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some of the mystery of movement by looking at them. When a cell of E. coli or Salmonella swims smoothly, each flagellum forms a left-handed superhelix with an ~ 2.3 µm pitch. Rotation of these “propellors” at rates of 100 – 200 revolutions / s (100 – 200 Hz) or more1,2 _{in a}

counterclockwise direction, as viewed from the distal end of the flagellum, drives the bacterium forward in a straight line.3– 8 _{Several flagella rotate side-by-side as}

a bundle.4 _{The observed velocities of 20 – 60 µm / s are}

remarkably high in comparison with the dimensions of the bacteria. Also remarkable is the fact that a cell may travel straight for a few seconds, but then tumble aimlessly for about 0.1 s before again moving in a straight line in a different direction. The tumbling occurs when the flagellum reverses its direction of rotation and also changes from a left-handed to a right-handed superhelix, which has just half the previous pitch.

Such behavior raised many questions. What causes reversal of direction of the propellor? Why do the bacteria tumble? How does a bacterium “decide” when to tumble? How is the flagellum changed from a left-handed to a right-handed superhelix? How does this behavior help the bacterium to find food? Most intriguing of all, what kind of motor powers the

flagella? The answers are complex, more than 50 genes being needed to specify the proteins required for assembly and operation of the motility system of

E. coli or Salmonella typhimurium.9

1. The Structure and Properties of Bacterial Flagella

Twenty or more structural proteins are present from the base to the tip of a complete bacterial flagel- lum. However, over most of their length the long thin shafts (Figs. 1-1, 19-1) are composed of subunits of single proteins called flagellins. Flagellin molecules have a high content of hydrophobic amino acids and, in Salmonella, contain one residue of the unusual

Nε-methyllysine. The subunits are arranged in a helix of outside diameter ~ 20 nm in which they also form 11 nearly longitudinal rows or protofilaments.10– 12a _Each

subunit gives rise to one of the projections seen in the stereoscopic view in Fig. 19-1B. The flagella usually appear under the electron microscope to be super- coiled (Fig 19-1C–E) with a long “wavelength” (pitch) of ~ 2.5 µm. The supercoiled structure is essential for function, and mutant bacteria with straight flagella are nonmotile. Under some conditions and with some mutant flagellins, straight flagella, of the type shown in Fig. 19-1B, are formed. There is a central hole which is surrounded by what appears to be inner and outer tubes with interconnecting “spokes.” However, all of the 494-residue flagellin subunits presumably have identical conformations, and each subunit contributes to both inner and outer tubes as well as to the outer projections. Basal bodies (Fig. 19-2) anchor the flagella to the cell wall and plasma membrane and contain the protic motors (Fig. 19-3) that drive the flagella.14–16

Figure 19-1 (A) Axial view of a 5-nm thick cross-section of the flagellar filament shown in (B). The 11 subunits form two

turns of the one-start helix. (B) Stereoscopic oblique view of a 30-nm long section of a flagellum of Salmonella typhimurium. This is a straight flagellum from a nonmotile strain of bacteria. The structure was determined to a resolution of 0.9 nm by

electron cryomicroscopy. From Mimori et al.11 _{Courtesy of Keiichi Namba. (C) Electron micrograph of a cell of S. typhimurium}

showing peritrichous (all-around) distribution of flagella. Courtesy of S. Aizawa.3 _{(D) Dark-field light micrograph of a flagel-}

lated cell of S. typhimurium with flagella dispersed during tumbling (see text). Courtesy of R. M. Macnab.3 _{(E) Image of a cell}

of Vibrio alginolyticus obtained with dark-field illumination showing the single polar flagellum.13 _{Because the illumination}

was strong, the size of the cell body and the thickness of the flagellum in the image appear large. Courtesy of Michio Homma.

Quasiequivalence. There are two distinct types of straight flagella: one (R) in which the protofilaments have a right-handed twist (as in Fig. 19-1) and the other (L) in which the protofilaments have a left-handed twist. These arise from two different conformations of the subunit proteins. Native supercoiled flagella contain a mixture of flagellins in the R- and L-states with all subunits in a given protofilament being in the same state. The supercoiling of the filament cannot be explained by stacking of identical subunits but is thought to arise because of an asymmetric distribution of protofilaments in a given state around the fila- ment.17– 19a _{Here, as with the icosahedral viruses}

(Chapter 7), quasiequivalence permits formation of a structure that would be impossible with full equiva- lence of subunits. The corkscrew shape of the flagel- lum is essential to the conversion of the motor’s torque into a forward thrust.18 _{Certain mutants of Salmonella}

have “curly” flagella with a superhelix of one-half the normal pitch. The presence of p-fluorophenylalanine in the growth medium also produces curly flagella, and normal flagella can be transformed to curly ones by a suitable change of pH. More important for biological function, the transformation from normal to curly also appears to take place during the tumbling of bacteria associated with chemotaxis.17

A B C D E 5␮m 5␮m 5␮m 10 nm

Hook L ring P ring S ring M ring C ring

Figure 19-2 (A) Electron micrograph of a flagellum from

E. coli stained with uranyl acetate. The M-and S-rings are

seen at the end. Above them are the P-ring, thought to connect to the peptidoglycan layer, and the L-ring, thought to connect to the outer membrane or lipopolysaccharide layer (see Fig. 8-28). An arrow marks the junction between

hook and thinner filament. From DePamphilis and Adler.14

The hook is often bent to form an elbow. (B) Average of ~100 electron micrographs of frozen-hydrated preparations of basal bodies showing the cytoplasmic C-ring (see Fig. 19-3) extending from the thickened M-ring. From

DeRosier.16

A B

Growth of flagella. Iino added p-fluorophenyl- alanine to a suspension of bacteria, whose flagella had been broken off at various distances from the body.20

Curly ends appeared as the flagella grew out. Unlike the growth of hairs on our bodies, the flagella grew from the outer ends. Because no free flagellin was found in the surrounding medium, it was concluded that the flagellin monomers are synthesized within the bacterium, then pass out, perhaps in a partially unfolded form, through the 2- to 3-nm diameter hole10,12

in the flagella, and bind at the ends.21 _{Flagella of} Salmonella grow at the rate of 1 µm in 2 – 3 min initially,

then more slowly until they attain a length of ~ 15 µm. More recent studies have provided details. The hook region (Fig. 19-3) grows first to a length of ~ 55 nm by addition to the basal-body rod of ~ 140 subunits of protein FlgE. During growth a hook cap formed from subunits FlgD prevents the FlgE subunits from passing out into the medium.22,23 _{Hook subunits are added}

beneath the cap, moving the cap outward. Hook growth is terminated by protein FlgK (also called hook-associated protein Hap1). This protein displaces the hook cap and initiates growth of the main fila- ment.24 _{The first 10 – 20 subunits added are those of}

the FlgK (Fig. 19-3). These are followed by 10 – 20 subunits of FlgL (Hap3), a modified flagellin whose mechanical properties can accomodate the stress induced in the flagella by their rotation.25_{FlgJ is also}

needed for rod formation.25a

Growth of the flagellum to a length of up to 20 µm continues with subunits of FliC that are added at the tip, which is now covered by a dodecamer of the cap protein FliD (HAP2).24,26,26a,b _{Its 5-fold rotational}

symmetry means that this “star-cap” does not form a perfect plug for the 11-fold screw-symmetry of the flagellum, a fact that may be important in allowing new flagellin subunits to add at the growing tip. If the

cap protein is missing, as in some FliD mutants, a large amount of flagellin leaks into the medium.24

Still unclear is how the protein synthesis that is taking place on the ribosomes in the bacterial cytoplasm is controlled and linked to “export machinery” at the base of the flagellin. As indicated in Fig. 19-3, the genetically identified proteins FlhA, FliH, and FliI are involved in the process that sends the correct flagellin subunits through the growing flagellum at the appro- priate time. FliI contains an ATPase domain.26c _FliS

protein may be an export chaperone.26d