Jonathan McLatchie
The Bacterial Flagellum: A Motorized Nanomachine
Jonathan McLatchie McLatchie (B.Sc, M.Res) M.Res) September 2012 Last updated: 3/9/2013
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1. Introduction The bacterial flagellum is a reversible, self-assembling, rotary nano-motor associated with the majority of swimming bacteria. There exists a number of different models of this rotary motor (Pallen and Matzke, 2006; Soutourina and Bertin, 2003). Flagella are produced by a very tightly regulated assembly assembl y pathway (Chevance and Hughes, 2008; McCarter, 2006; Macnab, 2003; Aldridge and Hughes, 2002; Chilcott and Hughes, 2000), and the archetypical system for understanding flagellar assembly belongs to Salmonella enterica serovar Typhimurium, a rod-shaped gram negative bacterium of the family Enterobacteriaceae family Enterobacteriaceae.. Flagella receive feedback from the environment by virtue of an elegant signal transduction circuit and can adjust their course in response to external stimuli by a mechanism known as chemotaxis (Baker et (Baker et al., 2006 Bourret and Stock, 2002; Bren and Eisenbach, 2000). The most extensively studied chemotaxis system belongs to Escherichia to Escherichia coli. coli. By itself, the rotor is able to turn at a speed between 6,000 and 17,000 rotations per minute (rpm) but normally only achieves a speed of 200 to 1000 rpm when the flagellar filament (that is, the propeller) is attached. Its forward and reverse gears allow the motor to reverse direction within a quarter turn. The bacterial flagellum, which has been described as a “nanotechnological marvel” (Berg, 2003), has long been championed as an icon of the modern intelligent design movement and the flagship example of “irreducible complexity” (Behe, 1996). But even biologists outside of this community have been struck by the motor’s engineering elegance and intrinsic beauty. beauty . As one writer put it, “Since the flagellum is so well designed and beautifully constructed by an ordered assembly pathway, even I, who am not a creationist, get a n awe-inspiring feeling from its ‘divine’ beauty,” (Aizawa, 2009).
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The bacterial flagellum exhibits a tightly-orchestrated manufacturing process, and manifests a level of engineering that is far superior to humanity’s best achievements. Indeed, researchers have even modelled the assembly process in view of finding inspiration for enhancing industrial operations (McAuley et al.). al.). The mechanistic basis of flagellar assembly is so breathtakingly elegant and mesmerising that the sheer engineering brilliance of the flagellar fl agellar motor cannot be properly appreciated without, at minimum, a cursory knowledge of its underpinning operations. The purpose of this essay is to review these intricate processes. In writing this paper, a relatively relativel y high level of technicality has been necessary in order to convey the engineering sophistication of this nano-machine. The sheer beauty and elegance of the bacterial flagellum has been largely hidden from the public view because of the time and patience it takes to understand and absorb the pertinent detail s of its assembly ass embly and operation. Without such detail, however, it is all too easy to under-appreciate the engineering grandeur of the flagellar apparatus.
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2. Flagellar Structure A simplified diagram of flagellar structure is given in figure 1. The flagellar motor contains as many as 40 distinct types of proteins. Flagella are polymers comprised of protein subunits (molecular weight 53kd), called flagellin, which form a helical structure with a hollow core, measuring roughly 15µm in length and 15nm in diameter (Berg et al., 2006). Each subunit contributes a small propeller blade to the outside of the structure.
Figure 1: Simplified diagrammatic representation of gram negative bacterial flagellar structure.
As can be seen from figure 1, the rotor (i.e. the tube-like structure that provides rotary motion) is embedded in the inner cell membrane, and rotates within the stator. The rotor passes through the membrane to the outside of the cell, and attaches to the flagellar filament via the hook (universal joint). The hook is required to be 55nm in length, and this is controlled by an elegant molecular mechanism (Erhardt et al., 2011; Erhardt et al., 2010;
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Keener et al., 2010; Waters et al., 2007; Hirano et al., 1994), a fact that we shall return to when we discuss assembly. The hook structure is shown in figure 2.
Figure 2: Diagrammatic representation of bacterial flagellar hook (universal joint) and filament (propeller).
The part of the flagellum embedded in the cell wall is known as the basal body, and this is comprised of the MS ring, rod, and L- and P- rings, as shown in figure 1. In gram-negative bacteria, an outer ring (the L ring) is anchored in the lipopolysaccharide layer. Another ring (the P ring) is anchored in the peptidoglycan layer of the cell wall. A third set of rings (the MS and C rings) are respectively found within the cytoplasmic membrane and the cytoplasm. The MS (membrane and supermembrane) ring is formed by the proteins FliG , FliM , and FliN; the C ring is comprised of the proteins FliN and FliM. Gram positive bacteria, by contrast, do not have an outer membrane, and thus only possess the inner pair of ri ngs. A series of proteins, called Mot proteins (MotA and MotB), surround the inner ring and
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are anchored in the cytoplasmic membrane. Approximately 11 MotA-MotB pairs create a ring structure around the flagellar base. MotA poss esses four transmembrane helices and a cytoplasmic domain. MotB possesses a single transmembrane helix and a periplasmic domain. The structures of the L, P, C and MS rings, in combination with the central rod (the motor that turns the flagellum), are collectively r eferred to as the “basal body”. An electron micrograph of the flagellar basal body is given in fi gure 3.
Figure 3: Electron micrograph of flagellar hook-basal-body (HBB).
3. A Proton Motive Force Drives the Flagellar Motor The flagellar motor is driven by a proton gradient across t he plasma membrane (Meister et al., 1987; Manson et al., 1977). FliG combines with the MotA-MotB pair to form a proton channel. Protons are transported across the cell membrane by the rotor, and this process drives the rotor’s rotation. Moreover, “[e]ach MotA-MotB pair is conjectured to form a structure that has two half-channels; FliG serves as the rotating proton carrier, perhaps with the participation of some of the charged residues identified in crystallographic studies. In this 6
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scenario, a proton from the periplasmic space passes into the outer half-channel and is transferred to an [ sic] FliG subunit. The MS ring rotates, rotating the flagellum with it and allowing the proton to pass into the inner half-channel and into the cell, ” (Berg et al., 2006). Some organisms (certain alkalophilic and marine bacteria such as Bacillus and Vibrio species) utilise sodium ions instead (Imae and Atsumi, 1989). Bacillus subtilis is a micro-organism capable of biofilm formation (Lemon et al., 2008). A biofilm is an antibiotic-resistant aggregate of bacteria, embedded within a matrix of extracellular polymeric substances (EPS) such as proteins and polysaccharides, in which microorganisms adhere to one another. In a biofilm, bacterial motility is switched off. This raises the question of how this inhibition of motility is achieved. What determines whether the engine of a car is connected to the components that spin the wheels of the vehicle? The answer is the clutch, which ensures that the engine and gears are disengaged. In the flagella of Bacillus subtilis, the analogue to the clutch of a car is a protein called EpsE (Blair et al., 2008), which is carried on the same operon as the genes nec essary for EPS formation. EpsE makes contact with the flagellum’s rotor. The protein responsible for polymerising into the rotor is called FliG. As stated above, the FliG subunits also convert the proton flux energy into the flagellum’s rotational energy. When EpsE interacts with FliG, it triggers a conformational change, causing it to bend such that the rotor is dis engaged from the proton powered engine of the flagellum. EpsE is bifunctional, for it “interacts with the flagella rotor to inhibit motility and also cooperates with other enzymes to synthesise the EPS matrix,” (Guttenplan et al., 2010).
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A diagrammatic illustration of the function of this molecular clutch is given in figure 4.
Figure 4: Diagrammatic representation of the function of the molecular flagellar clutch in Bacillus subtilis.
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4. Flagellar Assembly Readers may find it helpful to refer to figure 5 when reading the description that follows.
Figure 5: Schematic diagram of the location of proteins involved in flagellar assembly and function. 4.1 Assembly of Integral Membrane Components
Assembly of the flagellum’s integral membrane components is the first stage of the flagellum’s construction by the cell. This involves the integration of the cytoplasmic membrane proteins into the membrane by the Sec pathway (for a review of the Sec pathway, see Mori and Ito, 2001). It is thought that the export and MS ring proteins assemble in a coordinated way, and this hypothesis gains support from intergenic suppression data which shows that the MS ring protein FliF interacts with export protein FlhA (Kihara et al., 2001), as well as data on temperature-sensitive mutants that shows that the export proteins FliP and FliR are somehow sequestered unless the temperature-sensitive protein is the MS ring protein (Jones and Macnab, 1990a).
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The functions of the MS ring, a component which is critical to the flagellum’s function and assembly, is given in three stages: “Fir st, it acts as the mounting plate for the rotor/switch. Second, it connects via FliE to the entire axial structure and thus enables flagellar rotation. Third, it acts as a housing for the export apparatus,” (Macnab, 2003). 4.2 Assembly of Type III Export Apparatus and Rotor/Switch
The next thing to be assembled after the MS ring i s the flagellum-specific type III export apparatus and the rotor/switch. Many of the flagellar substructures are built from proteins that are secreted through the type III export pathway, and these proteins are added at t he distal end by diffusion down the Type III export pathway’s central narrow channel (Macnab, 2004). The addition of these subunits is often controlled by a capping structure. The type III export pathwa y is also used for other critical purposes during assembly, including export of the hook-length control protein FliK and the anti-sigma factor FlgM, which we will return to. There is a protein called FliE which is a Type III export substrate (Hirano et al., 2003). This protein is also needed for the export of other substrates (Minamino and Macnab, 1999) and is part of the basal body apparatus (Muller et al., 1992). 4.3 Rod Assembly
The next step is to assemble the proximal and distal rod. The four rod proteins are FlgB, FlgC, FlgF and FlgG (Homma et al.,1990). Another protein, called FlgJ, is bifunctional. The C-terminal domain of FlgJ possesses peptidoglycan hydrolysing (muramidase) activity, which is important for rod formation (Nambu et al ., 1999). This is because the peptidoglycan layer has to be locally digested to allow for the penetration of the rod. In fact, experiments with mutant FlgJ proteins with a broken C-terminal (muramidase) domain res ult in a basal
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body lacking the L ring and hook (Hirano et al ., 2001). Penetration of the peptidoglycan la yer is a prerequisite for rod formation (Dijkstra and Keck, 1996; Fein, 1979). The C-terminal muramidase domain of FlgJ, however, may be dispensable, since the muramidase domain is absent in homologs of FlgJ in some bacterial phyla, and it appears that the rod structure "occasionally and fortuitously finds a hole in the peptidoglycan layer by chance; when it does so, it proceeds to assemble a flagellum that includes the outer membrane L ring," (Hirano et al ., 2001). FlgJ also possesses binding affinity for rod proteins (Hirano et al., 2001). It is thought that FlgJ is the first protein to assemble since its N-terminal serves as a capping protein for the rod structure. Besides the genes that encode the rod subunit proteins (FlgB, FlgC, FlgF, and FlgG), more than 10 genes are necessary for rod assembly (Kubori et al., 1992). One study reported that "In the enteric bacteria, flgJ null mutants fail to produce the flagellar rods, hooks, and filaments but still assemble the integral membrane-supramembrane (MS) rings. These mutants are nonmotile," (Zhang et al., 2012). 4.4 L and P Ring Assembly
The next step is to assemble the L (lipoprotein) and P (periplasmic) rings. These are formed by assembly of the FlgH and FlgI proteins respectively around the rod (Jones et al., 1989). FlgH and FlgI are exported by the Sec pathway, as evidenced from the fact that they both undergo signal peptide cleavage (Jones and Macnab, 1990b; Homma et al., 1987). Two reasons have been proposed for why they use this pathway: “The first is that they are destined for finite compartments, and so there is no risk of infinite dilution. The second is that they have to assemble circumferentially around the nascent rod rather than by distal addition. They could probably be exported even before formation of the MS ring, but they would need to remain as monomer until the appropriate point in rod assembly. The unassembled P-ring
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protein would be in the periplasmic space, a hostile environment from the point of view of proteolysis; this is probably why FlgI has a periplasmic chaperone, FlgA,” (Macnab, 2003). 4.5 Hook Assembly
The next stage of the process is assembly of the hook. As stated previously, an elegant molecular mechanism ensures that the hook is 55nm in length (Erhardt et al., 2011; Erhardt et al., 2010; Keener et al., 2010; Waters et al., 2007; Hirano et al., 1994). The two proteins critical for hook-length determination are FliK and FlhB. FliK is critical for both the ability to switch and export filament and the hook-length control. FliK mutants result in polyhooks without filaments, while polyhook-filaments result from second-site suppressor mutations in FlhB (Williams et al., 1996). The hook-length determining protein FliK serves as a “secreted molecular ruler” (Erhardt et al., 2011) inasmuch as it “takes measurements of rod-hook length while being intermittently secreted through the assembly process of the HBB [Hook basal-body] complex and the number of secreted FliK ruler molecules per time it takes to complete the HBB defines the ultimate length of the flagellar hook,” (Erhardt et al., 2010). FliK is responsible for inducing a substrate specificity switch to filament-type substrate secretion upon completion of the hook structure. Moreover, “hook length is measured by secretion of a FliK molecule and hook polymerization will continue until a secreted FliK molecule is in close proximity and provided with sufficient time for a productive interaction with the FlhB component of the type III secretion apparatus at the base of the flagellum to flip a switch in secretion specificity,” (Erhardt et al., 2011). In other words, the N-terminal domain of FliK functions as a molecular sensor and transmitter of information on hook length. When the hook reaches the appropriate length, the information is transmitted to the C-terminal domain, resulting in a conformational change which in turn results in the C-terminus binding to the C-terminus of FlhB. This, in turn, 12
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results in a conformational change in the C-terminus of FlhB, which causes the substrate specificity switch. 4.6 Capping Proteins & Filament Assembly
The capping protein for the hook structure is FlgD (Ohnishi et al., 1994), and this is discarded following hook completion and replaced by hook-associated proteins, namely, the first hook-filament junction protein (FlgK), the second hook-filament junction protein (FlgL), and filament-capping protein (FliD) (Ikeda et al., 1989; Ikeda et al., 1987; Homma et al., 1985). The filament capping protein, FliD, migrates outwards as flagellin monomers are progressively added. The first and second hook-filament junction proteins remain in place and serve to connect the hook to the filament. Of t he capping proteins involved in construction of the rod, hook and filament (FlgJ, FlgD and FliD respectively), only FliD remains at the tip of the filament in the finished product. One study examined, using electron microscopy, the structure of the cap-fil ament complex and isolated cap dimer, reporting that “five leg-like anchor domains of the pentameric cap flexibly adjusted their conformations to keep just one flagellin binding site open, indicating a cap rotation mechanism to promote the flagellin self-assembly. This represents one of the most dynamic movements in protein structures,” (Yonekura et al., 2000).
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Figure 6: Reconstructed three-dimensional image of the flagellar filament capping protein (also known as FliD). Figure credit: Maki-Yonekura et al. (2003)
The cap rests on top of the hollow flagellar filament, and possesses five leg domains that point downwards and insert into cavities at the distal tip of the nascent filament (Maki et al ., 1998). The flagellin subunits (which are exported out the type III system and assemble at the distal-growing end of the filament) form a helical structure and possess 5.5 subunits per turn, and thus 5.5 subunits at the distal end of the growing filament. Since there are only 5 leg-like domains (leading to a symmetry mismatch), this means t hat there is always a small crevice or space at one point between the filament and cap plate. This indentation acts as a folding chamber for the newly-exported flagellin monomers which are added at this site (Yonekura et al., 2000). When a flagellin monomer has been incorporated into this space, the cap complex rotates and moves up, and a new indentation is created adjacent to the previous one that was just occupied (Minamino and Namba, 2004; Maki-Yonekuru et al ., 2003). The rotation rate of the filament cap is approximately 600 rotations per minute, or 10 per second, and 50 flagellin monomers are added per second.
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The hollow channel through which the flagellin monomers travel is extremely narrow -- onl y two nanometers in diameter. Since flagellin subunits have a kink in the middle, they are too large to travel through the tube folded, and thus they pass through the tube unfolded. They are thus unable to fold properly by themselves, and it is the FliD cap that ultimately facilitates the folding, and proper positioning and assembly, of the flagellin monomers. FliD is critical to filament assembly. Without the presence of FliD, the flagellin monomers are lost (Kim et al., 1999). As one paper explained, “A FliD-deficient mutant becomes nonmotile because it lacks flagellar filaments and leaks flagellin monomer out into the medium,” (Ikeda et al., 1996). 4.7 A Regulation Cascade of Flagellar Gene Expression
Bacterial flagellar genes are grouped in clusters on chromosomes. Escherichia coli Salmonella typhimurium require nearly 50 different genes for assembly and function, which are respectively organised across 15 and 17 operons (Komeda et al., 1980; Kutsukake et al., 1988). A regulatory cascade determines the timing of expression of flagellar genes (Chevance and Hughes, 2008; McCarter, 2006; Macnab, 2003; Aldridge and Hughes, 2002; Chilcott and Hughes, 2000). There are three classes of genes involved in this regulatory cascade: Class I genes, class II genes and class III genes, as shown in figure 6 (Kalir et al., 2001).
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Figure 7: Organisation of flagellar genes into three distinct classes. Class I contains only two genes in one operon (called flhD and flhC ). Class II consists of 35 genes across eight operons (including genes involved in the assembly of the hook-basal-body and other components of the flagellum, as well as the export apparatus and two regulatory genes called " fliA" and " flgM "). Those genes which are involved in the synthesis of the filament are controlled by the Class III promoters. Figure credit: Kalir et al. (2001).
At the top of the hierarchy are class I genes ( flhD and flhC ), which form the flhDC master operon. This encodes a transcriptional activator of the class II genes, called FlhD 4C2, a hetero-hexameric complex formed from FlhD and FlhC (Wang et al., 2006). This transcriptional activator is itself under the control of the inhibitor protein FliT, and its corresponding anti-regulator, the flagellar filament cap protein, FliD (Aldridge et al., 2010). There are 35 class II genes, spread across eight different operons. These includes genes involved in assembly of the hook-basal body and other flagellar components, in addition to the export apparatus and two regulatory genes, fliA (which codes for a sigma factor called σ28) (Ohnishi et al.,1990) and flgM (which codes for the anti-sigma factor) (Kutsukake et al.,1994; Ohnishi et al., 1992). We shall return to sigma factors and anti-sigma factors 16
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shortly. Expression of the flagellar genes is controlled b y a corresponding set of promoters – class I, class II and class III. Promoters are akin to a kind of molecular toggle switch which can initiate gene expression when recognised by RNA polymerase and an ass ociated specialised protein called a “sigma factor.” The enteric master regulator FlhD4C2 is responsible for turning on the class II promoters in association with a sigma factor, σ70. Mutations in this master regulator result in the shutting down of the entire regulon. The class II promoters ar e then responsible for the gene expression of the hook-basal-body subunits and its regulators, including the sigma factor σ28 (encoded by fliA) and its anti-sigma factor, flgM (anti-sigma factors, as their name suggests, bind to sigma factors to inhibit their transcriptional activity). σ28 is required to activate the class III promoters (Ohnishi et al.,1990). But here we potentially run into a problem. It makes no sense to assemble the flagellin monomers before completion of the hook-basal-body construction. Thus, in order to inhibit σ28, the anti-sigma factor FlgM prohibits it from interacting with the RNA polymerase holoenzyme complex (Saini et al., 2011; Kutsukake et al., 1994; Ohnishi et al., 1992). Upon completion of the hook-basal-body, the anti-sigma factor FlgM is secreted through the flagellar structures that are produced by the expressi on of the class II hook-basal-body genes. The class I II promoters (which are responsible for the expression of flagellin monomers, the chemotaxis system and the motorforce generators) are then activated by σ28 and the flagellum can be completed. There is, of course, variation on this flagellar setup from species to species. Indeed, “these systems differ from each other by the existence of specific sigma factors and transcriptional activators, by motive force and the efficiency of motors,” (Soutourina and Bertin, 2003). Figure 7 (Soutourina and Bertin, 2003), compares lateral (Enterobacteriaceae famil y) and polar (Pseudomonadaceae, Vibrionaceae families) flagellation cascades.
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Figure 8: Comparison of lateral (Enterobacteriaceae family) and polar (Pseudomonadaceae, Vibrionaceae families) flagellation cascades. Figure credit: Soutourina and Bertin (2003).
5. Chemotaxis & Signal Transduction There is a significant amount of variation on chemotaxis systems. The pathway is best understood in the Gammaproteobacteria: a class of bacteria which includes Escherichia coli (the most extensively studied example) and Salmonella enterica. It is this class to which the following discussion will pertain. A substantially less amount of data is available for other species of bacteria. Although individual genes bear homology to their Gammaproteobacteria counterparts, the pathways are somewhat mechanistically different. Furthermore, while general features of excitation remain conserved among bacteria and archaea, many of the 18
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more specific features are fairly diverse. One considerably more complex system than that of Escherichia coli is that of Bacillus subtilis (Rao et al., 2004; Bischoff and Ordal, 1992). Before we can properly appreciate the details and technicalities of this system, it is necessary to take a step back and understand the foundational principles upon which it is based. Bacteria are able to move towards a food source, such as glucose, by a process known as "chemotaxis." A requisite for this process to work is the ability of the bacterial flagellar motor to literally shift gears so that it switches from spinning counter-clockwise to rotating clockwise (Larsen et al., 1974). This change in rotation is brought about in response to chemical stimuli from the cell's exterior. These chemical signals are detected by a twocomponent signal transduction circuit that operates to induce the switch in flagellar rotation (Wadhams and Armitage, 2004; Falke et al., 1997). In general, a two-component regulatory system comprises an integral membrane protein known as a "histidine protein kinase," and a cytoplasmic protein known as a "res ponse regulator,” as shown in f igure 8.
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Figure 9: A diagrammatic representation of a two-component regulatory system.
The histidine protein kinase has two domains: an input domain and a transmitter domain (Mascher et al., 2006; Wolanin et al., 2002). The former is located on the outside of the cell, and is ideally situated to detect incoming environmental signals. The latter is situated on the cytoplasmic face of the cell membrane, and is positioned such that it can interact wit h the response regulator. An external stimulus causes a conformational change in the histidine protein kinase. This causes the transfer of phosphoryl groups (autophosphorylation) from ATP to a conserved histidine residue. This phosopho-group is then transferred to an aspartate residue of the response regulator. This enables the response regulat or to bind to the DNA in order to regulate the transcription of its target genes. What I have thus far described represents a very basic two-component regulatory system. It was, however, necessary to look at the system in principle before we describe its application 20
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in the case of chemotaxis. It is to the latter that I now turn. Readers may find it helpful to refer to figure 9 while reading the descriptions that follow.
Figure 10: A diagrammatic representation of the mechanisms underlying bacterial chemotaxis.
How do bacteria detect a chemical gradient? The answer lies in a certain class of transmembrane receptors called methyl-accepting chemotaxis proteins (hereafter, MCPs). Different MCPs can detect different types of molecules, and are able to bind attractants or repellents (Kehrys and Dahlquistg, 1982). These receptors then communicate with -- and activate -- the so-called "Che proteins" (Grebe and Stock, 1998). Proteins called CheA and CheW are bound to the receptor (McNall y and Matsumura, 1991; Conley et al., 1989). The former is the histidine kinase for this system. Upon activation of the receptor, the CheA's conserved histidine residue undergoes autophosphorylation (Shi et al., 2011; Zhang et al., 2005). There are two response regulators, called CheB and CheY. There is a transfer of a phosphoryl group to their conserved aspartate residue from CheA (Li et al., 1995). CheY subsequently interacts with the flagellar switch protein call ed FliM (Bren and Eisenbach, 1998). This induces the switching in flagellar direction from counter-clockwise to clockwise.
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This clockwise rotation upsets the entire flagella bundle and causes it to break up. The result is that the bacterium "tumbles" (Larsen et al., 1974). This means that bacteria are able to redirect their course and repeatedly re-evaluate and adjust their bearings in response to environmental stimuli such as food or poisons. When the other response regulator, CheB, is activated by the histidine kinas e CheA, it operates as a methylesterase (Stewart and Dahlquist, 1988). This means that it actively removes methyl groups from glutamate residues on the receptor's cytoplasmic surface. Meanwhile, another protein (called CheR) actively adds methyl residues to these same glutamate residues: that is to say, it works as a methyltransferase (Springer and Koshland, 1977). At this point the engineering shows a stroke of genius. If the stimulus is at a high level, there will be a corresponding decline in the level of phosphorylation of the CheA protein: and, as a consequence, of the response regulators CheY and CheB as well. Remember that the role of CheB is to remove methyl groups from glutamate residues on the receptor's cytoplasmic surface. But now, phosphorylated CheB is not available and so this task is not performed. The degree of methylation of the MCPs will thus be raised. When the MCPs are fully methylated, the cell will swim continuously because the MCPs are no longer responsive to the stimuli. This entails that the level of phosphorylated CheA and CheB will increase even when the level of attractant remains high and the cell will commence the process of tumbling. But now, the phosphorylated CheB is able to demethylate the MCPs, and the receptors are again able to respond to the attracting chemical signals. In the case of repellents, the situation is similar -except that it is the least methylated MCPs which respond least while the f ully methylated ones respond most. This kind of regulation also means that the bacterium has a memory system for chemical concentrations from the recent past and compares them to its currently
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receiving signals. It can thus detect whether it is moving towards or away from a chemical stimulus. Chemotaxis pathways vary among bacteria. A comparative study of chemotaxis systems in Escherichia coli and Bacillus subtilis reported that “ E. coli and B. subtilis bias their motion towards favorable conditions with nearly identical behavior by adjusting the frequency of straight runs and reorienting tumbles. Both pathways share five orthologous proteins with apparently identical biochemistry. How these individual orthologs contribute to the overall function, however, is different, as illustrated when s ynonymous orthologs are deleted in each organism. Deletion of the CheY response regulator causes E. coli to run exclusively and B. subtilis to tumble exclusively. When the CheR methyltransferase is deleted in E. coli, the cells are incapable of tumbles and only run. Likewise, when the CheB methylesterase is deleted, E. coli cells are incapable of runs and only tumble. In B. subtilis, cells still run and tumble when either CheB or CheR is deleted, though they no longer precisely adapt. Remarkably, both genes complement in the heterologous host. Deletion of the CheW adaptor protein in E. coli results in a run-only phenotype, whereas there is no change in phenotype for the synonymous deletion in B. subtilis. When the genes involved in regulating methylation are deleted (cheBR in E. coli and cheBCDR in B. subtilis), E. coli does not adapt, whereas B. subtilis either oscillates or partially adapts when exposed to attractants. These differences demonstrate that the pathways are different even though they involve homologous proteins,” [internal citations omitted] (Rao et al., 2004).
6. The Evolution of the Bacterial Flagellum Many of the sub-components found within the flagellar structure are known to be homologous to other bacterial organelles. For example, the stator proteins MotA and MotB are homologs of ExbB and ExbD, which form part of the TonB-dependent active transport 23
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system and which serve to energize transport of vitamin B12, and iron-chelating compounds called siderophores, across the outer membrane of Gram-negative bacteria. ExbB/D and MotA/B are also known to be homologous to TolQ/R, which play an important role in the maintenance of outer membrane stability. The energizing of these s ystems by proton movement across the inner membrane resembles the setup of the flagellum's stator, which couples proton transport across the inner membrane to the motor's rotati on. The rotor protein FliG is also homologous to the magnesium transporter MgtE. The sequence identities of FliG and MgtE, however, appear to be rather weak (<20% as reported by PSI-BLAST), although there is clear similarity to the N-terminal domain of MgtE. The most common response to the claim that the bacterial flagellum manifests irreducible complexity has been to point to the type III secreti on system (T3SS), a needle-like syringe used by certain bacteria (e.g. the archetype for this system Yersinia pestis) to inject toxins into organisms, as a possible evolutionary predecessor. There are a number of problems, however, with this hypothesis. For one thing, it sidesteps the need to also e xplain the components of the type III export machinery (including FlhA, FlhB, FliR, FliQ, FliP, FliI etc.), at least most of which are essential for its function. Indeed, one study “examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex,” finding that all six of the Type III secretion system components homologous to those listed above are required for the system’s function (Sukhan et al., 2001). One of the purposes of offering this description in such detail is to reveal the futility of mere appeals to biochemical homology of flagellar proteins to proteins involved in other cellular functions (Pallen and Matzke, 2006). Indeed, homology does nothing to demonstrate that the necessary transitions are evolutionarily feasible (Gauger and Axe, 2011), and it has been shown that the process of gene duplication and recruitment, as a source of evolutionary 24
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novelty, is extremely limited: If a duplicated gene has a slightly negative fitness cost, the maximum number of non-adaptive point mutations that a new innovation in a bacterial population can require is two or fewer; this number jumps to six or fewer if the duplication is selectively neutral (Axe, 2010). A further point that is worth noting is that certain irreducibly complex subsystems in flagella are also found in other organelles, and perform essentiall y identical roles. Pointing to such homologies, therefore, only succeeds in pushing the problem back a stage -- at some point, these systems require explanation. Take, for example, the proteins FliK and FlhB, which function in hook-length determination. FliK and FlhB are homologous to YscP and YScU respectively, which also regulate substrate specificity and needle-length of the type III secretion system in Yersinia (Wood et al., 2008; Edqvist et al., 2003). Furthermore, although there is room for debate, the present author is persuaded that the type III secretion injectisome evolved from the flagellar subunit exporters, largely based on, among other things, two key observations: 1. While type III injectisomes are found only in gram-negative ba cteria that possess both an inner and outer membrane, flagella are found also i n gram-positive bacteria that possess only a single membrane: Flagella are thus more widely distributed. 2. Multi-cellular organisms on which type III injectisomes are used emerged on the scene much later than bacteria, so evolutionary pressure for such an organelle would proceed the evolutionary pressure for motility. For a more thorough discussion of this subject, I refer readers to Abby and Rocha (2012), Saier et al. (2004), and Nguyen et al. (2000).
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Moreover, there are a number of flagellar components that are presently not known to have homologs in non-flagellar systems. Examples include the rod cap FlgJ, the L and P ring proteins FlgH and FlgI, the MS ring-rod junction protein FliE, the filament capping protein FliD, and the anti-sigma factor FlgM. A number of these components form part of irreducibly complex subsystems of the flagellum. One study critiques the feasibility of the flagellum’s evolution from the T3SS, reporting that “the potential for cross-recognition between type III exported proteins of different s ystems in the same cell carries several implications. First, these observations explain why segregation of these systems by specific environmental cues is necessary. For example, expression of a flagellum under host conditions would result in loss of polarized secretion of Yop proteins into target host cells. Additionally, display of flagellin to ma crophages by direct injection via the Ysc secretin would countermand the anti-inflammatory strat egy used by the Yersinia. Flagellin is a potent cytokine inducer. Further, because flagellin expression is controlled by such high expression promoters, it also suggests that flagellin, if expressed, may competitively interfere with virulence protein secr etion. Indeed, this latter suggestion may explain why an important subset of major human pathogens, including Y. pestis, Shigella spp., Bordetella pertussis and recent isolates of E. coli 0157:H7, have lost flagellar biosynthetic capacity altogether, even though they have the requisite flagellar genes,” [internal citation omitted] (Meyer and Minnich, 2004). Even in the event that it was somehow feasible to evolve the flagellar export apparatus and basal body by evolution, there is the problem of producing the filament. The filament cap needs to be of very specific structure in order to interact with, and direct the assembly of, the flagellin monomers that are exported. It is also crucial for flagellar assembly and one of the very last proteins to be added. How did a protein of such specific structure evolve and how long would it have taken? Surely, a filament capping protein (which serves no other purpose 26
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within the cell as far as we know) is of no use without the exporting of the flagellin monomers whose assembly it facilitates. But without the presence of such a capping protein, the flagellin monomers are of no value -- since they are lost into the medium and do not assemble into a filament. Moreover, it is essential that only a single filament capping protein, but many specifically structured flagellin monomers (a filament is typically comprised of around 20,000 monomers), be exported. It is also essential that their respec tive export be in the correct order -- FliD must be exported before the export of flagellin subunits. How long did it take for such a complex system to evolve? In any event, leaving aside the fact that the flagellar filament is assembled with the assistance of an essential capping protein encoded by fliD, the exported flagellin monomers need to stick both to each other and to the export machinery’s outer components (so that they are not lost from the cell into the surrounding medium). The specific and co-ordinated mutations required to facilitate such an innovation are likely to be well beyond the reach of a Darwinian process. Perhaps one could argue that FliD and the filament proteins (FliC), in addition to the rod and hook proteins, evolved from an ancestral adhesive pilin secreted from the primitive type III secretion system. But such a hypothesis only pushes the need for explanation back a step -- at some point, the elegant interaction between FliD and the flagellin monomers still needs to be accounted for. The motor itself exhibits irreducible complexity, and is dependent on the critical proteins FliG, MotA, MotB and FliM. Remove any one of those proteins and the motor will completely cease to function. Inducing mutations in FliG, for example, yields “a non-motile, or Mot-, phenotype, in which flagella are assembled but do not rotate ,” (Lloyd and Blair, 1997). A study that conducted a “deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium” found that “deletions at the N-terminus produced a counterclockwise switch bias, deletions in the cent ral region of the protein produced poorly 27
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motile or nonflagellate cells, and deletions near the C-terminus produced only nonflagellate cells,” (Toker et al.,1996). Many more examples could be given. But the bottom line is this: The bacterial flagellum exhibits remarkable design, and irreducible complexity at ever y tie. When so much of the assembly process and functional operation of the flagellum appears to resist explanation in evolutionary terms, perhaps it is time to lay aside such a paradigm and begin the search for alternatives.
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Blair, K. M., Turner, L., Winkelman, J. T., Berg, H. C., & Kearns, D. B. (2008). A Molecular Clutch Disables Flagella in the Bacillus subtilis Biofilm. Science, 320(5883), 16361638. Bourret, R. B., & Stock, A. M. (2002). Molecular Information Processing: Lessons from Bacterial Chemotaxis. Biochemistry, 277 (12), 2002. Bren, A., & Eisenbach, M. (2000). How Signals Are Heard during Bacterial Chemotaxis: Protein-Protein Interactions in Sensory Signal Propagation. Journal of Bacteriology, 182(24), 6865-6873. Bren, A., & Eisenbach, M. (1998). The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY. Journal of Molecular Biology, 278(3), 507-14. Chevance, F. F. V., & Hughes, K. T. (2008). Coordinating assembly of a bacterial macromolecular machine. Nature reviews. Microbiology, 6 , 455-465. Chilcott, G. S., & Hughes, K. T. (2000). Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiology and molecular biology reviews : MMBR, 64(4), 694 – 708. Conley, M. P., Wolfe, A. J., Blair, D. F., & Berg, H. C. (1989). Both CheA and CheW Are Required for Reconstitution of Chemotactic Signaling in Escherichia coli. Journal of Bacteriology, 171(9), 590-593. Dijkstra, A., & Keck, W. (1996). Peptidoglycan as a Barrier to Transenvelope Transport. Journal of Bacteriology, 178(19), 5555-5562. Edqvist, P. J., Olsson, J., Lavander, M., Sundberg, L., Wolf-watz, H., & Lloyd, S. A. (2003). YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System. Journal of Bacteriology, 185(7), 2259 – 2266. Erhardt, M., Hirano, T., Su, Y., Paul, K., Wee, D. H., Mizuno, S., Aizawa, & Hughes, K. T. (2010). The role of the FliK molecular ruler in hook-length control in Salmonella enterica. Molecular Microbiology, 75(5), 1272-1284. Erhardt, M., Singer, H. M., Wee, D. H., Keener, J. P., & Hughes, K. T. (2011). An infrequent molecular ruler controls flagellar hook length in Salmonella enterica. The EMBO Journal , 30, 2948-2961. Falke, J. J., Bass, R. B., Butler, S. L., Chervitz, S. A., & Danielson, M. A. (1997). THE TWO-COMPONENT SIGNALING PATHWAY OF BACTERIAL CHEMOTAXIS: A Molecular View of Signal Transduction by Receptors, Kinases, and Adaptation Enzymes. Annu Rev Cell Dev Biol., 13, 457-512. Fein, J. E. (1979). Possible Involvement of Bacterial Autolytic Enzymes in Flagellar Morphogenesis. Journal of Bacteriology, 137 (2), 933-946.
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Gauger, A. K., & Axe, D. D. (2011). The Evolutionary Accessibility of New Enzyme Functions : A Case Study from the Biotin Pathway. Bio-Complexity, 2011(1), 1-17. Grebe, T. W., & Stock, J. (1998). Bacterial chemotaxis: The five sensors of a bacterium. Current Biology, 8, 154-157. Guttenplan, S. B., Blair, K. M., & Kearns, D. B. (2010). The EpsE Flagellar Clutch Is Bifunctional and Synergizes with EPS Biosynthesis to Promote Bacillus subtilis Biofilm Formation. PLoS Genetics, 6 (12), 1-12. Hirano, T., Minamino, T., Namba, K., & Macnab, R. M. (2003). Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export. Journal of Bacteriology, 185(8), 2485-2492. Hirano, T., Minamino, T., & Macnab, R. M. (2001). The Role in Flagellar Rod Assembly of the N-terminal Domain of Salmonella FlgJ , a Flagellum- specific Muramidase. Journal of Molecular Biology, 359-369. Hirano, T., Yamaguchi, S., Oosawa, K., & Aizawa, S. (1994). Roles of FliK and FlhB in Determination of Flagellar Hook Length in Salmonella typhimunum. Journal of Bacteriology, 176 (17), 5439-5449. Homma, M., Komeda, Y., Iino, T., & Macnab, R. M. (1987). The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export. Journal of Bacteriology, 169(4), 1493-1498. Homma, M., Kutsukake, K., Hasebe, M., Iino, T., & Macnab, R. M. (1990). FlgB , FlgC , FlgF and FlgG A Family of Structurally Related Proteins in the Flagellar Basal Body of Salmonella typhimurium. Journal of Molecular Biology, 215(2), 465-477. Homma, M., Kutsukake, K., & Iino, T. (1985). Structural Genes for Flagellar HookAssociated Proteins in Salmonella typhimurium. Journal of Bacteriology, 163(2), 464471. Ikeda, T., Asakura, S., & Kamiya, R. (1989). Total Reconstitution of Salmonella Flagellar Filaments from Hook and Purified Flagellin and Hook-associated Proteins in Vitro. Journal of Molecular Biology, 209, 109-114. Ikeda, T., Homma, M., Iino, T., Asakura, S., & Kamiya, R. (1987). Localization and Stoichiometry of Hook-Associated Proteins within Salmonella typhimurium Flagella. Journal of Bacteriology, 169(3), 1168-1173. Ikeda, T., Oosawa, K., & Hotani, H. (1996). Self-assembly of the Filament Capping Protein , FliD , of Bacterial Flagella into an Annular Structure. Journal of Molecular Biology, 259, 679-686. Imae, Y., & Atsumi, T. (1989). Na+-driven bacterial flagellar motors. J Bioenerg Biomembr., 21(6), 705-16.
