A variety of experiments showed that the flagellum is a rigid propellor that is rotated by a “motor” at the base. For example, a bacterium, artificially linked by means of antibodies to a short stub of a flagellum of another bacterium, can be rotated by the second bacterium. Rotation of cells tethered to a cover slip has also been observed. Although it is impossible to see individual flagella on live bacteria directly, bundles of flagella and even single filaments (Fig. 19-1C) can be viewed by dark-field light microscopy.8,29 Normal
flagella appear to have a left-handed helical form, but curly Salmonella flagella, which have a superhelix of one-half the normal pitch, form a right-handed helix.5
Normal bacteria swim in straight lines but periodically “tumble” before swimming in a new random direction. This behavior is part of the system of chemotaxis by which the organism moves toward a food supply.30
Curly mutants tumble continuously. When bacteria tumble the flagella change from normal to curly. The pitch is reversed and shortened. A proposed mecha- nism for the change of pitch involves propagation of cooperative conformational changes down additional
rows of flagellin subunits.31
There are no muscle-type proteins in the flagella. By incubating flagellated bacteria with penicillin and then lysing them osmotically, Eisenbach and Adler obtained cell envelopes whose flagella would rotate in a counterclockwise fashion if a suitable artificial electron donor was added.32 This and other evidence
showed that ATP is not needed. Rather, the torque developed is proportional to the protonmotive force and, under some circumstances, to ∆ pH alone. It is the flow of protons from the external medium into the cytoplasm that drives the flagella.8 Movement of E. coli cells in a capillary tube can also be powered by
an external voltage.33 In alkalophilic strains of Bacillus
and some Vibrio species a sodium ion gradient will substitute.13 Several hundred protons or Na+ ions
must pass through the motor per revolution.8 Some
estimates, based on energy balance,29 are over 1000.
However, Na+-dependent rotation at velocities of up
to 1700 Hz has been reported for the polar flagellum of Vibrio alginolyticus. It is difficult to understand how the bacterium could support the flow of 1000 Na+ per
revolution to drive the flagellum.2
Flh A Fli H,I Peptidoglycan Inner membrane Outer membrane Mot B Mot A Flg E (Hook) Fli F (MS-ring) Fli G Flg H (L-ring) Flg I (P-ring) Periplasm
C-Ring: FliM, FliN Rotor Flg B, C, F, G, J Rod Flg L (HAP3) Flg K (HAP1) Fli C (filament, flagellin) Fli D (HAP2, cap)
Figure 19-3 Schematic drawing of bacterial flagellar motor. Based on drawings
of Berg,27 Zhou and Blair,28 and Elston and Oster.1
What kind of protic motor can be imagined for bacterial flagella? Electron microscopy reveals that the flagellar hook is attached to a rod that passes through the cell wall and is, in turn, attached to a thin disc, the M-ring (or MS-ring), which is embedded in the cyto- plasmic membrane both for gram- positive and gram-negative bacteria (Fig. 19-3). Two additional rings are present above the M-ring of flagella from gram-negative bacteria. The P-ring interacts with the pepti- doglycan layer, and the L-ring con- tacts the outer membrane (lipopoly- saccharide; Fig. 19-3). A logical possibility is that the M-ring, which lies within the plasma membrane, is the rotor, and a ring of surrounding protein subunits is the stator for the motor (Fig. 19-4).34,35 Glagolev
and Skulachev suggested in 1978 that attraction between – COO –
and – NH3+ groups provides the
force for movement.34 Protons
passing down an H+-conducting
pathway from the outer surface could convert –NH2 groups to –NH3+, which would then be
attracted to the – COO – groups on
the stator subunits. When these two oppositely charged groups meet, a proton could be transferred from – NH3+ to – COO – destroying the electrostatic
attraction. At the same time, movement of the M-ring would bring the next – NH2 group to the H+-conducting
pathway from the outside. The – COOH of the stator would now lose its proton through a conducting pathway to the inside of the bacterium, the proximity of the new – NH3+ assisting in this proton release.
Since that time, other models based on electrostatic interactions have been advanced.1,29,36
Approximately 40 genes are required for assembly of the flagella, but mutations in only five motility genes have produced bacteria with intact flagella that do not rotate. Among these genes are motA, mot B, FliG, FliM, and FliN.16,29,37,37a Infection with a lambda transducing
bacteriophage carrying functional motB genes restores motility to motB mutants by inducing synthesis of the motB protein. Block and Berg observed rotation of single bacteria tethered to a coverslip by their flagella. As the synthesis of the motB protein increased,the fla- gellar rotation rate increased in as many as 16 steps. This suggested that as many as 16 subunits of the motB gene product may contribute to the operation of the motor.38
Both the M-ring and the thin S-ring, which lies directly above it and is now usually referred to as the MS-ring, are formed from ~20 – 25 subunits of the 61- kDa FliF protein.39 Both the MotA and
MotB proteins are embedded in the inner bacterial membrane and appear to form a circular array of “studs” around the M-ring.16 MotA has a large cytosolic
domain as well as four predicted trans- membrane helices40 while MotB has a
large periplasmic domain and probably binds to the peptidoglycan.37,41,41a The
MotA and MotB proteins, which bind to each other, are thought to form the ~ 8 functional units in the stator of the motor.37 Proteins FliG, FliM, and FliN
are evidently parts of the rotor assembly. FliM and FliN form an additional ring, the cytoplasmic or C-ring, which had been difficult to see in early electron microscopy. As many as 40 of each of these subunits may be present in the ring.42,43 A ring of FliG subunits joins the
C-ring to the MS-ring (Fig. 19-3). FliE is also a part of the basal body.25a
From study of mutants it has been concluded that three charged residues of FliG, R279, D286, and D287 are directly involved in generation of torque by the motor.44 Side chains of these residues
may interact with the cytoplasmic domains of MotA and MotB. Residues R90 and E98 of MotA may be involved in
controlling proton flow through the motor units.28,44
The two prolines P173 and P122 are also essential for torque generation.28
There are obvious similarities between the flagellar motors and the protic turbines of ATP synthases (Fig. 18-14), but there are also substantial differences. It apparently takes about 12 protons for one revolution of the ATP synthase but about 1000, or ~ 125 per motor unit, for rotation of a bacterial flagellum. Elston and Oster propose an ion turbine more complex than that of ATP synthase. They suggest that the rotor might contain about 60 slanted rows of positively charged groups spaced as shown in Fig. 19-4. The motor is reversible, i.e., it can rotate in either direction. One possibility is that the subunits alter their conformations cooperatively in such a way that the slant of the rows of charged groups is reversed. Other possibilities for altering the constellation of charges via conformational changes can be imagined.1 See also Thomas et al.44a
Membrane Mot A Fli G C-Ring (FliM, FliN) H+ Rotor
Surface of circumference of C-ring H+ Stator (one of ~8) Outer membrane Periplasmic space Cytosol 3. Chemotaxis
The flagellar motor is reversible, and in response to some signal from the bacterium it will turn in the opposite direction. At the same time, the flagellin sub- units and those of the hook undergo conformational changes that change the superhelical twist. Perhaps synchronous conformational changes in the M-ring also are associated with the change in direction of rotation and are induced by interaction with a switch complex that lies below the M-ring. This consists of proteins FliG, FliM, and FliN.44b Mutations in any
one of these proteins lead to the following four pheno- types: absence of flagella, paralyzed flagella, or flagella with the switch biased toward clockwise or toward counterclockwise rotation.45
What signals a change in direction of rotation? The answer lies in the attraction of bacteria to com- pounds that they can metabolize. Bacteria will swim toward such compounds but away from repellent substances, a response known as chemotaxis. Cells of E. coli swim toward higher concentrations of L-serine
(but not of D-serine), of L-aspartate, or of D-ribose.
