Bacterial Motility - flagella and nanotechnology

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The diagram above shows a model of a close-up view of the basal components of a bacterial flagellum. These are the wheels at the roots of the flagella as mentioned in the introduction to bacteria, if you have not read this introduction then click here to learn the basics of bacterial structure. The diagrams below add some labels to this structure. Note that only a tiny portion of the filament is shown here, since this is the long helix-like propeller that we saw previously. Remember the flagellum rotates like a propeller to propel the bacterial cell along, as shown by the arrows in the diagram below.

Above: the flagellar motor rotates, causing the flagellum to rotate (and the cell to rotate in the opposite sense). Notice the scale bar: the line illustrates a real-life length of 20 nanometres (20 millionths of a millimetre!) so we are dealing with a minute machine - a nanomachine! These minute electric motors evolved by natural means, on Earth, long before human beings existed.

Above: the flagellum emerges from the bacterial surface or wall (or cell envelope)` which consists of three main layers: the outer membrane (OM), the periplasm (P) which contains a mesh of very strong fibrous material called peptidoglycan (or murein) and an inner cytoplasmic membrane (CM). (Click here to learn about cell membranes). Note how different this arrangement is from an animal cell which has a single cell membrane rather than this double membrane structure. Beneath these layers is the cell interior (the intracellular compartment) or protoplast (consisting of cytoplasm and nucleoid) and external to these layers is the extracellular (external) environment, such as the water the bacterium is swimming in. (Additional layers may exist outside the OM, including a slime capsule, but we shall look at these possibilities later). This type of wall structure is particular to some types of bacteria called Gram negative bacteria, but other wall structures occur, as we shall see later. The root of the flagellum consists of a series of rings (made of proteins) that anchor the structure in the cell envelope.

Above: the flagella basal complex with some outer structures (Mot proteins) removed to show the internal structure of the motor. The labels are shown in the figure below:

The basal structure consists of a series of rings connected by a rod, the rings are the L ring (embedded in the lipid bilayer of the outer membrane), the P ring (embedded in the periplasm), the S and the M rings (the rotating motor or rotor) and the C ring. The L and P rings act as a bushing (a bushing is a ring-like structure that constrains moving mechanical parts, in this case the rotating rod, and may also be lubricated to reduce friction). The motor proteins (Mot) conduct electric current carried by positively charged protons ('positive electricity' as opposed to negative electricity in which the current is carried by negatively charged electrons as in a metal wire) from the periplasm into the cell cytoplasm. The electric charge is thought to flow into the M (motor) ring where it is converted into rotary mechanical motion, causing the M-ring to rotate. The M ring is attached to the rod, causing the rod to rotate. The M ring acts against the fixed Mot (stator) ring, which rotates slowly in the opposite direction to the M ring - slowly because it is fixed to the bacterial cell wall and so causes the whole bacterial cell to rotate in a direction opposite to the M ring and rod. The S ring is now known to be part of the rotor, along with the M ring, and forms a socket for the rod, but is not part of the stator. Confusion may arise when the stator ring is referred to as the S-ring.

The rod is attached to the filament via a flexible hook (which acts as a universal joint, transferring rotary motion to the filament via the hook associated proteins (HAPs)). The filament is actually much too long to show more than a tiny segment of it in these diagrams, it is about 20 nanometers in diameter, but 10 - 15 micrometers (10 - 15 thousand nanometers) long, which is longer than a typical bacterial cell which is about 2 micrometers long. The filament is made up of about 30 000 subunits of a protein called flagellin and is a corkscrew or helix shape. (The flagellin is arranged into typically 11 strands that are twined together). This shape is important, mutants with straight filaments are immotile - the filament is the propeller driven by this remarkable microscopic electric motor! (The cell body may contribute to thrust in some forms in which the body is also helical). The helical filament is hollow, and flagellin is transported from inside the cell, through the C ring (which has a hole in its center) and along the filament, in its hollow core, to its tip to which they are added - the filament constantly grows, as it must do to compensate for breakages. A cap protein forms the tip and stabilizes the filament.

