Jonathan McLatchie

The Bacterial Flagellum: A Motorized Nanomachine

Jonathan McLatchie (B.Sc, M.Res) September 2012 Last updated: 3/9/2013

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Jonathan McLatchie

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 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. 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 al., 2006 Bourret and Stock, 2002; Bren and Eisenbach, 2000). The most extensively studied chemotaxis system belongs to Escherichia 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. 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 an awe-inspiring feeling from its ‘divine’ beauty,” (Aizawa, 2009).

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Jonathan McLatchie

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.). The mechanistic basis of flagellar assembly is so breathtakingly elegant and mesmerising that the sheer engineering brilliance of the flagellar 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 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 details of its assembly 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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the tube-like structure that provides rotary motion) is embedded in the inner cell membrane. 2011.e. and rotates within the stator.. measuring roughly 15µm in length and 15nm in diameter (Berg et al. Each subunit contributes a small propeller blade to the outside of the structure. 2010. Flagellar Structure A simplified diagram of flagellar structure is given in figure 1. 2006). The rotor passes through the membrane to the outside of the cell. The hook is required to be 55nm in length. and attaches to the flagellar filament via the hook (universal joint). The flagellar motor contains as many as 40 distinct types of proteins. which form a helical structure with a hollow core.. 4 . As can be seen from figure 1.. Figure 1: Simplified diagrammatic representation of gram negative bacterial flagellar structure. Erhardt et al. Flagella are polymers comprised of protein subunits (molecular weight 53kd). and this is controlled by an elegant molecular mechanism (Erhardt et al. called flagellin. the rotor (i.Jonathan McLatchie 2.

surround the inner ring and 5 . by contrast. called Mot proteins (MotA and MotB). rod. The MS (membrane and supermembrane) ring is formed by the proteins FliG.Jonathan McLatchie Keener et al. as shown in figure 1. A series of proteins. Hirano et al. A third set of rings (the MS and C rings) are respectively found within the cytoplasmic membrane and the cytoplasm. 2007. 2010. and L. FliM. 1994). Figure 2: Diagrammatic representation of bacterial flagellar hook (universal joint) and filament (propeller). and thus only possess the inner pair of rings. In gram-negative bacteria. an outer ring (the L ring) is anchored in the lipopolysaccharide layer. Waters et al. the C ring is comprised of the proteins FliN and FliM. Another ring (the P ring) is anchored in the peptidoglycan layer of the cell wall.and P. and FliN. Gram positive bacteria. do not have an outer membrane. a fact that we shall return to when we discuss assembly...rings.. and this is comprised of the MS ring. The part of the flagellum embedded in the cell wall is known as the basal body. The hook structure is shown in figure 2.

Moreover. Protons are transported across the cell membrane by the rotor.. are collectively referred to as the “basal body”. C and MS rings. and this process drives the rotor’s rotation. MotB possesses a single transmembrane helix and a periplasmic domain. FliG combines with the MotA-MotB pair to form a proton channel. Approximately 11 MotA-MotB pairs create a ring structure around the flagellar base. The structures of the L. Manson et al. 3. Figure 3: Electron micrograph of flagellar hook-basal-body (HBB). An electron micrograph of the flagellar basal body is given in figure 3. MotA possesses four transmembrane helices and a cytoplasmic domain. 1977). P. In this 6 .. “[e]ach MotA-MotB pair is conjectured to form a structure that has two half-channels. 1987. FliG serves as the rotating proton carrier. A Proton Motive Force Drives the Flagellar Motor The flagellar motor is driven by a proton gradient across the plasma membrane (Meister et al. in combination with the central rod (the motor that turns the flagellum). perhaps with the participation of some of the charged residues identified in crystallographic studies.Jonathan McLatchie are anchored in the cytoplasmic membrane.

