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Spinning tails
Spinning tails David J DeRosier Brandeis University, Waltham, USA The torque-generating, direction-reversing switch proteins of the bacterial flagellar rotary motor form a cytoplasmic extension of the bacterial flagellar basal body. 10,~, maps, obtained by electron cryomicroscopy, of the bacterial filament reveal an unusual alpha domain which forms the protein-subunit export channel. The details of subunit export, assembly, and assembly-monitoring machinery are becoming clearer. Current Opinion in Structural Biology 1995, 5:187-193
Introduction More than actomyosin or tubulokinesin, tile bacterial flagellum o f Salmonella typhimurium is the analogue o f a m a n - m a d e mechanical system (Fig. 1). Its heart is a 15 000 revolutions per minute, reversible rotary m o tor powered by the proton-motive gradient across the cell's inner membrane. Each revolution consumes about 1000 protons. A drive shaft, held by a bushing in the outer membrane, transmits torque across the cell's envelope. Attached to the drive shaft, a universal joint enables the m o t o r to drive the propeller, even when the drive shaft and propeller are not co-linear. A short junction joins the propeller to the drive shaft. T h e propeller, a long left-handed corkscrew, converts torque to thrust. A cap sits at the cell distal end o f the filament. By electron microscopy, the m o t o r associated parts and the bushing are seen to be rings ofsubunits, whereas the
drive shaft appears to be a helical assembly o f subunits. A b o u t four dozen genes are needed to build the flagellum. Some are required for regulation o f synthesis; some for export and assembly; some for the structure itselP, and a few are o f u n k n o w n function. Nineteen different proteins are k n o w n to be part o f the flagellar structure; it is t h o u g h t that there may be additional components. The location within the flagellum o f 17 o f these proteins has been determined [1",2"] (see Fig. 1 and Table 1). This article covers recent discoveries about the flagellum, with the emphasis being on the structure and function o f the flagellar components.
Turning tail to run T h e torque-generating proteins o f the m o t o r are MotA, MotB, FliG, FliM and FliN. MotA, a transmembrane
Table 1. The components of the flagellum of S. typhimurium. Name of part
Protein component(s)
Function Part of stator; proton channel
Studs
MotA, MotB
C-ring
FilM, FliN
Part of motor but not known if it is a component of the stator or the rotor
M-S ring
FliF, FliG
Part of rotor
L, P rings
FIgH, FIgl
Bushing permitting the rod to rotate while anchoring the flagellum
Rod Hook Junction
FIgB, FIgC, FIgF, FIgG FIgE FIgK, FIgL
Drive shaft: transmits torque from rod to filament, even when they are not coaxial Universal joint: transmits torque from rod to filament, even when they are not coaxial Joins hook to filament
Filament
FliC
Propeller
Cap
FliD
Assembles flagellin monomers into filament
Abbreviations HAP--hook-associated protein; L-type--left-handed; R-type--right-handed. © Current Biology lad ISSN 0959-440X
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Macromolecular assemblages
Cap
-
-
Filament
unction Exported flageilin subunit
\
. Hook Exterior L-ring
Outer membrane Peptidoglycan and periplasm Inner membrane
Rod
Studs C-ring
protein, forms the proton-conducting channel [3] and it joins with another membrane protein, MotB, to form a ring ofintramembranous studs [4]. MotB is thought to tether the studs to the peptidoglycan layer [5-7]. Thus, the evidence supports the M o t A - M o t B complex as being part of the stator (the fixed part of the motor). Do the remaining torque-generating proteins make up the rotor (the moving part of the motor)? Genetic studies suggest that FliG, FliM and FliN interact to form a complex known as the switch complex [8]. In addition to generating torque, the complex senses the signal to reverse the direction of motor rotation and then generates torque in the other direction. The intracellular signal for clockwise rotation is phosphorylation of Che Y, followed by binding of phospho-Che Y to FliM [9°]. Until recently, we had no information about the location of the switch complex, because isolated flagella lacked these proteins. In 1990, Driks and I [10] produced images of the basal body of the flagellum sporting a new cytoplasmic feature: a hollow cylindrical structure called the C-ring. Khan et al. [11] showed that this feature appeared more labile in cells having temperature-sensitive mutations in the switch proteins, which suggested that the C-ring might contain the switch complex. Francis et al. [12] reported on a motile but non-chemotactic
Cytoplasm
Fig. 1. Schematic of the parts of the bacterial flagellum of 5. typhimurium. The name, protein component(s), and function of each part is shown. The flagellum consists of several sets of rings (the studs and the L, P, S-M and C rings) coaxial with the filamentous component, which begins with the rod and ends with the cap at the tip of the filament. Most of the axial component is extracellular. Its center is a 30 ,g, wide channel through which flagellin monomers travel from the cell to their site of assembly at the cap. The flagellum can be over 1011m in length but is less than 0.1 ram in width at its widest point. The flagellum has a mass of over 1010 Da.
mutant having a FliF-FliG fusion protein. This mutant provided evidence that FliG is not part of the C-ring but is likely to be part o f the rotor. Francis et al. [13 °°] obtained preparations o f flagella lacking the C-ring but possessing the FliF-FliG fusion protein. Using antibodies against FliG, they located FliG on the cytoplasmic face of the M-ring, which is the part of the flagellum built from subunits of FliE Thus FliG appears to be intimately associated with FliE and is not a part of the C-ring. Is FliG part of the stator or the rotor? In the mutant, FliG is covalently attached to FliF; if the FIiF rotates, then FliG too must rotate. It is argued that FliF rotates because of its connection to the rod (drive shaft), which is connected to the hook illament structure. Thus, we think that FliG and FliF make up the rotor, which drives the rod, hook and filament. It is not a major part o f the C-ring, but FliM and FliN could be. Francis et al. [13 °'] showed that preparations of flagella having C-rings (Fig. 2) contain all three switch proteins. Electron micrographs o f preparations decorated with antibodies specific to the switch proteins suggested that FliM and FliN are part of the C-ring. Recalling that FliG, FliM and FliN interact to form the switch corn-
Spinning tails DeRosier plex, we suppose that these interactions are reponsible, at least in part, for attachment o f the C-ring through FliG to FliF and thus to the rest of the flagellum. Although FliM and FliN form a complex with FliG, it is unclear whether they are also part o f the rotor. Alternatively, they might be part of the stator, and their complex with FliG may be a structural intermediate in force generation rather like that formed between actin and myosin in the absence of ATE
comprise the axial component of the flagel]um. Each o f the nine different proteins o f the axial component appears to make up a particular segment o f the structure (Fig. 1). These components lie outside the cell's inner membrane; the hook and filament lie outside the outer membrane, as well. There is evidence for a flagellar-specific export apparatus that is different from the general, SecA-dependent protein export pathway [14]. The flagellar apparatus exports the proteins needed for assembly o f the axial structure. Export is stage specific: while the hook is being assembled, the flagellum exports hook subunits but not flagellins, which make up the flagellar filament (cited in [15]). The cell uses changes in the ability of the flagellmn to export particular proteins to sense the state of flagellar assembly [t6,17]. The flagellar protein FlgM, an anti-sigma factor, represses transcription of several genes, including the gene for flagellin. When the hook is completed, the flagellum begins to export FlgM. Upon export, the cytoplasmic concentration of FlgM decreases, which in turn removes the repression of flagellin synthesis and enables filament assembly to start. Thus, completion of the hook is a checkpoint used to couple parts of the assembly pathway. What makes the assembly of the axial component unusual is that components flow through an export channel contained m the partially assembled structure. Thus, flagellm subunits made inside the cell move down a channel inside the filament to the site of assembly, the cell-distal end of the filament [18,19], which can be several microns from the cell body. Export of the hook and flagellin subunits through the channel, however, does not suffice for assembly. At the distal end of the filament lies the FliD protein cap, which is required for addition o f flagellin subunits to the filament i, vivo [20,21]. Subunits ill the channel insert between the cap and tile end o f the filament so that the cap is displaced distally as the filament elongates. Without the cap, the subunits escape into the cell's surroundings. 111 vitro, the reverse is true. With the cap present, exogenous flagellin cannot add to the filament, but without the cap, it can.