Above: illustrating the flow of protons (carrying positive electric charge) across the Mot ring from the periplasm into the cell, powering the motor.

Above a diagram of the bacterial flagellum showing the detailed structure of the flagellum basal complex (in a Gram negative bacterium). The units of protein FliG (about 25-45 units) form a ring extension to the M-ring (the M ring is shown in section here) or an extension on top of the C-ring according to the model. Units of the proteins FliM (about 35 units) and FliN (about 110 units) form the 45 nm diameter C ring, which together with the M and S rings forms the rotor. This rotor drives the rod, which is a rotor-shaft connected through the centre of the L and P rings to the hook.

The proteins MotA and MotB form a ring of 10 studs embedded in the cytoplasmic membrane, forming the stator, which is tethered by connections to the rigid Peptidoglycan layer (PG). The L and P rings act as bushing.The C ring is made up of the proteins FliM and FliN (and perhaps also FliG) and the Mot proteins consist of two subunits: Mot A and MotB. Studies suggest that 4 MotA subunits complex with 2 MotB subunits and that mostly MotA, but also part of MotB contributes to the formation of a proton channel.

The hook associated proteins HAP3 and HAP1 are also known as FlgL and FlgK respectively. The rod is made up of a variety of proteins and the protein FliF spans the region between the M and S rings. It is thought that as protons (H+ or hydrogen nuclei) move through the Mot ring complex, MotA undergoes a shape change, exerting a torque (rotary force) on FliG which connects to the M ring.

The basal complex anchors the flagellum into the surface of the cell. There are two structural variations according to the type of bacterial wall it is anchored in. Gram positive bacteria (stain purple with Gram's stain) like Bacillus possess a thick peptidoglycan wall (about 80 nm) overlying the bilipid cytoplasmic membrane (CM). In this case the basal body has three rings, the 26 nm diameter M (membrane) ring embedded in the CM, the S (supramembrane) ring or socket attached to the inner surface of the peptidoglycan wall by techoic acids and the C (cytoplasmic) ring. The S ring is an extension of the M-ring, to which it is attached, and both are composed of the same ring of protein FliF, thus the M and S rings are sometimes considered to be a single double-ring, the MS ring. A rod (7nm diameter) passes into the S ring socket and its other (distal) end attaches to the hook. More details of these structures are illustrated below:

Redrawn from DeRosier, 1998.

These electron density maps are typically constructed using electron cryomicroscopy. Samples are rapidly frozen, for example in liquid nitrogen, and sectioned whilst frozen, avoiding many of the fixation artefacts associated with chemical fixation. Many sections are averaged together to produce the final image.

Note that it is difficult to map proteins from their predicted or measured shapes to electron density maps, such as those generated by electron cryomicroscopy. Some newer models have FliG as the top of the C-ring, the U-shaped structure at top in yellow in the fig. above, rather than as part of the M ring, whilst other models have it spanning the C and M rings. Whatever its exact position its interactions with Mot are thought to drive the rotor, including both the C and MS rings, which must, therefore, be connected in some way. The flagellin subunits have at least two chief domains: D1 lines the inner hollow core of the filament and D3 lines the outside. The hollow core allows the passage of flagellin subunits to the tip of the flagellum when it is being constructed or repaired.

There are some 25 or 26 FliG molecules in the flagellum base and about 11 Mot complexes, each containing 4 MotA and 2 MotB. It should also be realized that different bacterial species have different flagellum base architectures. An updated and more detailed model is shown below:

The table below summarizes the various substructures shown in the figures above and their encoding genes.

Note that the M, S and C rings form the rotor and hence spin on their axes like wheels. The rod is the drive shaft and hence also rotates, driving rotation of the flagellum. The hook acts as a universal joint transferring torque from the rotor to the filament, which may lie along a different axis. P and L rings act as bushings and do not rotate.