This raises the question of how this inhibition of motility is achieved. which is carried on the same operon as the genes necessary for EPS formation. The protein responsible for polymerising into the rotor is called FliG. the analogue to the clutch of a car is a protein called EpsE (Blair et al. 7 . Some organisms (certain alkalophilic and marine bacteria such as Bacillus and Vibrio species) utilise sodium ions instead (Imae and Atsumi. EpsE makes contact with the flagellum’s rotor. rotating the flagellum with it and allowing the proton to pass into the inner half-channel and into the cell. In the flagella of Bacillus subtilis. the FliG subunits also convert the proton flux energy into the flagellum’s rotational energy. A biofilm is an antibiotic-resistant aggregate of bacteria.. The MS ring rotates. 1989). causing it to bend such that the rotor is disengaged from the protonpowered engine of the flagellum.” (Guttenplan et al. embedded within a matrix of extracellular polymeric substances (EPS) such as proteins and polysaccharides.” (Berg et al. 2006). a proton from the periplasmic space passes into the outer half-channel and is transferred to an [sic] FliG subunit. which ensures that the engine and gears are disengaged. in which microorganisms adhere to one another. As stated above. bacterial motility is switched off. 2008). for it “interacts with the flagella rotor to inhibit motility and also cooperates with other enzymes to synthesise the EPS matrix. When EpsE interacts with FliG.. it triggers a conformational change. Bacillus subtilis is a micro-organism capable of biofilm formation (Lemon et al.. 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.Jonathan McLatchie scenario. 2008).. In a biofilm. EpsE is bifunctional. 2010).

Figure 4: Diagrammatic representation of the function of the molecular flagellar clutch in Bacillus subtilis.Jonathan McLatchie A diagrammatic illustration of the function of this molecular clutch is given in figure 4. 8 .

This involves the integration of the cytoplasmic membrane proteins into the membrane by the Sec pathway (for a review of the Sec pathway. 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. 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. 9 . 1990a). It is thought that the export and MS ring proteins assemble in a coordinated way.Jonathan McLatchie 4. 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.. see Mori and Ito. Flagellar Assembly Readers may find it helpful to refer to figure 5 when reading the description that follows. 2001). Figure 5: Schematic diagram of the location of proteins involved in flagellar assembly and function.

The addition of these subunits is often controlled by a capping structure. is given in three stages: “First. and these proteins are added at the distal end by diffusion down the Type III export pathway’s central narrow channel (Macnab. 2003). which is important for rod formation (Nambu et al. including export of the hook-length control protein FliK and the anti-sigma factor FlgM. 2003). called FlgJ. experiments with mutant FlgJ proteins with a broken C-terminal (muramidase) domain result in a basal 10 . The C-terminal domain of FlgJ possesses peptidoglycan hydrolysing (muramidase) activity.. it acts as the mounting plate for the rotor/switch. Many of the flagellar substructures are built from proteins that are secreted through the type III export pathway.Jonathan McLatchie The functions of the MS ring. FlgF and FlgG (Homma et al. This protein is also needed for the export of other substrates (Minamino and Macnab.. Third. 4. Second. Another protein. 4. This is because the peptidoglycan layer has to be locally digested to allow for the penetration of the rod. 1992). it connects via FliE to the entire axial structure and thus enables flagellar rotation. 2004).2 Assembly of Type III Export Apparatus and Rotor/Switch The next thing to be assembled after the MS ring is the flagellum-specific type III export apparatus and the rotor/switch. it acts as a housing for the export apparatus...1990). is bifunctional. The type III export pathway is also used for other critical purposes during assembly. 1999) and is part of the basal body apparatus (Muller et al. In fact. There is a protein called FliE which is a Type III export substrate (Hirano et al. a component which is critical to the flagellum’s function and assembly. which we will return to. The four rod proteins are FlgB.” (Macnab. FlgC.3 Rod Assembly The next step is to assemble the proximal and distal rod. 1999).

but they would need to remain as monomer until the appropriate point in rod assembly. Two reasons have been proposed for why they use this pathway: “The first is that they are destined for finite compartments... as evidenced from the fact that they both undergo signal peptide cleavage (Jones and Macnab.. 2001).. 2001). hooks. it proceeds to assemble a flagellum that includes the outer membrane L ring." (Zhang et al. It is thought that FlgJ is the first protein to assemble since its N-terminal serves as a capping protein for the rod structure. FlgF. more than 10 genes are necessary for rod assembly (Kubori et al.Jonathan McLatchie body lacking the L ring and hook (Hirano et al. may be dispensable. These are formed by assembly of the FlgH and FlgI proteins respectively around the rod (Jones et al. 1979). The unassembled P-ring 11 . Besides the genes that encode the rod subunit proteins (FlgB. Fein. when it does so." (Hirano et al. FlgJ also possesses binding affinity for rod proteins (Hirano et al. 1987). FlgC. They could probably be exported even before formation of the MS ring. however... and so there is no risk of infinite dilution.4 L and P Ring Assembly The next step is to assemble the L (lipoprotein) and P (periplasmic) rings. 1996. These mutants are nonmotile. and it appears that the rod structure "occasionally and fortuitously finds a hole in the peptidoglycan layer by chance. Homma et al. 2001). One study reported that "In the enteric bacteria. Penetration of the peptidoglycan layer is a prerequisite for rod formation (Dijkstra and Keck. 4. flgJ null mutants fail to produce the flagellar rods. 1992). and filaments but still assemble the integral membrane-supramembrane (MS) rings. The C-terminal muramidase domain of FlgJ.. and FlgG). The second is that they have to assemble circumferentially around the nascent rod rather than by distal addition. 1990b. 2012). since the muramidase domain is absent in homologs of FlgJ in some bacterial phyla. 1989). FlgH and FlgI are exported by the Sec pathway.

2007. FliK is responsible for inducing a substrate specificity switch to filament-type substrate secretion upon completion of the hook structure. a hostile environment from the point of view of proteolysis... FliK is critical for both the ability to switch and export filament and the hook-length control. Moreover. 2010. The two proteins critical for hook-length determination are FliK and FlhB. the N-terminal domain of FliK functions as a molecular sensor and transmitter of information on hook length.” (Erhardt et al. the information is transmitted to the C-terminal domain. 2011. while polyhook-filaments result from second-site suppressor mutations in FlhB (Williams et al. in turn. Erhardt et al. Hirano et al.. In other words. Waters et al.. 4. Keener et al. 2003). “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. 12 .” (Erhardt et al. FlgA.5 Hook Assembly The next stage of the process is assembly of the hook. As stated previously. This. 2010. When the hook reaches the appropriate length..” (Macnab. 2011). 2011) inasmuch as it “takes measurements of rod-hook length while being intermittently secreted through the assembly process of the HBB [Hookbasal-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. 1994). FliK mutants result in polyhooks without filaments... 1996). 2010).. resulting in a conformational change which in turn results in the C-terminus binding to the C-terminus of FlhB.Jonathan McLatchie protein would be in the periplasmic space. this is probably why FlgI has a periplasmic chaperone. an elegant molecular mechanism ensures that the hook is 55nm in length (Erhardt et al. The hook-length determining protein FliK serves as a “secreted molecular ruler” (Erhardt et al..

4. 13 . the structure of the cap-filament complex and isolated cap dimer.6 Capping Proteins & Filament Assembly The capping protein for the hook structure is FlgD (Ohnishi et al. and this is discarded following hook completion and replaced by hook-associated proteins. the second hook-filament junction protein (FlgL).. using electron microscopy. reporting that “five leg-like anchor domains of the pentameric cap flexibly adjusted their conformations to keep just one flagellin binding site open. This represents one of the most dynamic movements in protein structures. 1994). only FliD remains at the tip of the filament in the finished product...Jonathan McLatchie results in a conformational change in the C-terminus of FlhB. Homma et al. hook and filament (FlgJ. which causes the substrate specificity switch. FliD. Ikeda et al.” (Yonekura et al. namely.. indicating a cap rotation mechanism to promote the flagellin self-assembly. 1989. One study examined. migrates outwards as flagellin monomers are progressively added. 2000). The filament capping protein. 1985). and filament-capping protein (FliD) (Ikeda et al. FlgD and FliD respectively). Of the capping proteins involved in construction of the rod.. 1987. The first and second hook-filament junction proteins remain in place and serve to connect the hook to the filament. the first hook-filament junction protein (FlgK).

Jonathan McLatchie Figure 6: Reconstructed three-dimensional image of the flagellar filament capping protein (also known as FliD). and thus 5. (2003) The cap rests on top of the hollow flagellar filament.. 14 .. the cap complex rotates and moves up. 2004. or 10 per second. This indentation acts as a folding chamber for the newly-exported flagellin monomers which are added at this site (Yonekura et al. The rotation rate of the filament cap is approximately 600 rotations per minute. 1998).5 subunits per turn.. and a new indentation is created adjacent to the previous one that was just occupied (Minamino and Namba. Since there are only 5 leg-like domains (leading to a symmetry mismatch). Figure credit: Maki-Yonekura et al. and 50 flagellin monomers are added per second.5 subunits at the distal end of the growing filament. When a flagellin monomer has been incorporated into this space. 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. 2003). Maki-Yonekuru et al. and possesses five leg domains that point downwards and insert into cavities at the distal tip of the nascent filament (Maki et al. this means that there is always a small crevice or space at one point between the filament and cap plate. 2000).

2008. 1988). and it is the FliD cap that ultimately facilitates the folding. FliD is critical to filament assembly. Aldridge and Hughes. 1999). as shown in figure 6 (Kalir et al.only two nanometers in diameter. of the flagellin monomers. 1996). Macnab. Escherichia coli Salmonella typhimurium require nearly 50 different genes for assembly and function. A regulatory cascade determines the timing of expression of flagellar genes (Chevance and Hughes. “A FliD-deficient mutant becomes nonmotile because it lacks flagellar filaments and leaks flagellin monomer out into the medium. and thus they pass through the tube unfolded. 2000). 2002. 2003. they are too large to travel through the tube folded. the flagellin monomers are lost (Kim et al.Jonathan McLatchie The hollow channel through which the flagellin monomers travel is extremely narrow -. 1980. and proper positioning and assembly.” (Ikeda et al. 2006. 2001). Without the presence of FliD.. Kutsukake et al. As one paper explained. McCarter. Since flagellin subunits have a kink in the middle. class II genes and class III genes... which are respectively organised across 15 and 17 operons (Komeda et al. They are thus unable to fold properly by themselves. 15 .. Chilcott and Hughes. There are three classes of genes involved in this regulatory cascade: Class I genes..7 A Regulation Cascade of Flagellar Gene Expression Bacterial flagellar genes are grouped in clusters on chromosomes. 4.

At the top of the hierarchy are class I genes (flhD and flhC).1990) and flgM (which codes for the anti-sigma factor) (Kutsukake et al.1994. We shall return to sigma factors and anti-sigma factors 16 . Those genes which are involved in the synthesis of the filament are controlled by the Class III promoters.. 2010). Class I contains only two genes in one operon (called flhD and flhC).. Figure credit: Kalir et al. and its corresponding anti-regulator. the flagellar filament cap protein. These includes genes involved in assembly of the hook-basal body and other flagellar components. This encodes a transcriptional activator of the class II genes. FliD (Aldridge et al. spread across eight different operons. 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. This transcriptional activator is itself under the control of the inhibitor protein FliT.. 1992). as well as the export apparatus and two regulatory genes called "fliA" and "flgM"). Ohnishi et al. 2006). in addition to the export apparatus and two regulatory genes..Jonathan McLatchie Figure 7: Organisation of flagellar genes into three distinct classes. a hetero-hexameric complex formed from FlhD and FlhC (Wang et al. (2001). which form the flhDC master operon.. There are 35 class II genes. fliA (which codes for a sigma factor called σ28) (Ohnishi et al. called FlhD4C2.

Indeed. Kutsukake et al. including the sigma factor σ28 (encoded by fliA) and its anti-sigma factor.. variation on this flagellar setup from species to species. by motive force and the efficiency of motors. The class II promoters are then responsible for the gene expression of the hook-basal-body subunits and its regulators. 17 .. But here we potentially run into a problem. Thus. 1994.1990). 2011.” The enteric master regulator FlhD4C2 is responsible for turning on the class II promoters in association with a sigma factor.. Figure 7 (Soutourina and Bertin. There is. Mutations in this master regulator result in the shutting down of the entire regulon. class II and class III. Vibrionaceae families) flagellation cascades. compares lateral (Enterobacteriaceae family) and polar (Pseudomonadaceae. the anti-sigma factor FlgM is secreted through the flagellar structures that are produced by the expression of the class II hook-basal-body genes. Upon completion of the hook-basal-body. bind to sigma factors to inhibit their transcriptional activity). the chemotaxis system and the motorforce generators) are then activated by σ28 and the flagellum can be completed. Ohnishi et al. 1992). The class III promoters (which are responsible for the expression of flagellin monomers. “these systems differ from each other by the existence of specific sigma factors and transcriptional activators. 2003). in order to inhibit σ28. It makes no sense to assemble the flagellin monomers before completion of the hook-basal-body construction. as their name suggests. flgM (anti-sigma factors. Expression of the flagellar genes is controlled by a corresponding set of promoters – class I. σ28 is required to activate the class III promoters (Ohnishi et al. the anti-sigma factor FlgM prohibits it from interacting with the RNA polymerase holoenzyme complex (Saini et al. σ70. of course. 2003).” (Soutourina and Bertin. Promoters are akin to a kind of molecular toggle switch which can initiate gene expression when recognised by RNA polymerase and an associated specialised protein called a “sigma factor..Jonathan McLatchie shortly.

Chemotaxis & Signal Transduction There is a significant amount of variation on chemotaxis systems. while general features of excitation remain conserved among bacteria and archaea. A substantially less amount of data is available for other species of bacteria. the pathways are somewhat mechanistically different. Figure credit: Soutourina and Bertin (2003). 5. Although individual genes bear homology to their Gammaproteobacteria counterparts.Jonathan McLatchie Figure 8: Comparison of lateral (Enterobacteriaceae family) and polar (Pseudomonadaceae. 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. many of the 18 . Vibrionaceae families) flagellation cascades. Furthermore.

. Before we can properly appreciate the details and technicalities of this system. 1992). These chemical signals are detected by a twocomponent signal transduction circuit that operates to induce the switch in flagellar rotation (Wadhams and Armitage. This change in rotation is brought about in response to chemical stimuli from the cell's exterior. 2004.” as shown in figure 8. One considerably more complex system than that of Escherichia coli is that of Bacillus subtilis (Rao et al. by a process known as "chemotaxis. Falke et al.Jonathan McLatchie more specific features are fairly diverse. a two-component regulatory system comprises an integral membrane protein known as a "histidine protein kinase. Bacteria are able to move towards a food source. In general. it is necessary to take a step back and understand the foundational principles upon which it is based. 2004." and a cytoplasmic protein known as a "response regulator.. such as glucose.." 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). 19 . 1997). Bischoff and Ordal.

The former is located on the outside of the cell. This phosopho-group is then transferred to an aspartate residue of the response regulator. This causes the transfer of phosphoryl groups (autophosphorylation) from ATP to a conserved histidine residue. 2006.. The histidine protein kinase has two domains: an input domain and a transmitter domain (Mascher et al. The latter is situated on the cytoplasmic face of the cell membrane. 2002).Jonathan McLatchie Figure 9: A diagrammatic representation of a two-component regulatory system. however. and is ideally situated to detect incoming environmental signals. An external stimulus causes a conformational change in the histidine protein kinase.. It was. and is positioned such that it can interact with the response regulator. necessary to look at the system in principle before we describe its application 20 . What I have thus far described represents a very basic two-component regulatory system. Wolanin et al. This enables the response regulator to bind to the DNA in order to regulate the transcription of its target genes.

These receptors then communicate with -. Upon activation of the receptor. Zhang et al. called CheB and CheY. 1982).. 1989). and are able to bind attractants or repellents (Kehrys and Dahlquistg. How do bacteria detect a chemical gradient? The answer lies in a certain class of transmembrane receptors called methyl-accepting chemotaxis proteins (hereafter. This induces the switching in flagellar direction from counter-clockwise to clockwise.. There are two response regulators. MCPs). 21 . 1991. the CheA's conserved histidine residue undergoes autophosphorylation (Shi et al. Proteins called CheA and CheW are bound to the receptor (McNally and Matsumura.the so-called "Che proteins" (Grebe and Stock. Conley et al. It is to the latter that I now turn. 1998).and activate -. There is a transfer of a phosphoryl group to their conserved aspartate residue from CheA (Li et al.. The former is the histidine kinase for this system. Different MCPs can detect different types of molecules. 1998). CheY subsequently interacts with the flagellar switch protein called FliM (Bren and Eisenbach. 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.. 1995). 2011.Jonathan McLatchie in the case of chemotaxis. 2005).

