Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding

Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the "neck," as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.     

Motor-Substrate Interactions in Mycoplasma Motility Explains Non-Arrhenius Temperature Dependence

Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by ∼400 “leg” proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10-40°C. This corresponds to an Arrhenius factor that decreases from ∼45 k_BT at 10°C to ∼10 k_BT at 40°C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction.     

Identification of a 349-kilodalton protein (Gli349) responsible for cytadherence and glass binding during gliding of Mycoplasma mobile

Several mycoplasma species are known to glide in the direction of the membrane protrusion (head-like structure), but the mechanism underlying this movement is entirely unknown. To identify proteins involved in the gliding mechanism, protein fractions of Mycoplasma mobile were analyzed for 10 gliding mutants isolated previously. One large protein (Gli349) was observed to be missing in a mutant m13 deficient in hemadsorption and glass binding. The predicted amino acid sequence indicated a 348,758-Da protein that was truncated at amino acid residue 1257 in the mutant. Immunofluorescence microscopy with a monoclonal antibody showed that Gli349 is localized at the head-like protrusion's base, which we designated the cell neck, and immunoelectron microscopy established that the Gli349 molecules are distributed all around this neck. The number of Gli349 molecules on a cell was estimated by immunoblot analysis to be 450 +/- 200. The antibody inhibited both the hemadsorption and glass binding of M. mobile. When the antibody was used to treat gliding mycoplasmas, the gliding speed and the extent of glass binding were inhibited to similar extents depending on the concentration of the antibody. This suggested that the Gli349 molecule is involved not only in glass binding for gliding but also in movement. To explain the present results, a model for the mechanical cycle of gliding is discussed.     

Triskelion Structure of the Gli521 Protein,Involved in the Gliding Mechanism of Mycoplasma mobile

Mycoplasma mobile binds to solid surfaces and glides smoothly and continuously by a unique mechanism. A huge protein, Gli521 (521 kDa), is involved in the gliding machinery, and it is localized in the cell neck, the base of the membrane protrusion. This protein is thought to have the role of force transmission. In this study, the Gli521 protein was purified from M. mobile cells, and its molecular shape was studied. Gel filtration analysis showed that the isolated Gli521 protein forms mainly a monomer in Tween 80-containing buffer and oligomers in Triton X-100-containing buffer. Rotary shadowing electron microscopy showed that the Gli521 monomer consisted of three parts: an oval, a rod, and a hook. The oval was 15 nm long by 11 nm wide, and the filamentous part composed of the rod and the hook was 106 nm long and 3 nm in diameter. The Gli521 molecules form a trimer, producing a “triskelion” reminiscent of eukaryotic clathrin, through association at the hook end. Image averaging of the central part of the triskelion suggested that there are stable and rigid structures. The binding site of a previously isolated monoclonal antibody on Gli521 images showed that the hook end and oval correspond to the C- and N-terminal regions, respectively. Partial digestion of Gli521 showed that the molecule could be divided into three domains, which we assigned to the oval, rod, and hook of the molecular image. The Gli521 molecule''s role in the gliding mechanism is discussed.Mycoplasmas are commensal and occasionally parasitic bacteria with small genomes that lack a peptidoglycan layer (31). Several mycoplasma species form membrane protrusions, such as the headlike structure in Mycoplasma mobile and the attachment organelle in Mycoplasma pneumoniae (15, 19, 21, 22, 25, 33, 34, 36). On solid surfaces, these species exhibit gliding motility in the direction of the protrusion; this motility is believed to be involved in the pathogenicity of mycoplasmas (12, 13, 16, 20, 21). Interestingly, mycoplasmas have no surface flagella or pili, and their genomes contain no genes related to other known bacterial motility systems. In addition, no homologs of motor proteins that are common in eukaryotic motility have been found (11).M. mobile, which was isolated from the gills of a freshwater fish in the early 1980s, is a fast gliding mycoplasma (14). It glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 μm/s, or three to seven times the length of the cell per second, exerting a force of up to 27 pN (8, 9, 24, 25, 32). Previously, we identified huge proteins involved in this gliding mechanism that are localized at the so-called cell neck, the base of the membrane protrusion (17, 26, 30, 35, 37, 39); we also visualized the putative machinery and the binding protein (1, 18, 23) and identified both the direct energy source used and the direct binding target (10, 27, 38). The force generated by the gliding machinery may be supported from inside the cell by a cytoskeletal “jellyfish” structure (28, 29). On the basis of these results, we proposed a working model, called the centipede or power stroke model, where cells are propelled by “legs” composed of Gli349 that repeatedly catch and release sialic acids fixed on the glass surface (5, 19, 21). These legs are driven by the force exerted by P42 through Gli521 molecules, which is supported by the jellyfish structure, based on energy from ATP hydrolysis.The Gli521 protein, which has an unusually high molecular mass (521 kDa), is suggested to have the role of force transmission, because a monoclonal antibody against this protein stops gliding, keeping the cells on a solid surface (35). About 450 molecules are estimated to be clustered in the gliding machinery with other component proteins, although their alignment has not been clarified (35, 37, 39). In this study, we isolated the Gli521 protein and studied its molecular shape using electron microscopy (EM) and biochemical analyses in order to understand the gliding mechanism.     