Function of the Hook

The hook functions as a universal joint on the molecular scale. A universal joint, as used in engineering, transfers 'rotational force' (strictly torque, in units of Nm, rather than force which is in N) from one shaft to another held at and angle to the first. The hook is rigid in rotation but flexible in bending and allows the flagellum to bend at the hook without affecting the direction of rotation. When we look at chemotaxis/chemokinesis then we shall see that teh flagellum needs to point in different directions to achieve useful movement.

Detailed Mechanism of Torque Generation - A Model

Quite a number of ingenious models have been proposed over the years, attempting to explain how the bacterial flagellum rotor generates torque. The most recent model can be summarized thus:

1. Protons flow (down their electrochemical gradients) through the MotA/B proton channels.
   
2. This induces a conformational change in MotA which extends charged amino acid residues towards charged residues on FliG (presumably without the protons the charged residues on MotA are internalized and screened).
   
3. The electrostatic force (Coulomb force) of repulsion pushes the MS and C rings to which the FliG is attached.
   

Certainly, electrostatic repulsion can easily generate sufficient force, as elementary calculations show.

Under typical conditions the filament (at least in Escherichia coli) is a left-handed super-helix (the flagellin protein subunits that make up the filament are arranged in a helix which is itself coiled into a larger helix). Counterclockwise rotation of this helix exerts force on the cell body (due to fluid viscosity resisting the moving filament) causing it to rotate as it is pushed along. In peritrichously flagellated bacteria, hydrodynamic forces draw the flagella into a bundle when they rotate counterclockwise. Clockwise rotation of the filaments causes them to fly apart in the bundles of peritrichous enteric bacteria or pulls the cell in reverse in polarly flagellated bacteria.

Performance of the bacterial flagella motor

Not all bacteria are motile and of those that are more than half use one or more helical flagella, like the one we have described above. Compared to other bacterial propulsion systems (which we shall look at later) the flagella propellers have the advantage of speed. For example, a cell of the bacterium Escherichia coli is about 2 micrometers (2 thousandths of a millimeter) long and has six flagella that originate from various points on the cell surface but the filaments come together to form a bundle which propels the cell at about 20 micrometers per second, or ten body lengths per second! This is a clear advantage that more than pays back the high cost of flagella - each flagellum contains about 1% of the bacterium's total protein and requires some 50 genes for its production (about 2 % of the genome of about 2 500 genes). The flagella enable swarmer cells to disperse from the biofilm (colony) and locate new sites for colonization. They enable the bacteria to swim to a source of nutrients and to avoid harmful irritants. The drawback of flagella is that they require a fluid medium in which to work most effectively, although bacteria can use them to move across moist surfaces. Flagella also fail to work well in highly viscous media, such as the muddy ooze at the bottom of ponds, and bacteria may employ different propulsion systems in these environments.

The helical filament rotates in a rigid manner as determined by attaching latex beads along the filaments of mutants in which the filaments are straight. This ruled out the possibility that they undulate like whips (as do the flagella of many non-bacterial cells) or that they move by winding and unwinding. More recent experiments using laser dark-field microscopy have enabled individual flagella to be directly observed rotating. They can flex, however, by virtue of the flexible hook. When the six flagella of Escherichia coli rotate counterclockwise (CCW) forces exerted on them by the surrounding water (hydrodynamic forces) force them to come together into a single bundle, trailing behind the tail end of the cell.

The flagella of Vibrio alginolyticus can rotate at about 1000 revolutions per second (rps) and propel the cells at up to 116 micrometers per second (compare to the flagellum of Escherichia coli with 270 rps and a top speed of 36 micrometers per second). However, removal of the filament (which loads the motor) may increase the engine rotation rate to 200 000 rps!

