Flagellar locomotion: assembly, rotation and variation

By Andres Hernandez Maduro

With the ever-fluctuating conditions of our world, it is crucial for biological organisms to be able to adapt and respond to external changes in their environment. This is especially true for bacteria, which face a constant struggle to procure limited resources and compete with the many other microscopic species around them. Their mechanisms for dynamic movement are principal in this – and the past decade has helped elucidate just how sophisticated they can be.

Universally present in bacteria, flagellar motion is the best understood mechanism of bacterial motility. Flagella are initially assembled at the inner membrane, where the MS-ring is first integrated and thereby attached to a cytoplasmic C-ring.1 A protein complex known as the flagellum-specific type three secretion (T3S) system then assembles in the centre of the two rings, at which point it allows protein subunits to bypass the inner membrane and form the flagellum’s proximal rod. These subunits include FlgE – which serves as an adaptor between the MS-ring and the axial rod – and a consequent stacking of FlgB, FlgC and FlgF proteins until the rod reaches the cell wall’s peptidoglycan layer.1

In Gram-negative bacteria, the assembling rod must be directed through further rings before exiting the outer membrane to prevent it from becoming angled and dysfunctional. These are made by proteins exported through the Sec secretory pathway into the periplasmic space, with FlgI forming a P-ring (in the peptidoglycan layer) and FlgH comprising a subsequent L-ring (integrated with the outer membrane’s lipid bilayer). This occurs in tandem with rod formation, and hence must be assisted by auxiliary proteins to prevent it from lagging. To elaborate on this, previous studies actually found that FlgA (transported alongside FlgI) works as a chaperone to polymerise FlgI in the cell wall, dissociating once the P-ring has been fully constructed.2

Once the proximal rod is complete, the distal rod is assembled through the P- and L- rings by the polymerisation of approximately 26 FlgG subunits, catalysed by the N-terminal scaffolding domain of FlgJ proteins at the tip of the rod. Being a dual-domain polypeptide, FlgJ is then able to use its C-terminal acetylmuramidase domain to essentially ‘drill’ through the outer membrane. The ensuing transition from rod to hook is not totally understood, but recent work has shown that the aforementioned L-ring formation most likely catalyses the dissociation of the FlgJ cap from the rod as it passes out of the cell, thereby inhibiting any further rod assembly outside.1 Consequently, a FlgD cap replaces it to act as a scaffold for the hook, made entirely of FlgE.

Finally, the process repeats itself after the hook is complete, with a FliD cap replacing the FlgD cap to form the ending filament of the flagellum (made of flagellins). Fascinatingly, however, this ultimate transition appears to be decided by what might be likened to a ‘molecular ruler’. Indeed, under normal conditions, researchers have observed the change to occur only when a hook has reached about 55nm in length.3 The molecule behind this is known as the ruler protein FliK. During hook polymerisation, FliK is secreted by the bacterium’s T3S system and unfolds to bind reversibly to both the inner surface of the hook and the FlhB making up the T3S system. In turn, hook formation is paused to allow the T3S system to experience a conformational change and stop the transmembrane transport of hook protein subunits.4 It then allows filament subunits to pass through, instead – including hook-associated proteins (HAPs) that connect the hook to the filament4 – and so we start to see the end of flagellar assembly.

With a fully established flagellum in place, a bacterium is able to swim through low-viscosity mediums via the thrust generated by its rotating flagellum. As with most mechanobiological pathways, this is powered by the chemiosmosis of protons in an electrochemical gradient. Specifically, protons in the bacterium’s periplasm diffuse into the cytoplasm by associating and then dissociating with transmembrane MotA stator units, located next to the rotor (a.k.a. the MS-ring).5 Since these are bound to the peptidoglycan layer by MotB intermediates, this action causes the stator units to rotate like gears around the C-ring, thereby applying torque to it and the rest of the flagellum. MotA rotation is further known to be clockwise – but we have an issue here. If a flagellum can spin both counterclockwise (CCW) and clockwise (CW), how can stator units produce parallel rotation within it? A recent cryo-electron microscopy study demonstrated that the C-ring changes in conformation to allow this. To rotate CCW, it contracts to allow MotA units to contact it from the outside; and, to rotate CW, the C-ring expands to allow the units to contact it from the inside.6 Since CW rotation causes flagella to ‘tumble’, this expansion is mediated by the C-ring binding to phosphorylated CheY proteins, making it critical in bacterial chemotaxis.

Current research is highlighting just how important it is to understand the structure and function of flagella, both in their locomotion and roles in bacterial survival and pathogenicity. Though most bacteria assemble their flagella from the same core constituents, some aspects do vary. Filament flagellins, for example, display massive structural variation between different species, which results in some bacteria being more susceptible in binding to the mucin proteoglycans in our mucus (e.g., P. aeruginosa, which is infectious and can cause pneumonia in humans).7 Hook and filament proteins also serve as potent antigens, and so there is much interest in using them as vaccine adjuvants.7 Given that the flagellum is one of the most intricate nanomachines in biology, it might also prove to be a wonderful inspiration for future nanotechnologies and a model for biochemistry research.

References:

  1. Cohen EJ, Hughes KT. Rod-to-hook transition for extracellular flagellum assembly is catalyzed by the L-ring-dependent rod scaffold removal. J Bacteriol 2014 Jul;196(13):2387-95. Available from: https://dx.doi.org/10.1128%2FJB.01580-14 
  2. Nambu T, Kutsukake K. The Salmonella FlgA protein, a putative periplasmic chaperone essential for flagellar P ring formation. Microbiology 2000 May;146(5):1171-1178. Available from: https://doi.org/10.1099/00221287-146-5-1171 
  3. Erhardt M, Singer HM, Wee DH, Keener JP, Hughes KT. An infrequent molecular ruler controls flagellar hook length in Salmonella enterica. EMBO J 2011 Jun 7;30(14):2948-61. Available from: https://doi.org/10.1038/emboj.2011.185 
  4. Minamino T, Moriya N, Hirano T, Hughes KT, Namba K. Interaction of FliK with the bacterial flagellar hook is required for efficient export specificity switching. Mol Microbiol 2009 Oct;74(1):239-251. Available from: https://doi.org/10.1111/j.1365-2958.2009.06871.x 
  5. Wadhwa N, Berg HC. Bacterial motility: machinery and mechanisms. Nat Rev Microbiol 2021. Available from: https://doi.org/10.1038/s41579-021-00626-4 
  6. Chang, Y., Zhang, K., Carroll, B.L. et al. Molecular mechanism for rotational switching of the bacterial flagellar motor. Nat Struct Mol Biol 2020:27;1041–1047. Available from: https://doi.org/10.1038/s41594-020-0497-2 
  7. Nedeljković M, Sastre DE, Sundberg EJ. Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure. International Journal of Molecular Sciences 2021; 22(14):7521. Available from: https://doi.org/10.3390/ijms22147521 

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