament, the bacterial organelle of motility, is the smallest rotary propeller known. It consists of 1), a basal body (part of which is the proton driven rotary motor), 2), a hook (universal joint-allowing for off-axial transmission of rotary motion), and 3), a filament (propeller-a long, rigid, supercoiled helical assembly allowing for the conversion of rotary motion into linear thrust). Helically perturbed (so-called "complex") filaments have a coarse surface composed of deep grooves and ridges following the three-start helical lines. These surface structures, reminiscent of a turbine or Archimedean screw, originate from symmetry reduction along the six-start helical lines due to dimerization of the flagellin monomers from which the filament self assembles. Using high-resolution electron microscopy and helical image reconstruction methods, we calculated three-dimensional density maps of the complex filament of Rhizobium lupini H13-3 and determined its surface pattern and boundaries. The helical symmetry of the filament allows viewing it as a stack of identical slices spaced axially and rotated by constant increments. Here we use the closed outlines of these slices to explore, in two dimensions, the hydrodynamic effect of the turbine-like boundaries of the flagellar filament. In particular, we try to determine if, and under what conditions, transitions from laminar to turbulent flow (or perturbations of the laminar flow) may occur on or near the surface of the bacterial propeller. To address these questions, we apply the boundary element method in a manner allowing the handling of convoluted boundaries. We tested the method on several simple, well-characterized cylindrical structures before applying it to real, highly convoluted biological surfaces and to simplified mechanical analogs. Our results indicate that under extreme structural and functional conditions, and at low Reynolds numbers, a deviation from laminar flow might occur on the flagellar surface. These transitions, and the conditions enabling them, may affect flagellar polymorphism and the formation and dispersion of flagellar bundles-factors important in the chemotactic response.

The structure of the helically perturbed flagellar filament of Pseudomonas rhodos: implications for the absence of the outer domain in other complex flagellins and for the flexibility of the radial spokes.

Bacterial flagella, the organelles of motility, are commonly divided into two classes: 'plain' and 'complex'. The complex filaments are pairwise, helically perturbed forms of the plain filaments and have been reported to occur only in Rhizobium and Pseudomonas. Previously, we reconstructed and analysed the structure of the complex filaments of Rhizobium lupini H13-3 and determined their unique symmetry and origin of the perturbations (Trachtenberg et al., 1986, J Mol Biol 190: 569-576; 1987, 195: 603-620; 1998, 276: 759-773; Cohen-Krausz and Trachtenberg, 1998, J Struct Biol 122: 267-282). Here, we analyse the structure of the flagellar filament of the other known complex filament, that of Pseudomonas rhodos, as reconstructed from electron microscope images. Compared with the filament of R. lupini, the filament of P. rhodos is more flexible, as implied from high-intensity darkfield light microscopy and, although constructed from flagellins of higher molecular weights (59 versus 41 kDa), has similar symmetry. Using cryonegative stained specimens and low-dose, field emission electron microscopy, we reconstructed and averaged 158 filaments each containing 170 statistically significant layer lines. The three-dimensional density maps of P. rhodos clearly suggest, when compared with those of R. lupini and the right-handed Salmonella typhimurium SJW1655, that R. lupini is missing the outer flagellin domain (D3), that the interior of the complex filament is rather similar to that of the plain filament and that the radial spokes (connecting domains D0 and D1), present in individual density maps, average out because of their variability and implied flexibility. Extending the three-start grooves and ridges on the propeller's surface, in the form of an Archimedean screw, may further improve the motility of the cell in viscous environments.

Mass distribution and spatial organization of the linear bacterial motor of Spiroplasma citri R8A2.

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, approximately 5 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.

Motility modes of Spiroplasma melliferum BC3: a helical, wall-less bacterium driven by a linear motor.