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 22 . of the response regulators CheY and CheB as well. the situation is similar -except that it is the least methylated MCPs which respond least while the fully methylated ones respond most. This means that it actively removes methyl groups from glutamate residues on the receptor's cytoplasmic surface. it operates as a methylesterase (Stewart and Dahlquist. another protein (called CheR) actively adds methyl residues to these same glutamate residues: that is to say. the phosphorylated CheB is able to demethylate the MCPs. CheB. 1974). But now. Remember that the role of CheB is to remove methyl groups from glutamate residues on the receptor's cytoplasmic surface. In the case of repellents. The result is that the bacterium "tumbles" (Larsen et al. and the receptors are again able to respond to the attracting chemical signals. 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. 1988). The degree of methylation of the MCPs will thus be raised. At this point the engineering shows a stroke of genius. When the MCPs are fully methylated. 1977).. But now. phosphorylated CheB is not available and so this task is not performed. as a consequence. Meanwhile. is activated by the histidine kinase CheA. If the stimulus is at a high level. there will be a corresponding decline in the level of phosphorylation of the CheA protein: and. When the other response regulator. it works as a methyltransferase (Springer and Koshland. 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.Jonathan McLatchie This clockwise rotation upsets the entire flagella bundle and causes it to break up.

however. 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. as illustrated when synonymous orthologs are deleted in each organism. when the CheB methylesterase is deleted. E. Both pathways share five orthologous proteins with apparently identical biochemistry. Deletion of the CheY response regulator causes E. It can thus detect whether it is moving towards or away from a chemical stimulus. coli to run exclusively and B. coli does not adapt. E. In B. coli and B. coli and cheBCDR in B. subtilis.. cells still run and tumble when either CheB or CheR is deleted. coli. 2004). though they no longer precisely adapt. For example. coli results in a run-only phenotype. 6. coli cells are incapable of runs and only tumble. subtilis to tumble exclusively. whereas B. Likewise. is different. whereas there is no change in phenotype for the synonymous deletion in B. When the CheR methyltransferase is deleted in E. Chemotaxis pathways vary among bacteria. subtilis. subtilis bias their motion towards favorable conditions with nearly identical behavior by adjusting the frequency of straight runs and reorienting tumbles. subtilis either oscillates or partially adapts when exposed to attractants. When the genes involved in regulating methylation are deleted (cheBR in E. which form part of the TonB-dependent active transport 23 .” [internal citations omitted] (Rao et al. How these individual orthologs contribute to the overall function.Jonathan McLatchie receiving signals. Remarkably. both genes complement in the heterologous host. subtilis). Deletion of the CheW adaptor protein in E. A comparative study of chemotaxis systems in Escherichia coli and Bacillus subtilis reported that “E. the cells are incapable of tumbles and only run. the stator proteins MotA and MotB are homologs of ExbB and ExbD. These differences demonstrate that the pathways are different even though they involve homologous proteins.

FliI etc.Jonathan McLatchie system and which serve to energize transport of vitamin B12. The sequence identities of FliG and MgtE. which couples proton transport across the inner membrane to the motor's rotation. 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. FlhB. and iron-chelating compounds called siderophores. which play an important role in the maintenance of outer membrane stability. For one thing. however. appear to be rather weak (<20% as reported by PSI-BLAST). 2011). 2001). Indeed. as a possible evolutionary predecessor.g. The most common response to the claim that the bacterial flagellum manifests irreducible complexity has been to point to the type III secretion system (T3SS). as a source of evolutionary 24 . There are a number of problems. it sidesteps the need to also explain the components of the type III export machinery (including FlhA. FliP. Indeed.” 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.. and it has been shown that the process of gene duplication and recruitment. homology does nothing to demonstrate that the necessary transitions are evolutionarily feasible (Gauger and Axe. with this hypothesis. The rotor protein FliG is also homologous to the magnesium transporter MgtE. 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. across the outer membrane of Gram-negative bacteria. 2006). a needle-like syringe used by certain bacteria (e. FliR. however. FliQ.). the archetype for this system Yersinia pestis) to inject toxins into organisms. at least most of which are essential for its function. ExbB/D and MotA/B are also known to be homologous to TolQ/R. although there is clear similarity to the N-terminal domain of MgtE. The energizing of these systems by proton movement across the inner membrane resembles the setup of the flagellum's stator.

