1. Mycoplasma mobile uses flexible legs, that literally walk on the surface. Better maybe to say, they run. While Usain Bolt runs 5.3 of its own body length per second,(5.3 bl/sec.) M. mobile runs a top burst speed of ~10 bl/sec
2. Four component proteins are directly involved in the mechanism, and at least 10 proteins are involved in the cytoskeleton. None of the four proteins has significant sequence similarities with proteins of any other bacterias.
3. Since none of these components can be dismissed without harming the motility function, this system is irreducible and interdependent, and on top, none of the components could be co-opted from other organisms.
Behe used, as an illustration, a moustrap, where all the components are required for its function, and as such, it is irreducibly complex. Mycoplasma genitalium is famous for having among the smallest genomes of self-replicating organisms. Mycoplasma mobile of the same family, deserves to become a star amongst bacterias as well. But for other reasons. The other gliding mycoplasma, M. pneumoniae, shares many gliding characteristics with M. mobile, but not similar amino acid sequences with proteins essential for gliding. Its motility is not related to any other known bacterial families. Its biomotility mechanism is unique. The gliding machinery exists at the base of the membrane protrusion, where many flexible legs project outside the cell and are supported by cytoskeletal structures from inside the cell. Four component proteins are directly involved in the mechanism, and at least 10 proteins are involved in the cytoskeleton. Leg and gear proteins have distinct molecular sizes and morphologies. None of the four proteins has significant sequence similarities with proteins of other bacteria, which means, they are no candidates to claim they could have been co-opted from another organism. Mycoplasma mobile is propelled by Gli349 protein “legs” which repeatedly catch and release on the surface where they walk, and are driven by the force exerted by a so-called P42 protein through Gli521 molecules, supported by surface structure on which they walk, based on the consumption of energy in the form of ATP. Each of the four proteins used for walking are essential.
1. Gli349 protein is the "leg" responsible for binding on the surface during gliding and is required to convey movement.
2. Gli521 is the motor or the gear-transmitting force from the motor to the leg.
3. Gli123 protein is necessary to stabilize Gli349 and Gli521 proteins.
4. P42 has ATPase activity. The energy for M. mobile gliding is supplied by ATP hydrolysis and that detachment from the solid surface is an energy-dependent process.
Each of these four proteins is essential for M.mobile motility. None can be co-opted since any has significant sequence similarities with proteins of other bacterias. On top of that, these are huge proteins. Gli349 protein is composed of 3181 amino acids. Gli521 has 4728 amino acids. Gli123 protein is composed of 1128 amino acids. P42 is a 356-amino-acid protein.
Mutation experiments in the last decade have demonstrated that these proteins, each, must be precisely and correctly sequenced, otherwise, the function ceases, and motility is not possible.
Prokaryotes have numerous means of locomotion built around distinct molecular mechanisms. A human running, a dog walking, an eagle flying, a fish swimming, a frog hopping, and a starfish crawling are each unique, but all share a root mechanism in the molecular properties of actin and myosin in muscle cells. For prokaryotes, however, their movements don’t just look different; they often use disparate molecular systems or organelles unique to a particular genus or even species. Some of the more-studied mechanisms of prokaryotic motility are flagellar-based swimming and swarming, type-IV pilus twitching motility, and the ‘adventurous’ motility of Myxococcus xanthus. This adventurous motility relies on both secreted polysaccharide and a helical motor that produces tread-like protrusions along the cell surface.