Fig. 2. The flagellar basal body and filament. (a) A section through the three-dimensional map of the flage{lar basal body. (b) A cutaway view of the three-dimensional map of the flagellar filament.
The assembly of the hook in vivo also requires a capping protein, FlgD. It adds to the distal end of the rod before hook assembly [22"]. Unlike the FliD cap, which remains with the filament, FlgD is released upon completion of the hook. The simplest role for these two different caps is to prevent subunit escape, but they may also be needed to stabilize subunits at the tip of the hook or filament. At 37°C , filaments do not readily polymerize from flagellin in vitro, whereas bacterial growth and flagellar assembly in vivo thrive at this temperature [211.
From head to tail Flagellar assembly: thereby hangs a tail The drive shaft (rod), universal joint (hook), junction (FlgK and FlgL), propeller (filament), and cap (FliD)
The order of the nine proteins in the axial structure (see Fig. 1), at least from the hook outwards, does not depend on the elaborate export and assembly pathways; it is determined by the bonding properties o f the individual
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Macromolecularassemblages proteins. For example, in vitro, FlgL will bind to FlgK that is in a hook-FlgK complex, but not to hook alone, and flagellin will assemble onto a FlgL-FlgK-hook complex or onto a filament, but not add onto hook or onto a FlgK-hook complex [23,24]. The specific bonding properties of six o f the nine proteins can also explain their stoichiometries in the completed structure. For example, in vitro, FlgK binds to the completed hook with a stoichiometry indistinguishable from that found in vivo. The explanation is that FlgK does not bind to itself, at least in the axial direction, and thus once it forms a cap on the hook, all the available FlgK-binding sites are covered and addition ceases [25]. Flagellin and hook protein are present in much larger numbers than is FlgK and their binding properties do not seem to limit their stoichiometries. A 101im micron long filament contains about 20000 flagellin subunits. Variations in the number o f flagellin subunits in vivo result from differences in the lengths of time of filament growth or from filament breakage; in any case, it is not known if there is such a thing as a completed filament. In contrast, hook assembly does have a beginning and an end. It starts with the addition o f hook-capping protein FlgD to the rod and ends when the hook reaches a length o f 550fii, corresponding to 125 subunits of the hook protein, FlgE. In vitro, hooks can be assembled from purified hook subunits, but they are much longer than wild-type hooks. In vivo, therefore, some mechanism regulates hook length even though assembly occurs outside the cell. Recent studies [26 °] of wild-type hook lengths reveal a standard deviation of 59 ill, corresponding to about a dozen subunits; some hooks have as many as 40 additional subunits. Overexpression of the hook protein can increase hook length. The variation in subunit stoichiometry must be due to a lack of strict regulation in the hook-assembly mechanism. The genes for hook protein (HgE), hook-assembly protein (FlgD) and the hook-proximal junction protein FlgK do not appear to be involved in length regulation, whereas the genes fliK andflhB do appear to be involved [26°,27°]. Mutations infliK produce abnormally long, filamentless hooks up to a micron in length. These mutants have defects in two steps of assembly: they fail to sense hook length, and they fail to start filament assembly. Second site revertants to partial motility produce long hooks, as infliK mutants, but with filaments. Thus the second site reversion corrects only one of the two steps. The second site mutation is inflhB. FlhB is a protein thought to be involved in subunit export and also in rod assembly [28]. Kutsakake et al. [27"] proposed that FliK transmits the hook-completed signal to FlhB, which in turn switches export capabilities so as to end hook growth and to start filament assembly (i.e. it switches on the export o f FlgM [29]). Is there in fact a signal indicating completion o f hook assembly? Could it be that the cell fixes hook length simply by fixing the length o f time available for hook assembly? This would explain the variability of subunit stoichiometry in the hook. If the state of hook assembly
is sensed, however, is there a structural ruler such as the gpH protein ruler used in T4 phage tail assembly [30]? Alternatively, is some other property of the hook sensed, which might vary with hook length? For example, the rate of subunit flow by diffusion along a tube decreases with increasing tube length. Perhaps the rate of subunit export or its effect on subunit concentration within the cell is sensed as is the case with FlgM. The answers will provide important insights into the strategies cells use for assembly o f machinery outside the cell.
Switching tails The hook and filament are dynamic structures. The hook is not straight, but adopts a superhelical, corkscrew shape [31], which changes continuously during rotation, as required of a universal joint. The filament also adopts a corkscrew shape [32,33], a shape essential to its role as a propeller. Unlike the hook, it must maintain its shape while the motor turns. Upon reversal of the motor, however, the hand and pitch o f the filament change. These changes appear to help the cell change its direction o f swimming [1"]. These polymorphisms result because the hook and filament subunits can adopt either of two conformational states. The shapes and dynamic polymorphisms can be described by the switching of rows of subunits between the two conformational states. Switching is induced by mechanical forces. Although parts of the same structure, the hook and filament switch independently of each other. Recently, Fahrner et al. [34 °] have found that mutations in FlgL (part of the hook-filament junction) affect the filament's ability to maintain its corkscrew shape. If the turning filament bumps into a solid object, the filament straightens out and ceases to propel the cell. Thus, FlgL (present at one end of a filament) increases the barriers between structural states of this long filament. Perhaps the FlgK-FlgL junction also acts as a buffer between the dynamic structural changes in hook and filament.
