Introduction
Some bacterial cells are stationary but the vast majority are able to move around in a liquid environment. This is called locomotion and this biologically important because it enables cells to change direction, thereby allowing them to move towards an attractant or away from a repellent (1). Locomotion is, therefore, crucial for bacterial survival and pathogenicity e.g. in host recognition and invasion (2). More than 70% of all bacteria are equipped with one or more flagella. These are proteinaceous structures that protrude from the cell surface and move the cell forward and backward through a propeller-like rotation (3). The number of flagella and the arrangement at the cell surface varies between bacterial species. Figure 1 (adapted from 1) shows electron micrographs of different bacteria, revealing the four different possibilities of how flagella are organized at the bacterial cell surface. These are: monotrichous; with a single flagellum (left top panel), amphitrichous; with a flagellum at each end (left bottom panel), lophotricous; with small bunches of flagella at one end (right top panel) and peritrichous; flagella all over (right bottom panel). The coordinated rotation of flagella enables polar flagellated cells of Thiospirillum to reach almost 90 µm/sec, while Pseudomonas aeruginosa swims at 73 µm/sec. In contrast, peritrichous rod-shaped bacteria swim much slower as evidenced by Escherichia coli that only reaches 17 µm/sec (1).
Functionally, the flagellum represents a nanomachine that is made up of about 30 different proteins, while, structurally, it can be divided into three main parts: the basal body, the hook and the filament (3,4). The basal body comprises a set of stacked rings that are firmly anchored in both the cell wall and plasma membrane and acts as rotary motor and protein export apparatus that facilitates the assembly of the flagellum’s distal parts (3,4). The filament is the main part of a flagellum and represents a thin propeller of up to 15 µm with a diameter of about 20 nm. It is made up of thousands of molecules of the same flagellin protein that form a supercoiled tubular helix (3,4). The hook is a curved, flexible joint that is attached to the basal body and connects the latter with the filament, thereby ensuring the transmission of motor torque to the filament and allowing it to rotate 360o (3,4). The inside of a flagellum consists of a central channel that runs along its entire length and is used for transport of hook and filament subunits towards tip of the growing flagellum. The core of a flagellum is a highly conserved molecular motor that comprises a plasma membrane-embedded rotor complex which is surrounded by numerous stator complexes (5,6). The energy for motor rotation is provided by the electrochemical potential of H+ or Na+ across the plasma membrane. The energy by this ion flow is utilized by stators to generate torque in the cytoplasmic part of the rotor complex (5,6). MotA and MotB (Motility protein A and B) are the typical stator elements of a bacterial motor complex. (5,6). Detailed structures of stator complexes are required to mechanistically explain how ion flow is coupled to torque-generating conformational changes. Importantly, high resolution structures of MotA/B complexes from different bacteria were reported recently (7), providing detailed molecular insight into the coupling of ion flow to flagellar rotation. Here, I will discuss these structures as well as their current functional understanding.
Molecular architecture of a bacterial flagellum
Bacteria such as E. coli and Salmonella have been used as model organisms to study the genetics, structure, assembly and function of a flagellum. This established that a typical bacterial flagellum is a large oligomeric complex that is produced by 40 genes with, in Salmonella, a mass of ~ 109 Da of which 99% is outside of the plasma membrane. The final flagellar structure comprises 30 different proteins and is schematically shown in the left panel of figure 2 (adapted from 6). Architecturally, it can be divided into three principal parts, namely: the basal body, the hook and the filament. The basal body functions as rotary motor enabling the flagellum to spin clockwise or counterclockwise at a speed of about 300 Hz in Salmonella or up to 1700 Hz in the marine bacterium Vibrio spp (5). An electron micrograph of the Salmonella basal body is shown in the right panel of figure 2, revealing that it comprises four stacked rings that are anchored in the cell wall and plasma membrane, respectively (8). The L ring is located in the outer membrane and is formed by 26 copies of the protein FlgH. The P ring is found in the peptidoglycan layer and is made up of 26 molecules of the protein FlgI. The MS ring is localized to the plasma membrane and comprises 26 copies of the protein FliF, while the C ring resides within the cytoplasm and is formed by three proteins FliG, FliM and FliN with a stoichiometry of probably 26:32:110 (4).
The export apparatus facilitates the transport of hook and filament subunits through a central channel towards the growing tip of the structure. It comprises a transmembrane export gate that is located within the MS ring and consists of six membrane proteins FlhA, FlhB, FliQ, FliP and FliR, as well as cytoplasmic ATPase complex made up three soluble proteins FliH, FliI and FliJ (4). The precise stoichiometry of the export apparatus is unknown but it has been reported that FliP assembles into a homohexameric ring (9). The flagellar motor consists of a reversable rotor comprising the MS and C rings (FliF, FliG, FliM and FliN) with FliG, FliM and FliN functioning as switch complex because these are responsible for switching the direction of rotation in response to external stimuli (4). In addition, the motor contains multiple stators that surround the rotor and couple the energy from ion flow across the plasma membrane to torque generation in the cytoplasmic portion of the C ring. Specifically, the stator represents an ion channel that is attached to the peptidoglycan layer and converts the ion flow across the channel into thrust required for flagellar motor rotation. In most bacteria, the stator is formed by a complex of MotA and MotB proteins that utilize the energy of proton transport to power the flagellar motor, while PomA and PomB, the homologues stator proteins of marine bacteria, employ the transport of Na+ for this purpose (5). The hook is a flexible curve-shaped joint that is connected to the basal body and the filament and smoothly transmits the torque of the rotary motor to the filament propeller. This allows the filament to rotate 360o. The hook is a relatively short helical segment made up of about 130 copies of FlgE that are organized into 11 protofilaments (3). The filament is a helical screw that propels the bacterial cell upon rotation by the motor complex. It is formed by more than 5000 molecules of the flagellin protein FliC that are, like the hook, organized into 11 protofilaments assuming an overall supercoiled tubular conformation (10). Owing to its tubular shape, the filament functions as propeller. A pentameric cap complex is located at the tip of the filament and is made up of FliD (11). This cap is crucial for filament assembly as well as adherence to surfaces
Flagellar assembly
A bacterial flagellum is built from the inside out, starting with the basal body followed by the hook and then the filament (12). The basal body therefore serves as a scaffold for the final flagellar structure. In order to obtain the basal body, the MS ring is assembled first from FliF. Subsequently, FliG, FliM and FliN are added, resulting in the C ring. These initial steps don’t require assistance of other proteins. Next, the export apparatus is constructed from FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ and FliR. This complex is required for the construction of the flagellar components beyond the plasma membrane – hook, filament and cap. To this end, subunits of the axial components are transported posttranslationally from the cytoplasm by the export apparatus through the central channel of the flagellum towards the tip of the growing structure (12). Flagellar protein export is driven by ATP hydrolysis and the proton motive force across the plasma membrane. The FliI subunit of the export apparatus displays ATPase activity and fulfills therefore a crucial role in powering of flagellar protein export. This process is, moreover, assisted by cytoplasmic chaperones that prevent the aggregation of flagellar subunits. Upon completion of the basal body, the export apparatus switches substrate specificity from hook subunits to those required for assembly of the filament (12).
Biochemical features of the flagellar stator complex
Bacteria employ two different ion-powered rotary motors to drive a flagellum, namely: one that collects the energy from H+ flow or one that collects the energy from Na+ flow across the plasma membrane. E. coli and Salmonella typically possess a H+-driven rotary motor, while marine bacteria use a Na+-driven rotary motor. A flagellar motor comprises a plasma membrane-embedded rotor complex which is surrounded by numerous stator complexes. Stators couple the energy of ion flow across the plasma membrane to the generation of torque by the rotary portion of the rotor complex which is converted into thrust by the helical flagellar filament. In most bacteria, the stator is formed by a complex of MotA and MotB proteins that utilize the energy of proton transport to power the flagellar motor, while PomA and PomB, the homologues stator proteins of marine bacteria, employ the transport of Na+ for this purpose (5). E. coli MotA and MotB are two plasma membrane proteins of 32 and 34 kDa, respectively. Their topology is schematically shown in figure 3 (adapted from REF), revealing that MotA contains four potential transmembrane helices (TMHs) and a large cytoplasmic domain between TMH2 and 3, whereas MotB contains one predicted TMH, a short cytosolic N-terminal region and a C-terminal peptidoglycan-binding domain (PGB). The segment that directly precedes the TMH of MotB is known as the plug.
Structural features of the flagellar stator complex
It is thought that a complex containing multiple copies of MotA and MotB function as proton channel. Specifically, a MotA4B2 complex with two proton-conducting pathways has been proposed. It has, moreover, been shown that within this complex, a highly conserved aspartate residue (Asp32) within the TMH of E. coli MotB as well as a highly conserved proline residue (Pro173) within the third TMH of E. coli MotA are in close proximity and are part of a proton-conducting pathway. To explain the coupling of ion transport to the formation of mechanical force, it has been suggested that passage of ions triggers conformational changes in the cytoplasmic domain of MotA that, in turn, generate torque in the rotor. MotA probably contacts FliG that is the part of the rotor’s C ring and through this interaction the proposed conformational changes of the stator drive rotation of the rotor. To mechanistically understand how a stator unit functions, a detailed structure of this complex is needed. It is therefore important to note that recently high- resolution structures of MotA/B complexes with the different ion specificities from various Gram-positive and Gram-negative bacteria were obtained by cryo-EM (7).
These include the stator complex of Bacillus subtilis at 3.5 Å resolution and Clostridium sporogenes at 3.4 Å, which are shown in figure 4 in surface representation with MotA and MotB indicated. Both stators display a pentagonal architecture with a complex made up of five A subunits and two B subunits that is in contrast to the previously suggested stoichiometry of 4:2. A top view of the complex is presented in the top and bottom right panels and reveal an asymmetric structure with the five-membered MotA ring in the membrane that houses the MotB dimer and forms a seal around it.
Figure 5 shows the stator complex of Bacillus subtilis in ribbon representation (top left panel) as well as the structure of a single MotA and MotB subunit (bottom panels). The four TMHs of MotA are clearly resolved and are organized in two layers with TMH3 and 4 lining the central pore, while TMH1 and 2 are located at the edges and form a surrounding outer layer of helices. The PGB domain of MotB was, unfortunately, not resolved unlike its single TMH. The centrally localized MotB dimer protrudes with its TMHS out of the stator complex at the extracytoplasmic side and these are followed by a short helical segment that is located between TMH3 and 4 of MotA. The MotB TMHs comprise the so-called plug helices that are crucial for sealing the complex when switched off. As discussed above, the heptameric MotA ring forms a seal around the MotB dimer and, therefore, the plug helices are not the only block against ion leakage.
The TMHs of MotA and B contain several conserved residues that are important for functioning of the flagellar complex. These include Asp32 of MotB that represents ion-binding site as well as Thr180 and 209 of MotA (left panel of figure 6). Both aromatic residues are probably part of a Na+/H+ conducting pathway. Both copies of Asp32 (shown as red sticks) are located centrally inside a ring made up by Thr180 and Thr209 (in orange or magenta ball and sticks). Additionally, two conserved prolines of MotA – Pro173 and 222 – are important for torque generation. Pro222 interacts with other MotA subunits, while Pro173 (in aquamarine ball and sticks) forms another ring of conserved residues (right panel of figure 6). This ring contacts Trp26 (in blue sticks) of MotB, which is strictly conserved.
It is thought that activation of ion flow through the stator complex is triggered by association of the inactive stator complex with the flagellar C-ring via the cytoplasmic domains of MotA, resulting in release of the MotB plug helices. Based on the structures presented above, two potential routes were proposed. Firstly, the cytoplasmic N-termini of MotB interact with the inside of the MotA ring. This, in turn, triggers the movement of the C-ring that would be relayed to MotB, and thereby resulting in displacement of the plug. Secondly, movement of MotA’s TMH3 and 4 could change directly the conformation of the plug, resulting in its displacement.
Mechanism of for the generation of flagellar torque
The molecular mechanism of the stator complex probably involves rotation of the MotB dimer relative to the heptameric MotA ring, which is triggered by the binding of ions. A mechanistic model for coupling of ion flow to the generation of torque is shown in figure 7 (adapted from 7). Within the stator complex, Asp32 of MotB is located in such a way that it is ideally positioned to bind either H+ or Na+. Following association of MotB with the peptidoglycan layer through its PGB-domain, the stator complex is activated, which involves transition from the relaxed state to the tensioned state (upper panel), and results in opening of the ion channel. The 5:2 stoichiometry of the stator complex enables the Asp32 residues to alternately bind ions and interact with MotA. Specifically, opening of one ion channel triggers partial rotation of the MotA ring. This rotation would bring the second Asp32 in the appropriate position to interact with MotA subunits, and closes the first ion channel while opening the second. Each subsequent binding event would trigger a step-wise rotation of the MotA ring, thereby powering the flagellar motor. This raises the question of how rotation of the MotA ring is transduced into flagellar motion. Conceivably, docking of the C-terminal domain of FliG between two MotA subunits induces a conformational change in the stator complex that relays movement of the MotA ring to the C-ring, thereby coupling ion flow to flagellar rotation.
Summarizing conclusion
Bacterial swimming was reported for the first time by the Dutch microbiologist Antoni van Leeuwenhoek in 1676 (13) without a clear mechanistic understanding. It is now well established that more than 70% of all bacteria are equipped with one or more flagella, which enables them to move around in liquid environments (1,3). Locomotion enables bacterial cells to move towards an attractant or away from a repellent and is, therefore, crucial for bacterial survival and also pathogenicity. A bacterial flagellum represents a sophisticated nanomachine that is made up of about 30 different proteins and comprises three main parts: the basal body, the hook and the filament. The basal body functions as rotary motor enabling the flagellum to spin clockwise or counterclockwise (4,5,6). Bacteria employ two different ion-powered rotary motors to drive a flagellum, namely: one that collects the energy from H+ flow or one that collects the energy from Na+ flow across the plasma membrane. A flagellar motor comprises a plasma membrane-embedded rotor complex which is surrounded by numerous stator complexes. Stators couple the energy of ion flow across the plasma membrane to the generation of torque by the rotary portion of the rotor complex which is converted into thrust by the helical flagellar filament (4,5,6). In most bacteria, the stator is formed by a complex of MotA and MotB proteins that utilize the energy of proton transport to power the flagellar motor. Recent detailed structures of stator complexes from different Gram-positive and Gram-negative bacteria reveal an asymmetric complex made up of 5 MotA subunits and two MotB subunits (7). The MotB dimer is located centrally within a heptameric MotA ring. The molecular mechanism of the stator complex probably involves rotation of the MotB dimer relative to the heptameric MotA ring, which is triggered by the binding of ions. It is thought that binding of the C-terminal domain of FliG between two MotA subunits induces a conformational change in the stator complex that relays movement of the MotA ring to the C-ring, thereby coupling ion flow to flagellar rotation.
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