Flagella Bundles

Although many bacteria have a single flagellum at one end or side of the cell, or perhaps one at each end, so-called polar flagellation,  many also have multiple flagella that entwine together to form a rotating flagellum bundle. This is the type seen in Escherichia coli and Salmonella typhimurium. These bacteria have peritrichous falgellation - they have several or many flagella dispersed over the cell's surface. Obviously if flagellum rotated independently then the cell would go nowhere fast! Instead the many flagella bundle together at the rear of the cell when the cell needs to 'run' in a straight line. In order to turn around the flagella change the sense of their rotation, rotating in the opposite direction, and this mysteriously causes the flagella bundle to fly apart and the cell tumbles, randomly changing direction, before the flagella switch rotation direction again and come together as a bundle and the cell moves off in a new direction. A flagella bundle creates the opportunity for more locomotive force, since more motors are working together to propel the cell and are perhaps also useful for moving through more viscous fluids.

Above: the flagella of a peritrichously flagellated bacterium, such as Escherichia coli or Salmonella,  forming a bundle at the back of the cell for propulsion forwards (to the left) in a straight-line. Note the filaments have a constant amplitude and wavelength (pitch of the helix).

Above: one or more flagella leave the bundle, causing the cell to rotate to a new random bearing.

Such a bacterium will undergo a series of runs and tumbles as it finds its way around.

What causes flagella to bundle or to fly apart - a hydrodynamic model?

It is said that 'hydrodynamic forces' cause the flagella to bundle, for example during CCW (counter-
clockwise) rotation in the left-handed flagella of Escherichia coli the flagella bundle together. Recall that a helix or screw can be either left-handed or right-handed. If you look down the axis of a left-handed helix from behind and then rotate it CCW then it will move forwards as it rotates. A right-handed helix, on the other hand, will move forwards when rotating CW (clockwise) when viewed in the same manner.

We propose a model using elementary fluid mechanics. Perhaps we can picture what could be happening with a simple diagram. The diagram below is the view we would have looking from behind a bacterium with 6 flagella, like Escherichia coli, as it swims forward straight away from us (into the page). The six left- handed (LH) flagella rotate CCW. Since these are LH helices, the flagella will drive forward into the page (X marks the tail-end of their velocity vectors). Remember that we have a very low Reynolds number (Re) for such a small organism and so the water medium is behaving as a very viscous (thick and sticky) fluid.

Vortices generated by the 6 rotating flagella of Escherichia coli as viewed from behind the cell looking forward along its direction of locomotion.

Inside the 'circle' of flagella, driven by their CCW rotation, a CW vortex would be established. At this low
Re we would expect no turbulence, so these are orderly vortices and the central CW vortex will spiral outward toward us (the central dot indicates the head of its velocity vector coming out of the page toward us). This core of fluid will be displaced, essentially drilled out of the viscous medium and the 6 flagella will close together to fill the void it leaves behind it (fluid flow from outside the flagella, passing in-between them to fill this void would not readily occur, since the flows of neighboring vortices tend to cancel midway between them) and the flagella will bundle together. Fluid will continue to spiral CW around the bundle as it rotates CCW, propelling the cell.

We have to ask the question: do the flagella or the cell body generate most of the propulsive thrust? For a spherical or rod-shaped bacteria powered by a single CCW rotating flagellum the cell body will rotate CW. This rotation of the cell body will not, by itself, generate thrust since, as already discussed, in order to generate thrust in a high viscous fluid it is necessary to have time-non-reversible flow, which requires a helix of definite handedness. Perhaps the spiral S-layer of protein that encases many bacteria (forming an outer layer around the cell envelope) helps break the symmetry and generate thrust. In coma (vibrioid) and spiral forms, the cell body will indeed displace fluid in a non-reversible manner and so contribute to the thrust, effectively drilling its way through the medium.

So, what happens when the flagella rotate CW?

If the bundle remained intact then it would begin to pull the cell backwards by displacing a helix of fluid toward the cell body. However, the external fluid will flow towards the end of the flagellum bundle and perhaps this drives the bundle apart by forcing its way between the flagella. If this was so, might the cell be observed to reverse momentarily before tumbling? This raises the question as to how many flagella must rotate CW for the bundle to separate. If one flagellum only reverses direction, then its flow would couple with the neighboring flagellar vortices.

Consider two neighboring vortices, one rotating CW and one rotating CCW, as shown below:

A pair of oppositely rotating vortices couple together and move downwards in this case.

This vortex pair will drive fluid in one direction between them and around them – the two vortices are coupled together. Viscosity will cause the vortex pair to move in the direction of this flow. Some of the pairs will try to move toward the center (which they can not do since other flagella in the bundle will block their path) and other pairs will tend to move away from the bundle: the bundle will disperse.

In addition to changing rotation sense, the flagella also change pitch and this may also contribute to the decoupling of the flagella and dispersal of the bundle. Indeed, in Escherichia coli, a CW rotating filament switches from the left-handed (LH) helix of the CCW rotating state to a right-handed (RH) helix.

The Versatile Flagella Filament

The flagella filament is more than a rigid rotor, it is a smarter device. It is made up of 11 helical protofilaments of flagellin protein monomers. These protofilaments can switch between a RH helix (R-state) and a LH-helix (L-state). The R-state is slightly shorter. To form the LH-helical filament required in the normal running state, some of the protofilaments (those on the outside of the helical curve) must be longer and hence in the L-state with those on the shorter side of the helical curve in the R-state. Thus the normal helical filament is in a supercoiled states: it is a helix of helices.

This ability to switch from one shape (conformation) to another, a conformational change, when a small amount of energy is supplied is a characteristic feature of proteins which thus have several stable states, depending on energy. This is one of the key features of proteins that living systems exploit.

If all the protofilaments were in the same state, either all R or all L, then there would be no supercoiling and the filaments would be straight. The flagella filament does have other helical modes that may be used in locomotion. If more of the protofilaments switch to the shorter R-state then the angle of the helix increases, it becomes more tightly coiled with a shorter length and a smaller pitch (wavelength) and a larger helical diameter (amplitude). There are one or more curly states in which the diameter and pitch decrease but in which the length remains the same or increases.

Swarmer Cells

Most bacteria alternate between a single-celled stage and some form of multicellular stage during their life-cycle. Although bacteria rarely form true multicellular organisms, in which cells are in intimate contact and communicating directly with their neighbors through cell-to-cell junctions, most do form multicellular aggregates in which cells are separately embedded in a common slime layer, which is attached to a solid surface and forming a biofilm. Cells in the biofilm are often immotile and certainly lose their ability to swim as they shed their flagella. Swarmer cells, however, are a dispersive stage and these may float, swim, crawl or glide away from the biofilm before eventually settling down, adhering to a surface and establishing a new microcolony which may eventually develop into a biofilm. Sometimes the flagella assist in adhesion by sticking to a suitable surface they come into contact with. However, soon after adhering the swarmer cells lose their flagella and establish a microcolony. Bacteria in both the swarmer cell stage and the biofilm stage maintain a degree of individuality and both are capable of dividing and multiplying.

Biofilms also disperse by shedding slimy streamers, containing cells, downstream. Generally, dispersal is more efficient if the biofilm sits upstream of fluid flow. How do the swarmer cells, which are often released into the stream some 400 or so micrometers above the surface swim in these currents? Research has shown that Escherichia coli, at least, has a neat trick. In those strains under study it was found that swimming Escherichia coli (swarmer cells) preferentially circle to the left until they find a sheltered crevice in a surface, where flow is less, and they swim upstream to establish a new microcolony and biofilm!

Swarming and crawling

The normal swimming flagellated cells that we have considered so far are described as swarmer cells, or planktonic cells, distinguishing them from the non-flagellated cells that are integral residents in biofilms. However, 'swarming' is usually used for yet another, though closely related, phenomenon: the mass migration of colonies of bacteria across a solid surface. This swarming behaviour is regulated by quorum sensing and in Escherichia coli and Salmonella typhimurium involves a switch to a multinucleoid elongate filamentous hyperflagellated phenotype. These multinucleoid cells, called filaments, are up to 50 micrometres long and have several nucleoids, dispersed at intervals throughout the filament (nucleoid division and cell wall growth have continued in the absence of cell division). They move as a colony with the outer layers spiraling outward with the evacuated space inside the colony becoming filled with new cells. This can give rise to fast colony expansion at rates up to ~3 mm/s (1 cm/h). Spiral and 2D-branching patterns of colonial growth result and probably acts to optimize nutrient uptake on a solid substrate in which diffusion is limited (in a solid sheet that completely covers the surface, many cells may become starved of nutrients due to competition with their neighbours: similar considerations have shown to predict the branching growth of sponges in 3D in computer models utilizing the diffusion equation).