Spiroplasma are members of the Mollicutes (Mycoplasma, Acholeplasma and Spiroplasma) - the simplest, minimal, free-living and self-replicating forms of life. The mollicutes are unique among bacteria in completely lacking cell walls and flagella and in having an internal, contractile cytoskeleton, which also functions as a linear motor. Spiroplasma are helical, chemotactic and viscotactic active swimmers. The Spiroplasmal cytoskeleton is a flat ribbon composed of seven pairs of fibrils. The ribbon is attached to the inner side of the cell membrane along its innermost (shortest) helical line. The cell's geometry and dynamic helical parameters, and consequently motility, can be controlled by changing differentially and in a co-ordinated manner, the length of the fibrils. We identified several consistent modes of cell movements and motility originating, most likely, as a result of co-operative or local molecular switching of fibrils: (i). regular extension and contraction within the limits of helical symmetry (this mode also includes straightening, beyond what is allowed by helical symmetry, and reversible change of helical sense); (ii). spontaneous and random change of helical sense originating at random sites along the cell (these changes propagate along the cell in either direction and hand switching is completed within approximately 0.08 second); (iii). forming a deformation on one of the helical turns and propagating it along the cell (these helical deformations may travel along the cell at a speed of up to approximately 40 microm s-1); (iv). random bending, flexing and twitching (equivalent to tumbling). In standard medium (viscosity = 1.147 centipoise) the cells run at approximately 1.5 microm s-1, have a Reynolds number of approximately 3.5 x 10-6 and consume approximately 30 ATP molecules s-1. Running velocity, duration, persistence and efficiency increase with viscosity upon adding ficoll, dextran and methylcellulose to standard media. Relative force measurements using optical tweezers confirm these findings.

The bacterial linear motor of Spiroplasma melliferum BC3: from single molecules to swimming cells.

Spiroplasma melliferum BC3 are wall-less bacteria with internal cytoskeletons. Spiroplasma, Mycoplasma and Acholeplasma belong to the Mollicutes, which represent the smallest, simplest and minimal free-living and self-replicating forms of life. The Mollicutes are motile and chemotactic. Spiroplasma cells are, in addition, helical in shape. Based on data merging, obtained by video dark-field light microscopy of live, swimming helical Spiroplasma cells and by cryoelectron microscopy, unravelling the subcellular structure and molecular organization of the cytoskeleton, we propose a functional model in which the cytoskeleton also acts as a bacterial linear motor enabling and controlling both dynamic helicity and swimming. The cytoskeleton is a flat, monolayered ribbon constructed from seven contractile fibrils (generators) capable of changing their length differentially in a co-ordinated manner. The individual, flat, paired fibrils can be viewed as chains of tetramers approximately 100 A in diameter composed of 59 kDa monomers. The cytoskeletal ribbon is attached to the inner surface of the cell membrane (but is not an integral part of it) and follows the shortest helical line on the coiled cellular tube. We show that Spiroplasma cells can be regarded, at least in some states, as near-perfect dynamic helical tubes. Thus, the analysis of experimental data is reduced to a geometrical problem. The proposed model is based on simple structural elements and functional assumptions: rigid circular rings are threaded on a flexible, helical centreline. The rings maintain their circularity and normality to the centreline at all helical states. An array of peripheral, equidistant axial lines forms a regular cylindrical grid (membrane), by crossing the lines bounding the rings. The axial and peripheral spacing correspond to the tetramer diameter and fibril width (100 A) respectively. Based on electron microscopy data, we assign seven of the axial grid lines in the model to function as contractile generators. The generators are clustered along the shortest helical paths on the cellular coil. In the model, the shortest generator coincides with the shortest helical line. The rest, progressively longer, six generators follow to the right or to the left of the shortest generator in order to generate the maximal range of lengths. A rubbery membrane is stretched over (or represented by) the three-dimensional grid to form a continuous tube. Co-ordinated, differential length changes of the generators induce the membranal cylinder to coil and uncoil reversibly. The switch of helical sense requires equalization of the generators' length, forming a straight cylindrical tube with straight generators. The helical parameters of the cell population, obtained by light microscopy, constitute several subpopulations related, most probably, to cell size and age. The range of molecular dimensions in the active cytoskeleton inferred from light microscopy and modelling agrees with data obtained by direct measurements of subunit images on electron micrographs, scanning transmission electron microscopy (STEM) and diffraction analysis of isolated ribbons. Swimming motility and chemotactic responses are carried out by one or a combination of the following: (i). reciprocating helical extension and compression ('breathing'); (ii). propagation of a deformation (kink) along the helical path; (iii). propagation of a reversal of the helical sense along the cell body; and (iv). irregular flexing and twitching, which is functionally equivalent to standard bacterial tumbling. Here, we analyse in detail only the first case (from which all the rest are derived), including switching of the helical sense.

The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili.

Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g. reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition. Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion. The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin. The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments. The polymerization mode and small diameter may indicate the absence of a central channel in the filament. Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A. The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1. The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell. There is no evidence for a central channel. On the basis of the homology with the oligomerization domain of type IV pilin and the density distribution of the filament map, we propose a structure for the central core.