The tale's end After analyzing the amino acid sequence of flagellins, Federov and Efimov [35] proposed that the flagellin subunit has an outer globular domain, consisting mostly of ]~-sheet, and an inner c~-domain, which consists of two four-helix bundles. The pairs o f four-helix bundles arise from N- and C-terminal segments of the protein (Fig. 3). The four-helix bundles o f the flagellin subunits are arranged radially about the filament axis rather like those in tobacco mosaic virus. On the basis of sequence homology between part of the Salmonella paratyphi flagellin sequence and the subtilisin sequence, Grewel and Salunke [36 °] constructed a model for flagellin residues 186 to 464 (Fig. 3). The predicted fold is a typical 0t/[3 domain consisting o f a central parallel ~-sheet covered by 0~-helices. By combining their model with that o f Fedorov and Efimov, the authors suggested a model for
Spinning tails DeRosier the whole flagellin subunit. How do these predicitions compare to observations of the structure? Structural studies are easiest on filaments from mutants in which all flagellin subunits are locked into the same conformational state [37]. These filaments are straight and have either a slight right-handed (R-type) or lefthanded (L-type) twist, depending on the conformational state of the protein. Mutants possessing these straight filaments do not swim, because their propellers cannot push fluid. Recently, two - 1 0 A maps o f the filament were completed. One (DG Morgan, C Owen, LA Melanson, DJ DeRosier, unpublished data; see Note added in proof) was of the L-type filament and the other (Y Mimori et al., personal conmmnication) of the R-type filament. Earlier studies of the same two P,.- and L-type filaments suggested a dramatic conformational change associated with the two states of the subunit [38]. The two maps, however, appear nearly identical, suggesting that the conformational switching involves very subtle changes. The filament structure (Fig. 3) consists of an outer globular domain (D3) and a middle (D2) domain, which attaches to the inner domains (D1 and DO). The inner domains consist of two coaxial cylinders connected by spokes.oThe center of the inner cylinder is a channel about 30A in diameter. Despite the similarities of the maps, the two groups interpret their maps differently. Morgan et al. find that 11 columns or rods appear to make up the innermost cylinder. The outer cylinder has 22 columns of rods, which abut domain D2. The spokes between the inner and outer cylinders are short rodshaped features. Morgan et al. tentatively identifed the rods as a-helices. Each asymmetric unit of the filament contains four a-helices: one i~ the inner domain, one
in the spoke, and two in the outer domain. In assigning features to domains, they adapted the scheme proposed by Namba et al. [39] (Fig. 3): the proteolytic fragment F27 segment makes up domain D3, the parts of proteolytic fragment F40 not common to F27 make up D2, and D1 (most of the outer ring) and DO (the inner ring) arise from the remaining N- and C-terminal segments. Earlier work on the structure of the Caulobacter cresce,tus filament supports the assignment of F27 to D3 [40]. Caulobacter flagellin is smaller than S. t y p h i m u r i , , i flagellin, lacking the segment of sequence that approximately corresponds to the F27 segment (Fig. 3), and its filament has a D2 domain but no D3 domain. The assignment of the N- and C-terminal segments to the domains D1 and DO attempts to take account of an analysis of the other axial proteins. All share heptad repeats of hydrophobic residues (a property of sequences involved in or-helical coiled-coils) in their N - and C-terminal segments. This suggests that the common fold in this shared structure is an ct-domain [41]. The structure of the hook reveals a single cylindrical inner domain [42]. Compared to hook protein, flagellin has an extended N-ternfinal segment. A plausible interpretation is that the extra residues generate the additional (innermost) cylinder. In their model, Morgan et al. (DG Morgan, C Owen, LA Melanson, DJ De Rosier, unpublished data; see Note added in proof) assign the N-terminal 70 residues to the inner 0t-helix, the spoke, and one of the outer two 0t-helices, and assign the C-ternfinal 30 residues to the fourth or-helix. In the filament, the {x-helices from one subuuit form bundles by interacting with the 0t-helices from adjacent subunits. The sharing of sequences from different subunits to form a domain is a theme found in viral capsids, It forms an interlocking design that should have the extra stability needed for extracellular assemblies. The pair of concentric cylinders explains why the
1
I I
t
(c)
(d)
[
I
]
(e)
[ (g) (h)
I ,o+D,
I DO + D1
D2
I I
D2 + D3
I
I DO + D1
I I
Fig. 3. A schematic showing the parts of flagellin protein sequence of Salmonella typhimurium. (a) The 494 amino acid residues of the entire polypeptide chain. (b) The F40 proteolytic fragment. (c) The F27 proteolytic fragment. (d) The alignment of the smaller C. crescentus sequence of flagellin with the S. typhimurium sequence. (e) The segment of S. paratyphi flagellin that is homologous to subtilisin. (f) Grewel and Salunke's predictions [36"] for the positions of the two segments that each give rise to a four-helix bundle. (g) The assignment of sequence to domain (D) used by Morgan et al. (DG Morgan, C Owen, LA Melanson, DJ DeRosier, unpublished data; see Note added in proof). (h) The assignment of sequence to domain used by Mimori et al. (Y Mimori et al., personal communication).
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Macromolecularassemblages cylinder is more rigid than the hook, which has a single cylinder. In their map, Y Mimori et al. (personal communication) also find rod-like structures in the inner and outer cylinders. They do not assign specific features to or-helices, but rather take up the issue of which parts o f the amino acid sequence make up the four domains. On the basis of the observed volume fractions for each o f the four domains, they suggest that domains D3 and D2 arises from the F27 segment (see Fig. 3) and that most o f domain D1 arises from the remainder of the F40 segment. The Nand C-ternainal segments make up DO (the inner cylinder) and the rest o f D1 (Fig. 3). The volumes of D3 and D2 do not seem to be big enough to accomodate all o f F40. Moreover, biophysical measurements [43] indicate that F27 contains two domains (D3 and D2?) and F40 contains three domains (D3, D2 and DI?). Mimori et al. (Y Mimori et al., personal communication) cite recent unpublished observations that deletion o f large parts o f the N - and C-terminal segments leads to filaments with a larger central channel than in wild-type. The two groups thus use different observations to support their interpretations. There is no evidence that is secure enough to decide which interpretation is right. For example, relative domain volumes may be calculated incorrectly if all features in the map do not have the correct weighting. Also, the sequence alignment of flagellin to other axial proteins may not be correct. High-resolution studies of the filaments made from flagellins lacking N- and/or C-terminal segments should help decide the matter. Both groups (Morgan et al. and Mimori et al.) agree that the or-helices run approximately parallel to the filament axis, an arrangement which disagrees with the model of Federov and Efimov, but which is in agreement with diffraction data [44]. The two groups also agree that the monomeric subunits, which are known to be partially° unfolded [45] are still too large to fit down the 30A diameter channel, and argue that the subunits unfold more to squeeze through. A colleague wondered if the walls of the protein channel might expand locally to permit subunit passage so that the filament would look like a snake swallowing a mouse. In summary, the proteins o f the switch complex, which together with MotA and MotB generate torque, cluster on the cytoplasmic face o f the flagellum adjacent to the ring of studs thought to contain MotA and MotB. To deduce the motor's mechanism, we will need more detailed structural studies o f the individual motor components and o f the whole assembly. Three-dimensional maps o f the motor with bound phospho-CheY will reveal the Che-Y binding sites, which are thought to be on FliM, and may reveal the structural changes that cause the motor to reverse its direction of rotation. The core o f the filament (or propellor) is a pair o f concentric tubes made largely o f axially packed 0~-helices. Higher resolution maps o f the filament and studies o f site directed mutants of flagellin should help in assigning the four flagellin domains (DO, D1, D2, D3) to spe-
cific segments o f the amino acid sequence. The assembly pathway o f the extracellular structures, especially the hook and the filament, remain mysterious. What identifies these proteins for export and how and where are the subunits inserted into the 30 A wide channel found in the hook and filament? An understanding of flagellar export and assembly will require identification o f all o f the export proteins, determination of their order of action in the export pathway, and localization o f them in the cell.