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the “carpet-model” and thus, provide important insights into their antimicrobial mechanisms.     

Towards nano-physiology of insects with atomic force microscopy

Little study of insects with modern nanotechnology tools has been done so far. Here we use one of such tool, atomic force microscopy (AFM) to study surface oscillations of the ladybird beetles (Hippodamia convergens) measured in different parts of the insect at picometer level. This allows us to record a much broader spectral range of possible surface vibrations (up to several kHz) than the previously studied oscillations due to breathing, heartbeat cycles, coelopulses, etc. (up to 5-10 Hz). Here we demonstrate three different ways with which one can identify the origins of the observed peaks - by physical positioning the probe near a specific organ, and by using biological or chemical stimuli. We report on identification of high frequency peaks associated with H. convergens heart, spiracular closer muscles, and oscillations associated with muscles activated while drinking. The method, being a relatively non-invasive technique providing a new type of information, may be useful in developing “nanophysiology” of insects.     

Analysis of protein aggregation kinetics using short amino acid peptide tags

Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10 mg/ml to 25.0 mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the “transient solubility (TS)”, the “aggregation initiation concentration (AIC)” and the “long-term solubility (LS)”. AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the “solubility” of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein.     

Detailed Analyses of Stall Force Generation in Mycoplasma mobile Gliding

Mycoplasma mobile is a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 μm/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP; the units catch and pull sialylated oligosaccharides fixed to solid surfaces. In this study, we measured the stall force of wild-type and mutant strains of M. mobile carrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 picoNewtons after the addition of 0–0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. After the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 picoNewtons. Considering the 70-nm step size, this small-unit force may be explained by the large gear ratio involved in the M. mobile gliding machinery.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp. in the inhibition of larval settlement

It has been suggested that bacteria associated with soft-bodied organisms are suggested to produce bioactive compounds against the attachment of invertebrate larvae and bacteria onto the surface of these organisms. Our recent study has demonstrated that epibiotic bacteria from the surface of the soft coral Dendronephthya sp. (Coelenterata: Octocoralia, Alcyonacea) inhibit the growth of bacteria commonly found in marine natural biofilms. In the present study, the effect of 11 epibiotic bacteria isolated from the surface of Dendronephthya sp. on larval settlement of the tubeworms Hydroides elegans was examined using laboratory bioassay. Among 11 bacterial isolates, 2 strains (18%) inhibited the larval settlement of H. elegans (Haswell), 4 strains (36%) were “inductive” to larvae and the remaining 5 strains (46%) were “non-inductive”. There was no correlation between the antifouling activities of bacterial isolates and their phylogenetic origin, i.e. closely related bacterial strains showed different effects on larval settlement of H. elegans. When all “inductive”, “non-inductive” and “inhibitive” bacterial isolates were mixed in a 1:1:1 ratio, the effect of the resultant multispecies film on larval settlement became “inhibitive”. Waterborne compounds of Vibrio sp. and an unidentified α-Proteobacterium, which suppressed the settlement of H. elegans and Bugula neritina (L.) larvae, were further investigated using size fractionation and bioassay-guided enzymatic analysis. It was found that antilarval settlement compounds from these bacteria were heat-stable polysaccharides with a molecular weight >100 kDa. The results indicate that the bacteria associated with the soft coral Dendronephthya sp. may contribute to the antifouling mechanisms of the soft-bodied organisms by producing compounds that are against bacterial growth and settlement of macrofoulers on the surface of their host.     