The hyperflagellated filaments use their many flagella to crawl over solid surfaces, such as agar in a petri dish. The flagella still bundle, but the cells do not tumble, instead when the flagella disperse the filament simply stops and the flagella may reform a bundle of the opposite sense resulting in a reversal of direction of movement. Synthesis of so many flagella appears to be triggered when the bacteria sense the presence of a highly viscous medium (such as the surface of an agar plate).

Update on Swarming

Research shows that bacteria only swarm when moving in groups and evidence suggests that they interlock their flagella to move as rafts. Cells may detach and reattach to the raft, but detached cells do not swarm but remain immotile instead. They can also secrete surfactant to reduce surface tension and facilitate gliding of the rafts. Research also suggests that both the flagella and pili can function as mechanoreceptors, sensing contact with a surface to trigger microcolony formation.

Above: a hyperflagfellated bacterial filament (this one is 4 times the normal length at about 8 micrometers and has 20 flagella). When fully developed this cell may have 100 flagella and measure about 50 micrometers in length!

Other Swimming Modes

Bacteria display a variety of locomotive methods, some not very well understood physically, including several different modes of swimming. Bacteria that have a single polar flagellum clearly can not employ the run-tumble-run method of peritrichous bacteria. One such example is Bdellovibrio and also Vibrio, Pseudomonas, Aeromonas and Shewanella:

Bdellovibrio, which predates other bacteria, is monotrichously flagellated: it has a single flagellum at one pole, the rear pole. Notice that the flagella helix is damped: it decreases in amplitude from the base to the tip. The flagellum appears thick because it is a sheathed flagellum in Bdellovibrio and Vibrio, meaning that it is covered in an extension of the outer cell membrane. Inside the sheath, the filament actually transitions from a larger diameter to a smaller diameter about half-way along its length and consists of 5 different flagellin types arrange din an ordered series from base to tip. The sheath possibly serves to increase thrust in higher viscosity media, which vibrioid bacteria are adapted for, but also protects the flagellin from host antibodies and other defences that recognize bacterial flagellins. The sheath can also be involved in blebbing-off outer membrane vesicles (OMVs) which carry cargo to other cells, including signaling molecules.

Shewanella putefaciens is a well-studied example of this flagellation type. CCW rotation of the filament results in forwards movement (flagellum pushing) whilst CW rotation causes reverse locomotion. For this to work the flagellum must maintain the same helicity rather than switching handedness when the rotation sense changes. Indeed, thinking of a corkscrew it is possible to see that the filament in Shewanella must be a LH helix in both CCW and CW rotation (recall that we are viewing the flagellum rotation sense by looking from the rear towards the front). However, during the CW to CCW transition, the filament flattens along its axis causing it to buckle in the hook region. This buckling causes a flicking motion that changes the orientation of the bacterium, causing it to turn.

More articles on bacterial motility:

Models of flagella rotation - how does the rotor work? Click here to find out!

Learn about motility in the strange spirochaetes which drill through materials (including people!).

Learn about gliding and other alternative mechanisms of locomotion in bacteria.

Download an illustrated essay on bacterial motility and navigation in pdf format: Prokaryotes_motility.

How can something as complex as a bacterial flagellum evolve?

Back to bacteria intro...

Page updated: 1/9/2013. Details about flagella bundles, swarming, swarmer cells and protein structure added. Errata concerning the nature of the stator and end-cap proteins applied.

Page updated: 24/8/2018. Recent discoveries on the mechanism of flagella rotation and the mechanism of swarming added.

Page updated: 26/6/2023.