Note added in proof The paper referred to in the text as DG Morgan, C Owen, LA Melanson, DJ Delkosier, unpublished data has now been accepted for publication [46"°].
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest Macnab RM: Flagella and motility. In Escherichia coil and Salmonella typhimufium: cellular and molecular biology, edn 2. Edited by Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE. Washington DC: American Society for Microbiology; t995. This is a comprehensive review of the flagellum and of flagellar-produced motility by one of the experts in the field. 1. •
2. Schuster SC, Khan S: The bacterial flagellar motor. Annu Rev • Biophys Biomol Struct 1994, 23:509-539. This is an excellent review of the flagellar motor by experts in the field. 3.
Blair DF, Berg HC: The MoIA protein of Eseherichia coil is a proton-conducting component of the flagellar motor. Cell 1990, 60:439-449.
4.
Khan S, Dapice M, Rcese TS: Effects of mot gene expression on the structure of the flagellar motor. J Mol Biol 1988, 202:575-584.
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Chun SY, Parkinson JS: Bacterial motility: membrane topology of the Escherichia coil MotB protein. Science 1988, 239:276-278.
6.
DeMot R, Vanderleyden J: The C-terminal sequence conservation between OmpA-related outer membrane proteins and MolB suggests a common function in both Gram.positive and Gram-negative bacteria, possibly in the interaction of these domains with peptidoglycan. Mol Microbiot 1994, 12:333-334.
7.
McCarter LL: MotY, a component of the sodium-type flagellar motor. J Bacteriot 1994, !76:4219-4225.
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Yamaguchi S, Aizawa S-I, Kihara M, Isomura M, Jones CJ, Macnab RM: Genetic evidence for a switching and energytransducing complex in the flagellar motor of Salmonella typhimurium. J Bacteriol 1986, 151:1172-1179.
Welch M, Oosawa K, Aizawa S-t, Eisenbach M: Phosphorylation-dependent binding of a signal molecule to the flageilar switch of bacteria. Proc Natt Acad Sci USA 1993, 90:8787-8791. This paper presents the first evidence of the binding of phospho-CheY to one of the switch proteins. This helps complete the chain of signalling from the cell's receptors (e.g. tsr) to the flagellar motor.
9. ,,
10.
Driks A, DeRosier DJ: Additional paris a.~ocialed with the bacterial flagellar basal body. J A4ol Biol 1990, 211:669-672.
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Francis NR, Irikura VM, Yamaguchi S, DeRosier DJ, Macnab RM: Localization of the Salmonella typhimurlum flagellar switch protein FIiG to the cytoplasmic M-ring face of the basal body. Proc Natl Acad Sci USA 1992, 89:6304-6308.
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Francis, NR, Sosinsky GE, Thomas D, DeRosier DJ: Isolation, characterization and structure of bacterial flageUar motors containing the switch complex. J Mol Biol 1994, 235:1261-1270. This paper presents evidence that the switch proteins are localized to the cytoplasmic structures in the flagellar basal body. it also presents three-dimensional maps of the basal body obtained by electron cryomicroscopy. 14.
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Fahrner KA, Block SM, Krishnaswamy S, Parkinson JS, Berg HC: A hook-associated protein (HAP3) facilitates torsionally induced transformations of the flagellar filament of Escherichia coll. J Mol Biol 1994, 238:173-186. This paper presents evidence that HAP3, a minor structural protein, is essential for the correct dynamic behavior of the filament. •
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36. •
Grewel N, Salunke DM: The antigenic domain of flagellin from S. paratyphi share a structural fold with subtilisin. FEBS Lett 1993, 322:111-114. This paper is interesting because it presents a model for part of the fold of flagellin on the basis of its homology to subtilisin. 37.
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Ohnisbi K, Ohto Y, Aizawa S-I, Macnab RM, lino T: FIgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurlum. J Bacteriol 1994, 176:2272-2281. The authors show that hook assembly requires a protein cap at the end of the hook. Subunits made inside the cell move down a protein channel in the hook and when arriving at the end are helped to assemble by FIgD.
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Vonderviszt F, Uedaira H, Kidokoro S-I, Namba K: Structural organization of flagellin. J Mol Biol 1990, 214:97-104.
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Astbury WT, Weibull C: Study of the structure of bacterial flagella. Nature 1949, 163:280-282.
45.
Aizawa S-I, Vonderviszt F, Ishima R, Akasaka K: Termini of Salmonella flagellin are disordered and become more organized upon polymerization into flagellar filament. J Mol Biol 1990, 211:673--677.
169:1168-1173.
21.
22. •
23.
Homma M, lino T, Kutsakake K, Yamaguchi S: In vitro reconstitution of flagellar filaments onto hooks of filamentous mutants of Salmonella typhimurium by addition of hook-associated proteins. Proc Nat/Acad Sci USA 1986, 83:6169~173.
24.
25.
Ikeda T, Asakura S, Kamiya R: Total reconstitution of Salmonella flagellar filaments from hook and purified flagellin and hook-associated proteins in vitro. J Mol Biol 1989, 209:109-114. Jones CJ, Macnab RM, Okino H, Aizawa F-I: Stolchiometric analysis of the flagellar hook-(basal-body) complex of Salmonella typhlmurium. J Mo/ Biol 1990, 212:377-387.
26.