Among the handful of other known prokaryotic motility mechanisms are those employed by the Mycoplasma, a genus in the class Mollicutes - Gram-positive bacteria (Firmicutes). The Mollicutes share several defining characteristics, most obviously the lack of cell wall that gives them their name (mollis is Latin for ‘soft’ or ‘pliable’). Therefore, although they phylogenetically fall in the Firmicutes, the Mollicutes have no cell wall for the Gram stain. The Mollicutes are some of the smallest cells known. These bacteria are often closely associated with host organisms, allowing the bacteria to attain drastically reduced genome sizes (The ~500,000 kilobases of M. genitalium is the lower limit for forming colonies). Mycoplasma a fascinating subject for study in a variety of fields. In particular, the mysterious motility observed for species of the M. pneumoniae cluster has inspired enthralling research that begins to illuminate novel mechanisms of prokaryotic motility. Surprisingly, this molecular system of motility appears specific for M. mobile. The gliding proteins of M. mobile are found in the closely related M. pulmonis, but nowhere else known. 2 The complex system of proteins that powers this feat is nearly unique to this microbe, so far as scientists know. The engine apparatus of M. mobile is found only in it and one related species. Mycoplasma species are the smallest bacterias known.1
It’s first worth getting a sense of how fast these microbes are moving (with a nod to the wonderful series of “Microbial Olympics” essays to which our own Merry Youle contributed. If you’ve witnessed bacterial motility under a microscope, you’re perhaps most familiar with flagellar-mediated E. coli swimming, which generates speeds of ~10 – 35 mm/sec. This translates to a top burst speed of ~17.5 ‘body’ lengths (bl)/sec. To put this into perspective (based on numbers here), the world’s fastest fish, the sunfish, clocks in at a staggering speed of 90 bl/sec, a goldfish manages a healthy 4.5 bl/sec, and US Olympian Michael Phelps achieves a meager 1 bl/sec. Of the motile Mycoplasma, the swiftest known is M. mobile, a presumed pathogenic resident of gills in freshwater fish. Discovered in the late 1970’s, M. mobile has been studied as a potential biomotor with gliding speeds of 2 – 7 mm/sec. Considering their small size (~0.7 mm) this gives a top burst speed of ~10 bl/sec. This M. mobile gliding is about half as fast as E. coli swimming, but still quite respectable. Indeed, it is double the sprinting speed of world-record-holding, Jamaican Olympian Usain Bolt (5.3 bl/sec.)!
So then, M. mobile gets around. How does it do it? Mycoplasma species lack flagella, pili, or homologs to any known motility system, prokaryotic or eukaryotic. It turns out that their motility is closely related to their shape – again, form fits function. Species in the M. pneumoniae cluster (including M. mobile) have a light-bulb shaped morphology consisting of a body and a polar membrane protrusion (head) called the attachment or terminal organelle. Beneath this bulb-shaped morphology lies a complex cytoskeleton. So what is this cytoskeleton made of? While some Mycoplasma encode known prokaryotic cytoskeletal elements such as FtsZ and MreB, these are absent in M. mobile. Instead, researchers identified a set of at least ten unique proteins as making up the M. motile cytoskeleton structure. Three of the four identified proteins are found in the neck portion of the cell surface, corresponding to the region where the tentacle portion of the internal cytoskeleton resides. the gliding proteins form a leg-like structure extending from inside the cell (associated in some way with the cytoskeleton components) to a ‘foot’ that interacts with surface substrates. In this way, M. mobile is able to ‘walk’ across surfaces and manifest its visible gliding motility.
Mycoplasma mobile features a protrusion that enables it to glide smoothly on solid surfaces. 4 M. mobile glides by a repeated catch-pull-release of sialylated oligosaccharides fixed on a solid surface by hundreds of 50-nm flexible “legs” sticking out from the protrusion. This gliding mechanism may be explained by a possible directed binding of each leg with sialylated oligosaccharides, by which the leg can be detached more easily forward than backward. 50-nm-long leglike structures are observed to stick out from the base of the protrusion and attach to the solid surface at their distal ends. Huge proteins, Gli123, Gli349, and Gli521 localize on the machinery surface are involved in this gliding mechanism. It is composed of an internal structure of the machinery, named the “jellyfish” structure, which consists of a bell shape at the cell front connected by dozens of tentacular strands comprised of 20-nm particles at 30-nm intervals, as well as the component proteins of the machinery. there is a direct energy source, a direct binding target. Cells are propelled by flexible “legs” composed of Gli349 that, through repeated cycles, catch, pull, and release sialylated oligosaccharides (SOs) fixed on the glass surface via the distal “feet”.
Gene locus of M. mobile coding gliding proteins.
Four proteins involved in the gliding mechanism are coded tandemly on the genome. The typical role of each protein is presented in the parentheses. 6
None of the four proteins has significant sequence similarities with proteins of other bacteria
The Gli349 protein is composed of 3181 amino acids and features a transmembrane segment near the N-terminus. Gli349 is responsible for binding on the surface during gliding and is required to convey movement.
They contain an oval part 14nm long at a pole, which has been named the “foot”. 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, about 90 degrees. The leg protein, Gli349, is anchored to the cell membrane, and is composed of two rigid rods, a flexible part, and a foot, and catches and pulls SOs fixed on the solid surface through the foot.
Structure of Gli349, the leg protein
(a) Rotary shadowing electron microscopy image of an isolated Gli349 protein.
(b) Schematic of the molecule on the cell surface. The leg protein, Gli349, is composed of two short arms linked by a foldable hinge, a long filament, and a foot. Some monoclonal antibodies decrease the gliding speed and finally remove the mycoplasmas from the glass surface. Weak repeats can be observed in the amino acid sequence, as presented by gray ovals. Arrows indicate putative movements in gliding.
Left: Rotary shadowing electron microscopy image of isolated Gli349 protein. Right: The central part marked by a white circle was suggested by image averaging to be stable and rigid.
Gli521 codes for 4728 amino acids. It is the motor or the gear-transmitting force from the motor to the leg. Identification of ATPase activity in P42, coded downstream of Gli521 on the genome, suggests that the role of Gli521 is the transmission of force from the motor to the leg, i.e., a “gear”. This protein is divided into three domains. Each of these domains may correspond to the arms of the triad. TM segments are predicted from amino acid sequences at the N- and C-terminal domains, and the N-terminal TM is actually cleaved by processing between residues 43 and 44. The Gli521 protein Molecule is directly involved in movements of Mycoplasma mobiles gliding machinery 5 Cells are propelled by “legs” composed of Gli349 repeatedly catching and releasing sialic acids fixed on the glass surface and are driven by the force exerted by P42 through Gli521 molecules, supported by the jellyfish structure, based on the energy of ATP.
Schematic of the molecule on the cell surface.
The gear protein, Gli521, is composed of three domains connected by flexible hinges that form a trimer. Amino acid substitutions that influence Gli349 function are marked by red asterisks. Monoclonal antibodies against the target sites decreased the gliding speed and finally stops gliding. Arrows indicate putative movements in gliding.
The Gli123 protein is composed of 1128 amino acids. This protein is coded tandemly upstream of the Gli349 and Gli521 proteins on the genome. In a strain lacking this protein, the localization and stability of Gli349 and Gli521 proteins are significantly disturbed. Gli123 exists at the same cellular position as Gli349 and Gli521. This protein provides binding sites in the cell for other gliding proteins and forms a complex of gliding machinery.
P42 The gliding mechanism is coupled to ATP hydrolysis. Information about direct energy sources is essential for elucidation of biomotility systems. P42 is a 356-amino-acid protein encoded downstream of Gli521. This protein was found to have ATPase activity. The energy for M. mobile gliding is supplied by ATP hydrolysis and that detachment from the solid surface is an energy-dependent process.
Mechanical characteristics of gliding and binding
The movement originates from the ATP hydrolysis at P42, with the motor of this system transmitting through Gli521 and reaching Gli349, thus propelling the cell forward. Mycoplasma, a genus of pathogenic bacteria, forms a membrane protrusion at a cell pole. It binds to solid surfaces with this protrusion and then glides. The mechanism is not related to known bacterial motility systems, such as flagella or pili, or to conventional motor proteins, including myosin.The gliding machinery is composed of four huge proteins at the base of the membrane protrusion and supported by a cytoskeletal architecture from the cell inside. Many flexible legs approximately 50 nm long are sticking out from the machinery. The movements generated by the ATP hydrolysis cell inside are transmitted to the “leg” protein through a “gear” protein, resulting in repeated binding, pull, and release of the sialylgalactose fixed on the surface by the legs. 7
The gliding mechanism likely depends on repeated ATP-dependent binding of a protein to the scaffold fixed on solid surfaces. This mechanism is similar to that of conventional motor proteins such as myosin and kinesin. However, Mycoplasma gliding has the following unique features:
(a) The machinery is half-exposed on the outside of the cell;
(b) the ATPase and binding site are positioned approximately 50 nm apart;
(c) the rail, or fixed sialyl-galactose, has no polarity;
(d) the stall force is as low as 0.24 pN per unit if we assume that one-quarter of all gliding units are engaged at any moment; and
(e) the mechanism probably does not have a pushing step, as analyses of Gli349 have suggested that the leg is flexible.
Schematic of the assembled gliding machinery based on information about individual structures.
The movements may be transferred as indicated by numbered arrows. (i) Movement is generated through ATP hydrolysis by P42. (ii, iii) The movements transmit through the Gli521 molecule. (iv) Gli349 repeats to pull sialylgalactose.
The centipede (power stroke) model for Mycoplasma mobile gliding.
The Mycoplasma cell membrane is indicated by the yellow line. The cell is moving to the right with repeated binding of sialyl-galactose fixed on the solid surface (blue), through stages (a–d ) connected by four steps. The antibodies and mutations that reduce binding and speed may decelerate binding. The antibody and mutation that reduce speed may decelerate pull. The antibody that reduces binding may accelerate release and decelerate binding. The mutation that reduces binding and elevates speed may accelerate release.
Schematic illustration of gliding mechanism
(A) Shown is a working model for the gliding mechanism (based on previous experiments) focusing on the single unit of machinery. The unit consists of an internal structure (upper blue) and three huge proteins: Gli123 (purple), Gli349 (red), and Gli521 (green) on the cell surface. The mechanism is divided into stages (1) through (4). The gliding direction is shown by a yellow arrow. The leg composed of Gli349 catches an SO on the solid surface tightly after thermal fluctuation (1). The leg pulls the SO, resulting in the cell propulsion. The unit force was estimated at around 1.5 pN in this study (2). Continuous displacement of the cell caused by other legs pulls the leg after stroke, resulting in drag generation (3). The leg is detached from the SO by the continuous displacement (4).
(B) About 75 legs (red) sticking out from the cell can work simultaneously. The cell glides in the direction of the yellow arrow. Unbound legs are not illustrated. APO, apoenzyme; Pi, phosphate. 3
The gliding machinery.
Schematic illustration of unit of the gliding machinery. The unit is likely composed of four proteins, Gli42, Gli123, Gli349, and Gli521, which are involved in gliding mechanisms are localized near the cell membrane and aligned at the cell neck forming 450 units per cell. Gli349, the leg protein, sticks out from the C-terminal region; its distal end can bind to sialic acid fixed on the solid surface. The gliding units are supported from inside the cell by the jellyfish-like structure, as presented in the top illustration. The jellyfish structure is composed of latticed solid “head” part and flexible strings attached with particles about 20nm in diameter.
(a) A whole cell which can glide to the right. The gliding machinery appears as a membrane protrusion and is composed of surface and internal structures. The surface structure contains 450 molecules of each of Gli123 (pink), Gli349 (red), and Gli521 (green). The internal jellyfish structure (blue) is composed of a ‘bell’ and ‘tentacles’. A similar structure was output by a 3D printer as shown in Supplementary material A.
(b) Magnified image of a surface unit and part of the internal structure. The molecule of Gli349 (red), that is, the leg protein, resembles an eighth note and is anchored to the cell membrane at the N-terminal transmembrane segment [22–24]. From the N terminus, two short rigid arms, a long flexible part, and a C-terminal oval ‘foot’ are linked tandemly. Two short arms are linked by a foldable hinge. The foot has binding activity to the binding target, SOs [16,22]. Gli521 (green), a ‘crank’ protein, forms a trimer, showing a triskelion reminiscent of eukaryotic clathrin, through association at an end. The molecular shape of the Gli521 monomer consists of three parts connected by flexible hinges, an oval, a rod, and a hook from the N terminus. Gli521 is suggested to bind to Gli349 at the oval on the basis of analyses of mutants [16,34]. The bell of the jellyfish structure (blue), a solid structure, is located at the cell head and is filled with a hexagonal lattice of 12-nm periodicity. Dozens of tentacles are connected to the bell, and these tentacles are covered with particles 20 nm in diameter at intervals of approximately 30 nm. (c) Structure of the foot part of Gli349, modeled by Phyre2 , mainly based on a choline binding protein (PDB, 4cp6), with a sequence coverage of 95% and >90% confidence. The structure is similar to the leucine rich repeat represented by a Toll-like receptor, TLR4 , positioning the essential amino acid, the 2770th serine colored yellow at the site marked by a yellow arrow . The N terminus at the left is connected to the other parts of Gli349. The foot may be removed more easily to the direction of the right arrow than the left one because of the lever action around the supporting point marked by an open triangle.
Updated centipede model for M. mobile gliding.
(a) The leg (Gli349) catches an SO on the solid surface. The tension applied to the leg from the front triggers a conformational change in Gli349 by movements transmitted from the internal motor, causing the pull (stroke), using the energy coupled with the conversion of ATPase forms.
(b) Cell movements occurring as a result of other legs pull the units forward, inducing conformational changes.
(c) Continuous pulling in the forward direction removes the foot from the SO, coupled with the incorporation of new ATP. The foot can be removed
Surface and cytoskeletal structures of Mycoplasma mobile involved in gliding motility.
(a) In the localization of Gli349, the leg protein is visualized by immunofluorescence microscopy. Gli123 and Gli521 are colocalized with Gli349 at the cell neck. The direction of gliding is indicated by a blue arrow.
(b) Negatively stained electron microscopy (EM) image of an intact M. mobile cell. The filamentous structures marked by white arrowheads (inset) may be the resting state of the legs.
(c) Rapid freeze-fracture deep-etch replica EM of M. mobile. The outer leaflet of the lower cell membrane can be seen. A leg-like structure is marked by an arrowhead. The angle of the leg structures in relation to the cell axis varied widely.
(d ) Schematic of the cell surface structure. The gliding machinery is composed of approximately 450 units.
(e) The cytoskeletal structure visualized under negative-staining EM. The cell membrane and cytosol were removed by treatment with 0.1% Triton X-100. ( f ) Schematic of the cytoskeletal structure of a M. mobile cell.
1. Mycoplasmas glide in the direction of a membrane protrusion, with a unique biomotility mechanism.
2. Two groups of Mycoplasma function with apparently different mechanisms, and details regarding the mechanism of the fastest species, M. mobile, are being clarified.
3. The gliding machinery exists at the base of the membrane protrusion, where many flexible legs project outside the cell and are supported by cytoskeletal structures from inside the cell.
4. Four component proteins are directly involved in the mechanism, and at least 10 proteins are involved in the cytoskeleton. Leg and gear proteins have distinct molecular sizes and morphologies.
5. Gliding is assumed to occur by repeated binding, pull, and release on a solid surface.
6. The other group of gliding mycoplasmas, including M. pneumoniae, shares many gliding characteristics with M. mobile, but they do not share similar amino acid sequences with proteins essential for gliding.
6. Cell motility , Peter Lenz, page 159
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