Localization of P42 and F1-ATPase α-Subunit Homolog of the Gliding Machinery in Mycoplasma mobile Revealed by Newly Developed Gene Manipulation and Fluorescent Protein Tagging

Mycoplasma mobile has a unique mechanism that enables it to glide on solid surfaces faster than any other gliding mycoplasma. To elucidate the gliding mechanism, we developed a transformation system for M. mobile based on a transposon derived from Tn4001. Modification of the electroporation conditions, outgrowth time, and colony formation from the standard method for Mycoplasma species enabled successful transformation. A fluorescent-protein tagging technique was developed using the enhanced yellow fluorescent protein (EYFP) and applied to two proteins that have been suggested to be involved in the gliding mechanism: P42 (MMOB1050), which is transcribed as continuous mRNA with other proteins essential for gliding, and a homolog of the F₁-ATPase α-subunit (MMOB1660). Analysis of the amino acid sequence of P42 by PSI-BLAST suggested that P42 evolved from a common ancestor with FtsZ, the bacterial tubulin homologue. The roles of P42 and the F₁-ATPase subunit homolog are discussed as part of our proposed gliding mechanism.     

Characterisation of physiological and immunological differences between Pacific oysters (Crassostrea gigas) genetically selected for high or low survival to summer mortalities and fed different rations under controlled conditions

Within the framework of a national scientific program named “MORtalités ESTivales de l'huître creuse Crassostrea gigas” (MOREST), a family-based experiment was developed to study the genetic basis of resistance to summer mortality in the Pacific oyster, Crassostrea gigas. As part of the MOREST project, the second generation of three resistant families and two susceptible families were chosen and pooled into two respective groups: “R” and “S”. These two groups of oysters were conditioned for 6 months on two food levels (4% and 12% of oyster soft-tissue dry weight in algal dry weight per day) with a temperature gradient that mimicked the Marennes-Oléron natural cycle during the oyster reproductive period. Oyster mortality remained low for the first two months, but then rapidly increased in July when seawater temperature reached 19 °C and above. Mortality was higher in “S” oysters than in “R” oysters, and also higher in oysters fed the 12% diet than those fed 4%, resulting in a decreasing, relative order in cumulative mortality as follows; 12% “S” > 12% “R” > 4% “S” > 4% “R”. Although the observed mortality rates were lower than those previously observed in the field, the mortality differential between “R” and “S” oysters was similar. Gonadal development, estimated by tissue lipid content, followed a relative order yielding a direct, positive relationship between reproductive effort and mortality as we reported precedently by quantitative histology. Regarding hemocyte parameters, one of the most striking observations was that reactive oxygen species (ROS) production was significantly higher in “S” oysters than in “R” oysters in May and June, regardless of food level. The absence of known environmental stress under these experimental conditions suggests that the ROS increase in “S” oyster could be related to their higher reproductive activity. Finally, a higher increase in hyalinocyte counts was observed for”S” oysters, compared to “R” oysters, in July, just before mortality. Taken together, our results suggest an association of genetically based resistance to summer mortality, reproductive strategy and hemocyte parameters.     

P. aeruginosa PilT Structures with and without Nucleotide Reveal a Dynamic Type IV Pilus Retraction Motor

Type IV pili are bacterial extracellular filaments that can be retracted to create force and motility. Retraction is accomplished by the motor protein PilT. Crystal structures of Pseudomonas aeruginosa PilT with and without bound β,γ-methyleneadenosine-5′-triphosphate have been solved at 2.6 Å and 3.1 Å resolution, respectively, revealing an interlocking hexamer formed by the action of a crystallographic 2-fold symmetry operator on three subunits in the asymmetric unit and held together by extensive ionic interactions. The roles of two invariant carboxylates, Asp Box motif Glu163 and Walker B motif Glu204, have been assigned to Mg²⁺ binding and catalysis, respectively. The nucleotide ligands in each of the subunits in the asymmetric unit of the β,γ-methyleneadenosine-5′-triphosphate-bound PilT are not equally well ordered. Similarly, the three subunits in the asymmetric unit of both structures exhibit differing relative conformations of the two domains. The 12° and 20° domain rotations indicate motions that occur during the ATP-coupled mechanism of the disassembly of pili into membrane-localized pilin monomers. Integrating these observations, we propose a three-state “Ready, Active, Release” model for the action of PilT.     