Hirano "1-, Yamaguchi S, Oosawa K, Aizawa S-h Roles of
•
FliK and FIhB in determination of fiagellar hook length in
Salmonella typhimurium. ] Bacteriol 1994, 176:5439-5449. These authors describe the interactions of genes that regulate hook length. •
acterization
Morgan DG, Owen C, Me(anson LA, DeRosier DJ: The 11 structure of bacterial fiagellar filaments: packing of c~-helices. J MOI Biol 1995, in press. This paper presents a map of the flagellar filament in the left-handed state. It permits us to see the alpha domain that generates the protein channel.
28.
Kubori T, Shimamoto S, Yamaguchi S, Namba K, Aizawa S-I: Morphological pathway of flagellar assembly in Salmonella typhlmurium. ] Mol Biol 1992, 226:433-446.
DJ DeRosier, Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, MA 02254, USA.
27.
Kutsakake K, Minamino T, Yokoseki 3-'. Isolation and charof fliK-independent flagellation mutants from Salmonella typhimurium. J Bacteriol 1994, 176:7625-7629. This paper presents analysis of genes that control hook length and presents a model for their interactions.
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Introduction More than actomyosin or tubulokinesin, tile bacterial flagellum o f Salmonella typhimurium is the analogue o f a m a n - m a d e mechanical system (Fig. 1). Its heart is a 15 000 revolutions per minute, reversible rotary m o tor powered by the proton-motive gradient across the cell's inner membrane. Each revolution consumes about 1000 protons. A drive shaft, held by a bushing in the outer membrane, transmits torque across the cell's envelope. Attached to the drive shaft, a universal joint enables the m o t o r to drive the propeller, even when the drive shaft and propeller are not co-linear. A short junction joins the propeller to the drive shaft. T h e propeller, a long left-handed corkscrew, converts torque to thrust. A cap sits at the cell distal end o f the filament. By electron microscopy, the m o t o r associated parts and the bushing are seen to be rings ofsubunits, whereas the
drive shaft appears to be a helical assembly o f subunits. A b o u t four dozen genes are needed to build the flagellum. Some are required for regulation o f synthesis; some for export and assembly; some for the structure itselP, and a few are o f u n k n o w n function. Nineteen different proteins are k n o w n to be part o f the flagellar structure; it is t h o u g h t that there may be additional components. The location within the flagellum o f 17 o f these proteins has been determined [1",2"] (see Fig. 1 and Table 1). This article covers recent discoveries about the flagellum, with the emphasis being on the structure and function o f the flagellar components.
Turning tail to run T h e torque-generating proteins o f the m o t o r are MotA, MotB, FliG, FliM and FliN. MotA, a transmembrane
Table 1. The components of the flagellum of S. typhimurium. Name of part
Protein component(s)
Function Part of stator; proton channel
Studs
MotA, MotB
C-ring
FilM, FliN
Part of motor but not known if it is a component of the stator or the rotor
M-S ring
FliF, FliG
Part of rotor
L, P rings
FIgH, FIgl
Bushing permitting the rod to rotate while anchoring the flagellum
Rod Hook Junction
FIgB, FIgC, FIgF, FIgG FIgE FIgK, FIgL
Drive shaft: transmits torque from rod to filament, even when they are not coaxial Universal joint: transmits torque from rod to filament, even when they are not coaxial Joins hook to filament
Filament
FliC
Propeller
Cap
FliD
Assembles flagellin monomers into filament
Abbreviations HAP--hook-associated protein; L-type--left-handed; R-type--right-handed. © Current Biology lad ISSN 0959-440X
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Macromolecular assemblages
Cap
-
-
Filament
unction Exported flageilin subunit
\
. Hook Exterior L-ring
Outer membrane Peptidoglycan and periplasm Inner membrane
Rod
Studs C-ring
protein, forms the proton-conducting channel [3] and it joins with another membrane protein, MotB, to form a ring ofintramembranous studs [4]. MotB is thought to tether the studs to the peptidoglycan layer [5-7]. Thus, the evidence supports the M o t A - M o t B complex as being part of the stator (the fixed part of the motor). Do the remaining torque-generating proteins make up the rotor (the moving part of the motor)? Genetic studies suggest that FliG, FliM and FliN interact to form a complex known as the switch complex [8]. In addition to generating torque, the complex senses the signal to reverse the direction of motor rotation and then generates torque in the other direction. The intracellular signal for clockwise rotation is phosphorylation of Che Y, followed by binding of phospho-Che Y to FliM [9°]. Until recently, we had no information about the location of the switch complex, because isolated flagella lacked these proteins. In 1990, Driks and I [10] produced images of the basal body of the flagellum sporting a new cytoplasmic feature: a hollow cylindrical structure called the C-ring. Khan et al. [11] showed that this feature appeared more labile in cells having temperature-sensitive mutations in the switch proteins, which suggested that the C-ring might contain the switch complex. Francis et al. [12] reported on a motile but non-chemotactic
Cytoplasm
Fig. 1. Schematic of the parts of the bacterial flagellum of 5. typhimurium. The name, protein component(s), and function of each part is shown. The flagellum consists of several sets of rings (the studs and the L, P, S-M and C rings) coaxial with the filamentous component, which begins with the rod and ends with the cap at the tip of the filament. Most of the axial component is extracellular. Its center is a 30 ,g, wide channel through which flagellin monomers travel from the cell to their site of assembly at the cap. The flagellum can be over 1011m in length but is less than 0.1 ram in width at its widest point. The flagellum has a mass of over 1010 Da.
mutant having a FliF-FliG fusion protein. This mutant provided evidence that FliG is not part of the C-ring but is likely to be part o f the rotor. Francis et al. [13 °°] obtained preparations o f flagella lacking the C-ring but possessing the FliF-FliG fusion protein. Using antibodies against FliG, they located FliG on the cytoplasmic face of the M-ring, which is the part of the flagellum built from subunits of FliE Thus FliG appears to be intimately associated with FliE and is not a part of the C-ring. Is FliG part of the stator or the rotor? In the mutant, FliG is covalently attached to FliF; if the FIiF rotates, then FliG too must rotate. It is argued that FliF rotates because of its connection to the rod (drive shaft), which is connected to the hook illament structure. Thus, we think that FliG and FliF make up the rotor, which drives the rod, hook and filament. It is not a major part o f the C-ring, but FliM and FliN could be. Francis et al. [13 °'] showed that preparations of flagella having C-rings (Fig. 2) contain all three switch proteins. Electron micrographs o f preparations decorated with antibodies specific to the switch proteins suggested that FliM and FliN are part of the C-ring. Recalling that FliG, FliM and FliN interact to form the switch corn-
Spinning tails DeRosier plex, we suppose that these interactions are reponsible, at least in part, for attachment o f the C-ring through FliG to FliF and thus to the rest of the flagellum. Although FliM and FliN form a complex with FliG, it is unclear whether they are also part o f the rotor. Alternatively, they might be part of the stator, and their complex with FliG may be a structural intermediate in force generation rather like that formed between actin and myosin in the absence of ATE
comprise the axial component of the flagel]um. Each o f the nine different proteins o f the axial component appears to make up a particular segment o f the structure (Fig. 1). These components lie outside the cell's inner membrane; the hook and filament lie outside the outer membrane, as well. There is evidence for a flagellar-specific export apparatus that is different from the general, SecA-dependent protein export pathway [14]. The flagellar apparatus exports the proteins needed for assembly o f the axial structure. Export is stage specific: while the hook is being assembled, the flagellum exports hook subunits but not flagellins, which make up the flagellar filament (cited in [15]). The cell uses changes in the ability of the flagellmn to export particular proteins to sense the state of flagellar assembly [t6,17]. The flagellar protein FlgM, an anti-sigma factor, represses transcription of several genes, including the gene for flagellin. When the hook is completed, the flagellum begins to export FlgM. Upon export, the cytoplasmic concentration of FlgM decreases, which in turn removes the repression of flagellin synthesis and enables filament assembly to start. Thus, completion of the hook is a checkpoint used to couple parts of the assembly pathway. What makes the assembly of the axial component unusual is that components flow through an export channel contained m the partially assembled structure. Thus, flagellm subunits made inside the cell move down a channel inside the filament to the site of assembly, the cell-distal end of the filament [18,19], which can be several microns from the cell body. Export of the hook and flagellin subunits through the channel, however, does not suffice for assembly. At the distal end of the filament lies the FliD protein cap, which is required for addition o f flagellin subunits to the filament i, vivo [20,21]. Subunits ill the channel insert between the cap and tile end o f the filament so that the cap is displaced distally as the filament elongates. Without the cap, the subunits escape into the cell's surroundings. 111 vitro, the reverse is true. With the cap present, exogenous flagellin cannot add to the filament, but without the cap, it can.
Fig. 2. The flagellar basal body and filament. (a) A section through the three-dimensional map of the flage{lar basal body. (b) A cutaway view of the three-dimensional map of the flagellar filament.
The assembly of the hook in vivo also requires a capping protein, FlgD. It adds to the distal end of the rod before hook assembly [22"]. Unlike the FliD cap, which remains with the filament, FlgD is released upon completion of the hook. The simplest role for these two different caps is to prevent subunit escape, but they may also be needed to stabilize subunits at the tip of the hook or filament. At 37°C , filaments do not readily polymerize from flagellin in vitro, whereas bacterial growth and flagellar assembly in vivo thrive at this temperature [211.
From head to tail Flagellar assembly: thereby hangs a tail The drive shaft (rod), universal joint (hook), junction (FlgK and FlgL), propeller (filament), and cap (FliD)
The order of the nine proteins in the axial structure (see Fig. 1), at least from the hook outwards, does not depend on the elaborate export and assembly pathways; it is determined by the bonding properties o f the individual
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Macromolecularassemblages proteins. For example, in vitro, FlgL will bind to FlgK that is in a hook-FlgK complex, but not to hook alone, and flagellin will assemble onto a FlgL-FlgK-hook complex or onto a filament, but not add onto hook or onto a FlgK-hook complex [23,24]. The specific bonding properties of six o f the nine proteins can also explain their stoichiometries in the completed structure. For example, in vitro, FlgK binds to the completed hook with a stoichiometry indistinguishable from that found in vivo. The explanation is that FlgK does not bind to itself, at least in the axial direction, and thus once it forms a cap on the hook, all the available FlgK-binding sites are covered and addition ceases [25]. Flagellin and hook protein are present in much larger numbers than is FlgK and their binding properties do not seem to limit their stoichiometries. A 101im micron long filament contains about 20000 flagellin subunits. Variations in the number o f flagellin subunits in vivo result from differences in the lengths of time of filament growth or from filament breakage; in any case, it is not known if there is such a thing as a completed filament. In contrast, hook assembly does have a beginning and an end. It starts with the addition o f hook-capping protein FlgD to the rod and ends when the hook reaches a length o f 550fii, corresponding to 125 subunits of the hook protein, FlgE. In vitro, hooks can be assembled from purified hook subunits, but they are much longer than wild-type hooks. In vivo, therefore, some mechanism regulates hook length even though assembly occurs outside the cell. Recent studies [26 °] of wild-type hook lengths reveal a standard deviation of 59 ill, corresponding to about a dozen subunits; some hooks have as many as 40 additional subunits. Overexpression of the hook protein can increase hook length. The variation in subunit stoichiometry must be due to a lack of strict regulation in the hook-assembly mechanism. The genes for hook protein (HgE), hook-assembly protein (FlgD) and the hook-proximal junction protein FlgK do not appear to be involved in length regulation, whereas the genes fliK andflhB do appear to be involved [26°,27°]. Mutations infliK produce abnormally long, filamentless hooks up to a micron in length. These mutants have defects in two steps of assembly: they fail to sense hook length, and they fail to start filament assembly. Second site revertants to partial motility produce long hooks, as infliK mutants, but with filaments. Thus the second site reversion corrects only one of the two steps. The second site mutation is inflhB. FlhB is a protein thought to be involved in subunit export and also in rod assembly [28]. Kutsakake et al. [27"] proposed that FliK transmits the hook-completed signal to FlhB, which in turn switches export capabilities so as to end hook growth and to start filament assembly (i.e. it switches on the export o f FlgM [29]). Is there in fact a signal indicating completion o f hook assembly? Could it be that the cell fixes hook length simply by fixing the length o f time available for hook assembly? This would explain the variability of subunit stoichiometry in the hook. If the state of hook assembly
is sensed, however, is there a structural ruler such as the gpH protein ruler used in T4 phage tail assembly [30]? Alternatively, is some other property of the hook sensed, which might vary with hook length? For example, the rate of subunit flow by diffusion along a tube decreases with increasing tube length. Perhaps the rate of subunit export or its effect on subunit concentration within the cell is sensed as is the case with FlgM. The answers will provide important insights into the strategies cells use for assembly o f machinery outside the cell.
Switching tails The hook and filament are dynamic structures. The hook is not straight, but adopts a superhelical, corkscrew shape [31], which changes continuously during rotation, as required of a universal joint. The filament also adopts a corkscrew shape [32,33], a shape essential to its role as a propeller. Unlike the hook, it must maintain its shape while the motor turns. Upon reversal of the motor, however, the hand and pitch o f the filament change. These changes appear to help the cell change its direction o f swimming [1"]. These polymorphisms result because the hook and filament subunits can adopt either of two conformational states. The shapes and dynamic polymorphisms can be described by the switching of rows of subunits between the two conformational states. Switching is induced by mechanical forces. Although parts of the same structure, the hook and filament switch independently of each other. Recently, Fahrner et al. [34 °] have found that mutations in FlgL (part of the hook-filament junction) affect the filament's ability to maintain its corkscrew shape. If the turning filament bumps into a solid object, the filament straightens out and ceases to propel the cell. Thus, FlgL (present at one end of a filament) increases the barriers between structural states of this long filament. Perhaps the FlgK-FlgL junction also acts as a buffer between the dynamic structural changes in hook and filament.
The tale's end After analyzing the amino acid sequence of flagellins, Federov and Efimov [35] proposed that the flagellin subunit has an outer globular domain, consisting mostly of ]~-sheet, and an inner c~-domain, which consists of two four-helix bundles. The pairs o f four-helix bundles arise from N- and C-terminal segments of the protein (Fig. 3). The four-helix bundles o f the flagellin subunits are arranged radially about the filament axis rather like those in tobacco mosaic virus. On the basis of sequence homology between part of the Salmonella paratyphi flagellin sequence and the subtilisin sequence, Grewel and Salunke [36 °] constructed a model for flagellin residues 186 to 464 (Fig. 3). The predicted fold is a typical 0t/[3 domain consisting o f a central parallel ~-sheet covered by 0~-helices. By combining their model with that o f Fedorov and Efimov, the authors suggested a model for
Spinning tails DeRosier the whole flagellin subunit. How do these predicitions compare to observations of the structure? Structural studies are easiest on filaments from mutants in which all flagellin subunits are locked into the same conformational state [37]. These filaments are straight and have either a slight right-handed (R-type) or lefthanded (L-type) twist, depending on the conformational state of the protein. Mutants possessing these straight filaments do not swim, because their propellers cannot push fluid. Recently, two - 1 0 A maps o f the filament were completed. One (DG Morgan, C Owen, LA Melanson, DJ DeRosier, unpublished data; see Note added in proof) was of the L-type filament and the other (Y Mimori et al., personal conmmnication) of the R-type filament. Earlier studies of the same two P,.- and L-type filaments suggested a dramatic conformational change associated with the two states of the subunit [38]. The two maps, however, appear nearly identical, suggesting that the conformational switching involves very subtle changes. The filament structure (Fig. 3) consists of an outer globular domain (D3) and a middle (D2) domain, which attaches to the inner domains (D1 and DO). The inner domains consist of two coaxial cylinders connected by spokes.oThe center of the inner cylinder is a channel about 30A in diameter. Despite the similarities of the maps, the two groups interpret their maps differently. Morgan et al. find that 11 columns or rods appear to make up the innermost cylinder. The outer cylinder has 22 columns of rods, which abut domain D2. The spokes between the inner and outer cylinders are short rodshaped features. Morgan et al. tentatively identifed the rods as a-helices. Each asymmetric unit of the filament contains four a-helices: one i~ the inner domain, one
in the spoke, and two in the outer domain. In assigning features to domains, they adapted the scheme proposed by Namba et al. [39] (Fig. 3): the proteolytic fragment F27 segment makes up domain D3, the parts of proteolytic fragment F40 not common to F27 make up D2, and D1 (most of the outer ring) and DO (the inner ring) arise from the remaining N- and C-terminal segments. Earlier work on the structure of the Caulobacter cresce,tus filament supports the assignment of F27 to D3 [40]. Caulobacter flagellin is smaller than S. t y p h i m u r i , , i flagellin, lacking the segment of sequence that approximately corresponds to the F27 segment (Fig. 3), and its filament has a D2 domain but no D3 domain. The assignment of the N- and C-terminal segments to the domains D1 and DO attempts to take account of an analysis of the other axial proteins. All share heptad repeats of hydrophobic residues (a property of sequences involved in or-helical coiled-coils) in their N - and C-terminal segments. This suggests that the common fold in this shared structure is an ct-domain [41]. The structure of the hook reveals a single cylindrical inner domain [42]. Compared to hook protein, flagellin has an extended N-ternfinal segment. A plausible interpretation is that the extra residues generate the additional (innermost) cylinder. In their model, Morgan et al. (DG Morgan, C Owen, LA Melanson, DJ De Rosier, unpublished data; see Note added in proof) assign the N-terminal 70 residues to the inner 0t-helix, the spoke, and one of the outer two 0t-helices, and assign the C-ternfinal 30 residues to the fourth or-helix. In the filament, the {x-helices from one subuuit form bundles by interacting with the 0t-helices from adjacent subunits. The sharing of sequences from different subunits to form a domain is a theme found in viral capsids, It forms an interlocking design that should have the extra stability needed for extracellular assemblies. The pair of concentric cylinders explains why the
1
I I
t
(c)
(d)
[
I
]
(e)
[ (g) (h)
I ,o+D,
I DO + D1
D2
I I
D2 + D3
I
I DO + D1
I I
Fig. 3. A schematic showing the parts of flagellin protein sequence of Salmonella typhimurium. (a) The 494 amino acid residues of the entire polypeptide chain. (b) The F40 proteolytic fragment. (c) The F27 proteolytic fragment. (d) The alignment of the smaller C. crescentus sequence of flagellin with the S. typhimurium sequence. (e) The segment of S. paratyphi flagellin that is homologous to subtilisin. (f) Grewel and Salunke's predictions [36"] for the positions of the two segments that each give rise to a four-helix bundle. (g) The assignment of sequence to domain (D) used by Morgan et al. (DG Morgan, C Owen, LA Melanson, DJ DeRosier, unpublished data; see Note added in proof). (h) The assignment of sequence to domain used by Mimori et al. (Y Mimori et al., personal communication).