Figure 19-4 Schematic drawing of a hypothetical configuration of rotor
and one stator unit in a flagellar motor as proposed by Elston and Oster.1
The rotor can hold up to 60 positive charges provided by protons flowing from the periplasm through the stator motor units that surround the C-ring and hopping from one site to the next along the slanted lines. The rotor is composed of 15 repeating units, each able to accommodate four protons. Negative charges on the stator units are 0.5 nm from the rotor charges at their closest approach. For details see the original paper.
Phenol and Ni2+ ions are repellent.46– 48 By what
mechanism can a minuscule prokaryotic cell sense a concentration gradient? It is known that the plasma membrane contains receptor proteins, whose response is linked to control of the flagella. Since the dimen- sions of a bacterium are so small, it would probably be impossible for them to sense the difference in concentration between one end and the other end of the cell. The chemotatic response apparently results from the fact that a bacterium swims for a relatively long time without tumbling when it senses that the concentration of the attractant is increasing with time. When it swims in the opposite direction and the concentration of attractant decreases, it tumbles sooner.49
Koshland47 proposed that as the membrane
receptors become increasingly occupied with the attractant molecule, the rate of formation vf of some compound X, within the membrane or within the bacterium, is increased (Eq. 19-1). When [X] rises higher than a threshold level, tumbling is induced. At the same time, X is destroyed at a velocity of vd.
Subsequently, a readjustment of vf and vd occurs such that the concentration of X falls to its normal steady state level. X would act directly on the flagellar motor.
The receptors for L-serine50–51a and L-aspartate52,53
are 60-kDa proteins encoded by genes tsr and tar in
Salmonella or E. coli.46,54 These proteins span the inner
plasma membrane of the bacteria as shown in Figs. 11-8 and 19-5. The functioning of the receptor has been discussed in some detail in Chapter 11. However, there is still much that is not understood. The sym- metric head, whose structure is known (Fig. 11-8), has two binding sites, but the aspartate receptor binds only one aspartate tightly. There is substantial evidence that suggests a piston-type sliding of one helix toward the cytoplasm as part of the signaling mechanism.54a
While the flagella are distributed around the cell, the receptors appear to be clustered at the cell poles.55
Proteins encoded by genes cheA, cheW, cheY and
cheZ, cheB, and ChR are all involved in controlling
chemotaxis.48,56 Their functions are indicated in the
scheme of Fig. 19-5. All of the corresponding protein products have been isolated and purified, and the whole chemotaxis system has been reconstituted in phospholipid vesicles.57 Gene CheA encodes a 73-kDa
protein kinase, which binds as a dimer to the cyto- plasmic domains of the related aspartate, serine, and ribose/galactose receptors with the aid of a coupling protein, cheW (Fig. 19-5). A great deal of effort has been expended in trying to understand how binding of an attractant molecule to the periplasmic domain of the receptor can affect the activity of the CheA kinase, but the explanation is unclear. There is a consensus
vf vd
[X] (19-1)
that a small but distinct conformational alteration is transmitted through the receptor.58– 61a An apparently
α-helical region containing methylation sites (Fig. 19-5) appears to be critically involved in the signaling, responding not only to occupancy of the receptor site but also to intracellular pH and temperature and to methylation. Mutation of the buried Gly 278 found in this region to branched hydrophobic amino acids, such as Val or leucine, locks the receptor in state with a superactivated CheA kinase, while substitution of Gly 278 with aspartate leaves the kinase inactive.61
Occupancy of the normal receptor site with ligand (aspartate, serine, etc.,) dramatically decreases the kinase activity.
The CheA protein is an autokinase which, upon activation by the receptor, becomes phosphorylated on Nεof the imidazole ring of His 48. It then transfers this phospho group from His 48 to the carboxylate of Asp 57 of the 654-residue protein CheY, which is known as the response regulator.62– 65d The unregulated
flagellum rotates counterclockwise (CCW). Phospho-
CheY (CheY-P, which qualifies as X in Eq. 19-1) carries the message to the flagellar motor to turn clockwise (CW).
This is apparently accomplished through the binding of CheY-P to the N-terminal portion of protein FliM. This presumably induces a conformational change, which is propagated to FliG and to all of the proteins of the rotor and flagellar rod, hook, and filament.45,65,66,66a
The flagella fly apart, and the bacterium tumbles and heads randomly in a new direction.
Tumbling occurs most often when receptors are unoccupied, and the bacteria change directions often, as if lost. However, if a receptor is occupied by an attractant, the activity of CheY is decreased and less CheY-P will be made. The carboxyl phosphate linkage in this compound is labile and readily hydrolyzed, a process hastened by the phosphatase CheZ.67– 69
Consequently, in the presence of a high enough attractant concentration the tumbling frequency is decreased, CCW flagellar rotation occurs, and the bacterium swims smoothly for a relatively long time.
There are still other important factors. Occupancy of the receptor by a ligand makes the receptor protein itself a substrate for the chemotaxis-specific methyl- transferase encoded by the cheR gene.62,70,71 This
enzyme transfers methyl groups from S-adenosyl- methionine to specific glutamate side chains of the receptor to form methyl esters. In the aspartate receptor there are four such glutamate residues in a large cytoplasmic domain that includes the C terminus. Two of these glutamates are initially glutamines and can undergo methylation only if they are deaminated first.72 An esterase encoded by the cheB gene72 removes
the methyl ester groupings as methanol. The action of the CheR methyltransferase is apparently unregulated, but the esterase activity of CheB is controlled by the phosphorylation state of the
Aspartate bound to receptor CheY CheY, response regulator Pi CheA~P CheY~P H2O CheZ S-Adenosyl- methionine (AdoMet) CheR S-Adenosyl- homocysteine CH3OH H2O R C O O – (Receptor) R C OCH3 O Induction of CW rotation and tumbling FliM Methylation sites W CheA Coupling protein Histidine autokinase CheW Inner membrane CheB H2O CheB-P CheA Signalling path
Figure 19-5 Schematic represen-
tation of an important chemotactic system of E. coli, S. typhimurium, and other bacteria. The trans- membrane receptor activates the autokinase CheA, which transfers its phospho group to proteins CheY and CheB to form CheY-P and CheB-P. CheY-P regulates the direction of rotation of the flagella, which are distributed over the bacterial surface. CheR is a methyltransferase which methylates glutamate carboxyl groups in the receptor and modu- lates the CheA activity. CheZ is a phosphatase and CheB-P a methylesterase.
autokinase CheA. CheB competes with CheY (Fig. 19-5), and CheB-P is the active form of the esterase. After a chemotactic stimulus the level of CheA-P falls and so does the activity of the methylesterase. The number of methyl groups per receptor rises making the CheA kinase more active and opposing the de- crease in kinase activity caused by receptor occupancy. The system is now less sensitive to the attractant; the bacterium has adapted to a higher attractant concentra- tion.62,73,73a It tumbles more often unless the attractant
concentration rises; if it is headed toward food tum- bling is still inhibited. If it is headed away from the attractant the levels of both CheY-P and ChB-P rise. A high level of fumarate within the cell also acts on the
switch–motor complex and favors CW rotation.74
For some bacterial attractants such as D-galactose, D-ribose, maltose, and dipeptides75 the corresponding
binding proteins,38,76 which are required for the sugar
uptake (e.g., Fig. 4-18A), are also necessary for chemo- taxis. The occupied binding proteins apparently react with membrane-bound receptors to trigger the chemo- tactic response. The aspartate receptor (tar gene product) appears also to be the receptor for the maltose-binding protein complex,47 and both the aspartate and the serine
receptor (tsr gene product) also mediate thermotaxis and pH taxis.77,77a Clusters of identical receptors may
function cooperatively to provide high sensitivity and dynamic range.77b
B. Muscle
There is probably no biological phenomenon that has excited more interest among biochemists than the movement caused by the contractile fibers of muscles. Unlike the motion of bacterial flagella, the movement of muscle is directly dependent on the hydrolysis of ATP as its source of energy. Several types of muscle exist within our bodies. Striated (striped) skeletal