among other things. 2003). Multi-cellular organisms on which type III injectisomes are used emerged on the scene much later than bacteria. While type III injectisomes are found only in gram-negative bacteria that possess both an inner and outer membrane. 2. Saier et al. these systems require explanation. Edqvist et al. so evolutionary pressure for such an organelle would proceed the evolutionary pressure for motility. FliK and FlhB are homologous to YscP and YScU respectively. Take. (2004). Furthermore. and perform essentially identical roles.. Pointing to such homologies. 25 . for example. 2008. the maximum number of non-adaptive point mutations that a new innovation in a bacterial population can require is two or fewer. therefore.Jonathan McLatchie novelty. the present author is persuaded that the type III secretion injectisome evolved from the flagellar subunit exporters.at some point. which also regulate substrate specificity and needle-length of the type III secretion system in Yersinia (Wood et al. is extremely limited: If a duplicated gene has a slightly negative fitness cost. largely based on. A further point that is worth noting is that certain irreducibly complex subsystems in flagella are also found in other organelles. For a more thorough discussion of this subject. and Nguyen et al. which function in hook-length determination.. this number jumps to six or fewer if the duplication is selectively neutral (Axe. two key observations: 1. although there is room for debate. 2010). only succeeds in pushing the problem back a stage -. I refer readers to Abby and Rocha (2012). flagella are found also in gram-positive bacteria that possess only a single membrane: Flagella are thus more widely distributed. the proteins FliK and FlhB. (2000).

the MS ring-rod junction protein FliE. because flagellin expression is controlled by such high expression promoters. Bordetella pertussis and recent isolates of E. expression of a flagellum under host conditions would result in loss of polarized secretion of Yop proteins into target host cells. these observations explain why segregation of these systems by specific environmental cues is necessary. even though they have the requisite flagellar genes. have lost flagellar biosynthetic capacity altogether. Even in the event that it was somehow feasible to evolve the flagellar export apparatus and basal body by evolution. Additionally. and direct the assembly of. may competitively interfere with virulence protein secretion. 2004). Indeed. It is also crucial for flagellar assembly and one of the very last proteins to be added. First. a filament capping protein (which serves no other purpose 26 .. One study critiques the feasibility of the flagellum’s evolution from the T3SS. there are a number of flagellar components that are presently not known to have homologs in non-flagellar systems. it also suggests that flagellin. Flagellin is a potent cytokine inducer. the flagellin monomers that are exported. How did a protein of such specific structure evolve and how long would it have taken? Surely. The filament cap needs to be of very specific structure in order to interact with. there is the problem of producing the filament. if expressed.” [internal citation omitted] (Meyer and Minnich. Examples include the rod cap FlgJ. Shigella spp. coli 0157:H7. reporting that “the potential for cross-recognition between type III exported proteins of different systems in the same cell carries several implications. display of flagellin to macrophages by direct injection via the Ysc secretin would countermand the anti-inflammatory strategy used by the Yersinia. Further. pestis. the L and P ring proteins FlgH and FlgI. the filament capping protein FliD. this latter suggestion may explain why an important subset of major human pathogens. including Y. For example. A number of these components form part of irreducibly complex subsystems of the flagellum. and the anti-sigma factor FlgM.Jonathan McLatchie Moreover.

MotB and FliM. deletions in the central region of the protein produced poorly 27 . be exported.” (Lloyd and Blair. How long did it take for such a complex system to evolve? In any event. yields “a non-motile. the elegant interaction between FliD and the flagellin monomers still needs to be accounted for. The specific and co-ordinated mutations required to facilitate such an innovation are likely to be well beyond the reach of a Darwinian process. It is also essential that their respective export be in the correct order -.Jonathan McLatchie within the cell as far as we know) is of no use without the exporting of the flagellin monomers whose assembly it facilitates. the flagellin monomers are of no value -. Perhaps one could argue that FliD and the filament proteins (FliC). but many specifically structured flagellin monomers (a filament is typically comprised of around 20. leaving aside the fact that the flagellar filament is assembled with the assistance of an essential capping protein encoded by fliD. Moreover. 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. in addition to the rod and hook proteins. in which flagella are assembled but do not rotate. But without the presence of such a capping protein. Inducing mutations in FliG.000 monomers). 1997). MotA.FliD must be exported before the export of flagellin subunits. evolved from an ancestral adhesive pilin secreted from the primitive type III secretion system. Remove any one of those proteins and the motor will completely cease to function. or Mot-. But such a hypothesis only pushes the need for explanation back a step -.at some point. The motor itself exhibits irreducible complexity. phenotype. for example. 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). and is dependent on the critical proteins FliG. it is essential that only a single filament capping protein.since they are lost into the medium and do not assemble into a filament.

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