191
192
Macromolecularassemblages cylinder is more rigid than the hook, which has a single cylinder. In their map, Y Mimori et al. (personal communication) also find rod-like structures in the inner and outer cylinders. They do not assign specific features to or-helices, but rather take up the issue of which parts o f the amino acid sequence make up the four domains. On the basis of the observed volume fractions for each o f the four domains, they suggest that domains D3 and D2 arises from the F27 segment (see Fig. 3) and that most o f domain D1 arises from the remainder of the F40 segment. The Nand C-ternainal segments make up DO (the inner cylinder) and the rest o f D1 (Fig. 3). The volumes of D3 and D2 do not seem to be big enough to accomodate all o f F40. Moreover, biophysical measurements [43] indicate that F27 contains two domains (D3 and D2?) and F40 contains three domains (D3, D2 and DI?). Mimori et al. (Y Mimori et al., personal communication) cite recent unpublished observations that deletion o f large parts o f the N - and C-terminal segments leads to filaments with a larger central channel than in wild-type. The two groups thus use different observations to support their interpretations. There is no evidence that is secure enough to decide which interpretation is right. For example, relative domain volumes may be calculated incorrectly if all features in the map do not have the correct weighting. Also, the sequence alignment of flagellin to other axial proteins may not be correct. High-resolution studies of the filaments made from flagellins lacking N- and/or C-terminal segments should help decide the matter. Both groups (Morgan et al. and Mimori et al.) agree that the or-helices run approximately parallel to the filament axis, an arrangement which disagrees with the model of Federov and Efimov, but which is in agreement with diffraction data [44]. The two groups also agree that the monomeric subunits, which are known to be partially° unfolded [45] are still too large to fit down the 30A diameter channel, and argue that the subunits unfold more to squeeze through. A colleague wondered if the walls of the protein channel might expand locally to permit subunit passage so that the filament would look like a snake swallowing a mouse. In summary, the proteins o f the switch complex, which together with MotA and MotB generate torque, cluster on the cytoplasmic face o f the flagellum adjacent to the ring of studs thought to contain MotA and MotB. To deduce the motor's mechanism, we will need more detailed structural studies o f the individual motor components and o f the whole assembly. Three-dimensional maps o f the motor with bound phospho-CheY will reveal the Che-Y binding sites, which are thought to be on FliM, and may reveal the structural changes that cause the motor to reverse its direction of rotation. The core o f the filament (or propellor) is a pair o f concentric tubes made largely o f axially packed 0~-helices. Higher resolution maps o f the filament and studies o f site directed mutants of flagellin should help in assigning the four flagellin domains (DO, D1, D2, D3) to spe-
cific segments o f the amino acid sequence. The assembly pathway o f the extracellular structures, especially the hook and the filament, remain mysterious. What identifies these proteins for export and how and where are the subunits inserted into the 30 A wide channel found in the hook and filament? An understanding of flagellar export and assembly will require identification o f all o f the export proteins, determination of their order of action in the export pathway, and localization o f them in the cell.
Note added in proof The paper referred to in the text as DG Morgan, C Owen, LA Melanson, DJ Delkosier, unpublished data has now been accepted for publication [46"°].
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest Macnab RM: Flagella and motility. In Escherichia coil and Salmonella typhimufium: cellular and molecular biology, edn 2. Edited by Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE. Washington DC: American Society for Microbiology; t995. This is a comprehensive review of the flagellum and of flagellar-produced motility by one of the experts in the field. 1. •
2. Schuster SC, Khan S: The bacterial flagellar motor. Annu Rev • Biophys Biomol Struct 1994, 23:509-539. This is an excellent review of the flagellar motor by experts in the field. 3.
Blair DF, Berg HC: The MoIA protein of Eseherichia coil is a proton-conducting component of the flagellar motor. Cell 1990, 60:439-449.
4.
Khan S, Dapice M, Rcese TS: Effects of mot gene expression on the structure of the flagellar motor. J Mol Biol 1988, 202:575-584.
5.
Chun SY, Parkinson JS: Bacterial motility: membrane topology of the Escherichia coil MotB protein. Science 1988, 239:276-278.
6.
DeMot R, Vanderleyden J: The C-terminal sequence conservation between OmpA-related outer membrane proteins and MolB suggests a common function in both Gram.positive and Gram-negative bacteria, possibly in the interaction of these domains with peptidoglycan. Mol Microbiot 1994, 12:333-334.
7.
McCarter LL: MotY, a component of the sodium-type flagellar motor. J Bacteriot 1994, !76:4219-4225.
8.
Yamaguchi S, Aizawa S-I, Kihara M, Isomura M, Jones CJ, Macnab RM: Genetic evidence for a switching and energytransducing complex in the flagellar motor of Salmonella typhimurium. J Bacteriol 1986, 151:1172-1179.
Welch M, Oosawa K, Aizawa S-t, Eisenbach M: Phosphorylation-dependent binding of a signal molecule to the flageilar switch of bacteria. Proc Natt Acad Sci USA 1993, 90:8787-8791. This paper presents the first evidence of the binding of phospho-CheY to one of the switch proteins. This helps complete the chain of signalling from the cell's receptors (e.g. tsr) to the flagellar motor.
9. ,,
10.
Driks A, DeRosier DJ: Additional paris a.~ocialed with the bacterial flagellar basal body. J A4ol Biol 1990, 211:669-672.
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Khan IH, Reese TS, Khan S: The cytoplasmic component of the bacterial flagellar motor. Proc Natl Acad Sci USA 1992, 89:6965-5960.
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Francis NR, Irikura VM, Yamaguchi S, DeRosier DJ, Macnab RM: Localization of the Salmonella typhimurlum flagellar switch protein FIiG to the cytoplasmic M-ring face of the basal body. Proc Natl Acad Sci USA 1992, 89:6304-6308.
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Francis, NR, Sosinsky GE, Thomas D, DeRosier DJ: Isolation, characterization and structure of bacterial flageUar motors containing the switch complex. J Mol Biol 1994, 235:1261-1270. This paper presents evidence that the switch proteins are localized to the cytoplasmic structures in the flagellar basal body. it also presents three-dimensional maps of the basal body obtained by electron cryomicroscopy. 14.
Vogler AP, Homma M, Irikura VM, Macnab RM: Salmonella typhimurium mutants defective in flagellar filament regrowth and sequence similarity of Fill to FoF1, vacuolar and archaebacterial ATPase subunits. J Bacteriol 1991, 173:3564-3572.
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Kutsakake K: Excretion of the anti-slgma factor through the flagellar substructure couples flagellar gene expression with flagellar assembly in Salmonella typhimuriurn. Mol Gen Genet 1994, 243:605-612.
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Minamino T, lino T, Kutsakake K: Molecular characterization of the Salmonella typhimurlum flhB operon and its protein products. J Bacteriol 1994, 176: 7630-7637.
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Katsura I, Hendrix R: Length determination in bacteriophage lambda tails. Cell 1984, 39:691~98.
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Kato S, Okamoto M, Asakura S: Polymorphic transition of the flagellar polyhook from Escherichia colt and Salmonella typhimurium. ] Mol Biol 1984, 173:463-476.
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Asakura S: Polymerization of flagella and polymorphism of flagella. Adv Biophys (Japan) 1970, 1:99-155.
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Calladine CR: Construction of bacterial flagella. Nature 1975, 225:121-124.
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Fahrner KA, Block SM, Krishnaswamy S, Parkinson JS, Berg HC: A hook-associated protein (HAP3) facilitates torsionally induced transformations of the flagellar filament of Escherichia coll. J Mol Biol 1994, 238:173-186. This paper presents evidence that HAP3, a minor structural protein, is essential for the correct dynamic behavior of the filament. •
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