Gliding Motility and Por Secretion System Genes Are Widespread among Members of the Phylum Bacteroidetes

The phylum Bacteroidetes is large and diverse, with rapid gliding motility and the ability to digest macromolecules associated with many genera and species. Recently, a novel protein secretion system, the Por secretion system (PorSS), was identified in two members of the phylum, the gliding bacterium Flavobacterium johnsoniae and the nonmotile oral pathogen Porphyromonas gingivalis. The components of the PorSS are not similar in sequence to those of other well-studied bacterial secretion systems. The F. johnsoniae PorSS genes are a subset of the gliding motility genes, suggesting a role for the secretion system in motility. The F. johnsoniae PorSS is needed for assembly of the gliding motility apparatus and for secretion of a chitinase, and the P. gingivalis PorSS is involved in secretion of gingipain protease virulence factors. Comparative analysis of 37 genomes of members of the phylum Bacteroidetes revealed the widespread occurrence of gliding motility genes and PorSS genes. Genes associated with other bacterial protein secretion systems were less common. The results suggest that gliding motility is more common than previously reported. Microscopic observations confirmed that organisms previously described as nonmotile, including Croceibacter atlanticus, “Gramella forsetii,” Paludibacter propionicigenes, Riemerella anatipestifer, and Robiginitalea biformata, exhibit gliding motility. Three genes (gldA, gldF, and gldG) that encode an apparent ATP-binding cassette transporter required for F. johnsoniae gliding were absent from two related gliding bacteria, suggesting that the transporter may not be central to gliding motility.     

Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here, we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMHs) and show that conserved glutamate residues in GldL TMH2 are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane, suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

Motile bacteria usually rely on external apparatus like flagella or pili, but gliding bacteria do not rely on obvious surface appendages for their movement. This study shows that bacteria in the phylum Bacteroidetes use proton-dependent motors to power protein secretion and gliding motility.     

Contact- and Protein Transfer-Dependent Stimulation of Assembly of the Gliding Motility Machinery in Myxococcus xanthus

Bacteria engage in contact-dependent activities to coordinate cellular activities that aid their survival. Cells of Myxococcus xanthus move over surfaces by means of type IV pili and gliding motility. Upon direct contact, cells physically exchange outer membrane (OM) lipoproteins, and this transfer can rescue motility in mutants lacking lipoproteins required for motility. The mechanism of gliding motility and its stimulation by transferred OM lipoproteins remain poorly characterized. We investigated the function of CglC, GltB, GltA and GltC, all of which are required for gliding. We demonstrate that CglC is an OM lipoprotein, GltB and GltA are integral OM β-barrel proteins, and GltC is a soluble periplasmic protein. GltB and GltA are mutually stabilizing, and both are required to stabilize GltC, whereas CglC accumulate independently of GltB, GltA and GltC. Consistently, purified GltB, GltA and GltC proteins interact in all pair-wise combinations. Using active fluorescently-tagged fusion proteins, we demonstrate that GltB, GltA and GltC are integral components of the gliding motility complex. Incorporation of GltB and GltA into this complex depends on CglC and GltC as well as on the cytoplasmic AglZ protein and the inner membrane protein AglQ, both of which are components of the gliding motility complex. Conversely, incorporation of AglZ and AglQ into the gliding motility complex depends on CglC, GltB, GltA and GltC. Remarkably, physical transfer of the OM lipoprotein CglC to a ΔcglC recipient stimulates assembly of the gliding motility complex in the recipient likely by facilitating the OM integration of GltB and GltA. These data provide evidence that the gliding motility complex in M. xanthus includes OM proteins and suggest that this complex extends from the cytoplasm across the cell envelope to the OM. These data add assembly of gliding motility complexes in M. xanthus to the growing list of contact-dependent activities in bacteria.     

Codon sextets with leading role of serine create “ideal” symmetry classification scheme of the genetic code

The standard classification scheme of the genetic code is organized for alphabetic ordering of nucleotides. Here we introduce the new, “ideal” classification scheme in compact form, for the first time generated by codon sextets encoding Ser, Arg and Leu amino acids. The new scheme creates the known purine/pyrimidine, codon–anticodon, and amino/keto type symmetries and a novel A + U rich/C + G rich symmetry. This scheme is built from “leading” and “nonleading” groups of 32 codons each. In the ensuing 4 × 16 scheme, based on trinucleotide quadruplets, Ser has a central role as initial generator. Six codons encoding Ser and six encoding Arg extend continuously along a linear array in the “leading” group, and together with four of six Leu codons uniquely define construction of the “leading” group. The remaining two Leu codons enable construction of the “nonleading” group. The “ideal” genetic code suggests the evolution of genetic code with serine as an initiator.     

Morphology of isolated Gli349, a leg protein responsible for Mycoplasma mobile gliding via glass binding, revealed by rotary shadowing electron microscopy

Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed.