Jonathan McLatchie

1

The Bacterial Flagellum: A Motorized Nanomachine

Jonathan McLatchie (B.Sc, M.Res)

September 2012

Last updated: 3/9/2013

Jonathan McLatchie

2

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).

Jonathan McLatchie

3

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.

Jonathan McLatchie

4

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.

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;

Figure 1: Simplified diagrammatic representation of gram negative bacterial flagellar

structure.

Jonathan McLatchie

5

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.

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 rings. A

series of proteins, called Mot proteins (MotA and MotB), surround the inner ring and

Figure 2: Diagrammatic representation of bacterial flagellar hook (universal joint) and

filament (propeller).

Jonathan McLatchie

6

are anchored in the cytoplasmic membrane. Approximately 11 MotA-MotB pairs create a

ring structure around the flagellar base. MotA possesses 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 referred to as the “basal body”. An electron

micrograph of the flagellar basal body is given in figure 3.

3. A Proton Motive Force Drives the Flagellar Motor

The flagellar motor is driven by a proton gradient across the 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

Figure 3: Electron micrograph of flagellar hook-basal-body (HBB).

Jonathan McLatchie

7

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 necessary 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 disengaged 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).

Jonathan McLatchie

8

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.

Jonathan McLatchie

9

4. Flagellar Assembly

Readers may find it helpful to refer to figure 5 when reading the description that follows.

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 co-

ordinated 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).

Figure 5: Schematic diagram of the location of proteins involved in flagellar assembly and

function.

Jonathan McLatchie

10

The functions of the MS ring, a component which is critical to the flagellum’s function and

assembly, is given in three stages: “First, 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 is 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 the 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 pathway 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 result in a basal

Jonathan McLatchie

11

body lacking the L ring and hook (Hirano et al., 2001). Penetration of the peptidoglycan layer

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

Jonathan McLatchie

12

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,

Jonathan McLatchie

13

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 the 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-filament 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).

Jonathan McLatchie

14

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 that 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.

Figure 6: Reconstructed three-dimensional image of the flagellar filament capping protein

(also known as FliD). Figure credit: Maki-Yonekura et al. (2003)

Jonathan McLatchie

15

The hollow channel through which the flagellin monomers travel is extremely narrow -- only

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 non-

motile 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).

Jonathan McLatchie

16

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 FlhD4C2, 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

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).

Jonathan McLatchie

17

shortly. Expression of the flagellar genes is controlled by 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 associated

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 are 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 expression of

the class II hook-basal-body genes. The class III 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 family) and

polar (Pseudomonadaceae, Vibrionaceae families) flagellation cascades.

Jonathan McLatchie

18

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

Figure 8: Comparison of lateral (Enterobacteriaceae family) and polar (Pseudomonadaceae,

Vibrionaceae families) flagellation cascades. Figure credit: Soutourina and Bertin (2003).

Jonathan McLatchie

19

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 two-

component 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 "response

regulator,” as shown in figure 8.

Jonathan McLatchie

20

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 with 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 regulator 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

Figure 9: A diagrammatic representation of a two-component regulatory system.

Jonathan McLatchie

21

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.

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 (McNally 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 called FliM (Bren and

Eisenbach, 1998). This induces the switching in flagellar direction from counter-clockwise to

clockwise.

Figure 10: A diagrammatic representation of the mechanisms underlying bacterial

chemotaxis.

Jonathan McLatchie

22

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 re-

direct 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 kinase 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 fully 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

Jonathan McLatchie

23

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 synonymous 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

Jonathan McLatchie

24

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 systems 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 rotation. 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 secretion 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 explain 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

Jonathan McLatchie

25

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 essentially 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 bacteria that possess both

an inner and outer membrane, flagella are found also in 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).

Jonathan McLatchie

26

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 systems 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 macrophages by direct injection via

the Ysc secretin would countermand the anti-inflammatory strategy 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 secretion. 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

Jonathan McLatchie

27

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 respective 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 central region of the protein produced poorly

Jonathan McLatchie

28

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 every 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.

Literature Cited

Abby, S. S., & Rocha, E. P. C. (2012). The non-flagellar type III secretion system evolved

from the bacterial flagellum and diversified into host-cell adapted systems. PLoS

genetics, 8(9), e1002983.

Aizawa, S. I. (2009). What is Essential for Flagellar Assembly? In K. Jarrell (Ed.), Pili and

Flagella: Current Research and Future Trends (p. 91). Caister Academic Press.

Aldridge, P., & Hughes, K. T. (2002). Regulation of flagellar assembly. Current Opinion in

Microbiology, 5, 160-165.

Aldridge, C., Poonchareon, K., Saini, S., Ewen, T., Soloyva, A., Rao, C. V., Imada, K., et al.

(2010). The interaction dynamics of a negative feedback loop regulates flagellar number

in Salmonella enterica serovar Typhimurium. Molecular microbiology, 78(6), 1416–30.

Axe, D. D. (2010). The Limits of Complex Adaptation : An Analysis Based on a Simple

Model of Structured Bacterial Populations. Bio-Complexity, 2010(4), 1-10.

Baker, M. D., Wolanin, P. M., & Stock, J. B. (2006). Signal transduction in bacterial

chemotaxis. BioEssays, 28, 9-22.

Behe, M. (1996). Darwin’s Black Box: The Biochemical Challenge to Evolution. Free Press.

Berg, H. C. (2003). The Rotory Motor of Bacterial Flagella. Annual Review of Biochemistry,

72, 19-54.

Berg, J., Tymoczko, J., & Stryer, L. (2006). A Rotory Motor Drives Bacterial Motion.

Biochemistry (6th ed., p. 993). Sara Tenney.

Bischoff, D. S., & Ordal, G. W. (1992). Bacillus subtilis chemotaxis: a deviation from the

Escherichia coli paradigm. Molecular Microbiology, 6(1), 23-28.

Jonathan McLatchie

29

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), 1636-

1638.

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.

Jonathan McLatchie

30

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 Hook-

Associated Proteins in Salmonella typhimurium. Journal of Bacteriology, 163(2), 464-

471.

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.

Jonathan McLatchie

31

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), 1327-

1339.

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), 1655-

1662.

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.

Jonathan McLatchie

32

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 assembly. 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 Signal-

Transducing 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 Design. 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.

Jonathan McLatchie

33

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). Peptidoglycan-

Hydrolyzing 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 systems. 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.

Jonathan McLatchie

34

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.

Jonathan McLatchie

35

Yonekura, K., Maki, S., Morgan, D. G., DeRosier, D. J., Vonderviszt, F., Imada, K., &

Namba, K. (2000). The Bacterial Flagellar Cap as the Rotary Promoter of Flagellin Self-

Assembly. Science, 290, 2148-2152.

Zhang, K., Tong, B. a, Liu, J., & Li, C. (2012). A single-domain FlgJ contributes to flagellar

hook and filament formation in the Lyme disease spirochete Borrelia burgdorferi.

Journal of bacteriology, 194(4), 866–74.

Zhang, J., Xu, Y., Shen, J., Luo, X., Chen, J., Chen, K., Zhu, W., et al. (2005). Dynamic

Mechanism for the Autophosphorylation of CheA Histidine Kinase: Molecular

Dynamics Simulations. Journal of the American Chemical Society, 127(33), 11709–

11719.