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Jones, C. J., Homma, M., & Macnab, R. M. (1989). L- , P- , and M-Ring Proteins of the Flagellar Basal Body of Salmonella typhimurium : Gene Sequences and Deduced Protein Sequences. Journal of Bacteriology, 171(7), 3890-3900. Jones, C. J., & Macnab, R. M. (1990a). Flagellar Assembly in Salmonella typhimurium : Analysis with Temperature-Sensitive Mutants. Journal of Bacteriology, 172(3), 13271339. Jones, C. J., & Macnab, R. M. (1990b). Stoichiometric Analysis of the Flagellar Hook(Basal-Body) Complex of Salmonella typhimurium. Journal of Molecular Biology, 212, 377-387. Kalir, S., McClure, J., Pabbaraju, K., Southward, C., Ronen, M., Leibler, S., Surette, M. G., et al. (2001). Ordering Genes in a Flagella Pathway by Analysis of Expression. Science, 292(5524), 2080-2083. Keener, J. P. (2010). A molecular ruler mechanism for length control of extended protein structures in bacteria. Journal of Theoretical Biology, 263(4), 481-489. Kehrys, M. R., & Dahlquistg, F. W. (1982). The Methyl-accepting Chemotaxis Proteins of Escherichia coli. The Journal of Biological Chemistry, 257 (17), 10378-10386. Kihara, M. A. Y., Minamino, T., Yamaguchi, S., & Macnab, R. M. (2001). Intergenic Suppression between the Flagellar MS Ring Protein FliF of Salmonella and FlhA , a Membrane Component of Its Export Apparatus. Journal of Bacteriology, 183(5), 16551662. Kim, J. S., Chang, J. H., Chung, S. I., & Yum, J. S. (1999). Molecular Cloning and Characterization of the Helicobacter pylori fliD Gene , an Essential Factor in Flagellar Structure and Motility. Society, 181(22), 6969-6976. Kubori, T., Shimamoto, N., Yamaguchi, S., Namba, K., & Aizawa, S. (1992). Morphological Pathway of Flagellar Assembly in Salmonella typhimurium. Journal of Molecular Biology, 226 , 433-446. Kutsukake, K., Iyoda, S., Ohnishi, K., & Iino, T. ( 1994). Genetic and molecular analyses of the interaction between the flagellum-specific sigma and anti-sigma factors in Salmonella typhimurium. EMBO Journal , 13(19), 4568-4576. Kutsukake K, Ohya Y, Yamaguchi S, I. T. (1988). Operon structure of flagellar genes in Salmonella typhimurium. Mol Gen Genet., 214(1), 11-15. Larsen, S. H., Reader, R. W., Kort, E. N., Tso, W.-W., & Adler, J. (1974). Change in direction of flagellar rotation is the basis of the chemotactic response in Escherichia coli. Nature, 249, 74-77. Lemon, K. P., Earl, A. M., Vlamakis, H. C., Aguilar, C., & Kolter, R. (2008). Biofilm Development with an Emphasis on Bacillus subtilis. Curr Top Microbiol Immunol., 322, 1-16.
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Li, J., Swanson, R., Simon, M., & Weis, R. (1995). The response regulators CheB and CheY exhibit competitive binding to the kinase CheA. Biochemistry, 34(45), 14626-36. Lloyd, S. A., & Blair, D. F. (1997). Charged Residues of the Rotor Protein FliG Essential for Torque Generation in the Flagellar Motor of Escherichia coli. Journal of Molecular Biology, 266 , 733-744. Macnab, R. M. (2003). How Bacteria Assemble Flagella. Annual Review of Microbiology, 57 , 77-100. Macnab, R. M. (2004). Type III flagellar protein export and flagellar assem bly. Biochimica et Biophysica Acta, 1694, 207 - 217. Maki, S., Vonderviszt, F., Furukawa, Y., Imada, K., & Namba, K. (1998). Plugging interactions of HAP2 pentamer into the distal end of flagellar filament revealed by electron microscopy. Journal of Molecular Biology, 277 (4), 771 – 777. Maki-yonekura, S., Yonekura, K., & Namba, K. (2003). Domain movements of HAP2 in the cap – filament complex formation and growth process of the. Proceedings of the National Academy of Sciences, 100(26), 3 – 4. Manson, M. D., Tedesco, P. A. T., Berg, H. C., Haroldt, F. M., & Van der Drift, C. (1977). A protonmotive force drives bacterial flagella. Proceedings of the National Academy of Sciences, 74(7), 3060-3064. Mascher, T., Helmann, J. D., & Unden, G. (2006). Stimulus Perception in Bacterial SignalTransducing Histidine Kinases. Microbiology and Molecular Biology Reviews, 70(4), 910-938. McAuley, M. T., Khalil, R., Stockton, D. J., & Schilstra, M. J. Modeling the Flagellar Motor Assembly Process to Help Improve Industrial Operations Systems Desi gn. Retrieved September 22, 2012, from http://homepages.stca.herts.ac.uk/~erdqmjs/pdf/FlagellarMotorAssembly.pdf McCarter, L. L. (2006). Regulation of flagella. Current Opinion in Microbiology, 9(2), 180 – 6. Mcnally, D. F., & Matsumura, P. (1991). Bacterial chemotaxis signaling complexes : Formation of a CheA / CheW complex enhances autophosphorylation and affinity for CheY. Proceedings of the National Academy of Sciences, 88, 6269-6273. Meister, M., Lowe, G., & Berg, H. C. (1987). The Proton Flux through the Bacterial Flagellar Motor. Cell , 49, 643-650. Minnich, S. A., & Meyer, S. C. (2004). Genetic analysis of coordinate flagellar and type III regulatory circuits in pathogenic bacteria. (M. W. C. and C. A. Brebbia, Ed.) Proceedings of the Second International Conference on Design & Nature, Rhodes, Greece. WIT Press.
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Minamino, T., & Macnab, R. M. (1999). Components of the Salmonella Flagellar Export Apparatus and Classification of Export Substrates. Journal of Bacteriology, 181(5), 1388-1394. Minamino, T., & Namba, K. (2004). Self-Assembly and Type III Protein Export of the Bacterial Flagellum. Journal of Molecular Microbiology and Biotechnology, 7 , 5 – 17. Mori, H., & Ito, K. (2001). The Sec protein-translocation pathway. Trends in Microbiology, 9(10), 494-500. Muller, V., Jones, C. J., Kawagishi, I., Aizawa, S.-ichi, & Macnabl, R. M. (1992). Characterization of the fliE Genes of Escherichia coli and Salmonella typhimurium and Identification of the FliE Protein as a Component of the Flagellar Hook-Basal Body Complex. Journal of Bacteriology, 174(7), 2298-2304. Nambu, T., Minamino, T., Macnab, R. M., & Kutsukake, K. (1999). PeptidoglycanHydrolyzing Activity of the FlgJ Protein , Essential for Flagellar Rod Formation in Salmonella typhimurium. Society, 181(5), 1555-1561. Nguyen, L., Paulsen, I. T., Tchieu, J., Hueck, C. J., & Saier, M. H. (2000). Phylogenetic Analyses of the Constituents of Type III Protein Secretion Systems. J. Mol. Microbiol. Biotechnol., 2(2), 125-144. Ohnishi, K., Kutsukake, K., Suzuki, H., & Lino, T. (1992). A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an anti-sigma factor inhibits the activity of the flagellum-specific Sigma factor, σF. Molecular Microbiology, 6 (21), 3149-3157. Ohnishi, K., Kutsukake, K., Suzuki, H., & Iino, T. (1990). Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium. Mol Gen Genet., 221(2), 139-147. Ohnishi, K., Ohto, Y., Aizawa, S.-ichi, Macnab, R. M., & Iino, T. (1994). FlgD Is a Scaffolding Protein Needed for Flagellar Hook Assembly in Salmonella typhimurium. Journal of Bacteriology, 176 (8), 2272-2281. Pallen, M. J., & Matzke, N. J. (2006). From The Origin of Species to the origin of bacterial flagella. Nature Reviews Microbiology, 4, 784-790. Rao, C. V., Kirby, J. R., & Arkin, A. P. (2004). Design and Diversity in Bacterial Chemotaxis : A Comparative Study in Escherichia coli and Bacillus subtilis. PLoS Biology, 2(2), 239-252. Saier, M. H. (2004). Evolution of bacterial type III protein secretion s ystems. Trends in Microbiology, 12(3), 113 – 5. Saini, S., Floess, E., Aldridge, C., Brown, J., Aldridge, P. D., & Rao, C. V. (2011). Continuous control of flagellar gene expression by the σ28-FlgM regulatory circuit in Salmonella enterica. Molecular microbiology, 79(1), 264 – 78.
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Shi, T., Lu, Y., Liu, X., Chen, Y., Jiang, H., & Zhang, J. (2011). Mechanism for the Autophosphorylation of CheA Histidine Kinase: QM/MM Calculations. The Journal of Physical Chemistry, 115(41), 11895 – 11901. Soutourina, O. A., & Bertin, P. N. (2003). Regulation cascade of flagellar expression in Gram-negative bacteria. FEMS Microbiology Reviews, 27 , 505-523. Springer, W. R., & Koshland, D. E. (1977). Identification of a protein methyltransferase as the cheR gene product in the bacterial sensing system. Proceedings of the National Academy of Sciences, 74(2), 533-53. Stewart, R. C., & Dahlquist, F. W. (1988). N-terminal half of CheB is involved in methylesterase response to negative chemotactic stimuli in Escherichia coli. Journal of Bacteriology, 170(12), 5728-5738. Sukhan, A., Kubori, T., & Wilson, J. (2001). Genetic Analysis of Assembly of the Salmonella enterica Serovar Typhimurium Type III Secretion-Associated Needle Complex. Journal of Bacteriology, 183(4), 1159-1167. Toker, A. S., Kihara, M. A. Y., & Macnab, R. M. (1996). Deletion Analysis of the FliM Flagellar Switch Protein of Salmonella typhimurium. Journal of Bacteriology, 178(24), 7069-7079. Vogler, A. P., Homma, M., Irikura, V. M., & Macnab, R. M. (1991). Salmonella typhimurium Mutants Defective in Flagellar Filament Regrowth and Sequence Similarity of Fliu to F0F1 , Vacuolar , and Archaebacterial ATPase Subunits. Journal of Bacteriology, 173(11), 3564-3572. Wadhams, G. H., & Armitage, J. P. (2004). Making sense of it all: bacterial chemotaxis. Nature Reviews Molecular Cell Biology, 5, 1024-1037. Wang, S., Fleming, R. T., Westbrook, E. M., Matsumura, P., & McKay, D. B. (2006). Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription. Journal of Molecular Biology, 355(4), 798 – 808. Waters, R. C., & Toole, P. W. O. (2007). The FliK protein and flagellar hook-length control. Protein Science, 16 (5), 769-780. Williams, A. W., Yamaguchi, S., Togashi, F., Aizawa, S.-ichi, Kawagishi, I., & Macnab, R. M. (1996). Mutations in fliK and flhB Affecting Flagellar Hook and Filament Assembly in Salmonella typhimurium. Journal of Bacteriology, 178(10), 2960-2970. Wolanin, P. M., Thomason, P. A., & Stock, J. B. (2002). Histidine protein kinases : key signal transducers outside the animal kingdom. Genome Biology Review, 3(10), 1-8. Wood, S. E., Jin, J., & Lloyd, S. a. (2008). YscP and YscU switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein YscI. Journal of bacteriology, 190(12), 4252 – 62.
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