by Mike Delmont
Many things in our lives are so common that we take them for granted, forgetting their remarkable complexity. These can range from the social microbiology involved in cheese making, to the physics behind the internal combustion engine, or the anatomy/physiology behind taking a couple of steps. Most of us walk every day without ever thinking about it, which causes us to tend to think of it as a very simple thing. There’s even a song from the beloved Christmas film, Santa Claus is Comin’ to Town, where the ease of something is compared to walking – it’s as easy as “put[ting] one foot in front of the other” (Figure 1A). However, walking is far from a simple process – numerous muscles all need to work in concert to allow you to move one of foot in front of the other (Figure 1B), with the added complications of the roles played by tendons, ligaments, and other body parts. Bacteria are no different (Figure 1C). A lot of people are familiar with the idea of bacteria “squirming” around and being capable of movement – especially, I would imagine, readers of Small Things Considered – and many are aware of the complexity involved in bacterial movement. But, it turns out, there is still a lot to ponder about this amazing example of biological innovation. As Elio put it, “hardly anything in biology is more fascinating than this marvel of miniaturization.”
How do bacteria move?
When looking at a bacterium (Figure 2A), one can see the tail-like appendage known as a flagellum (a Latin word for “whip,” due to its appearance). The number and arrangement of flagella varies depending on the particular bacterial species being looked at (Figure 2B). Other kinds of bacterial motility, such as gliding, do not depend on flagella. But I will not deal with them here.
There are two main types of flagellar motility, swimming and swarming. Bacteria can swim as individual cells through liquid media or swarm cooperatively in groups across semisolid surfaces. Both of these types of movement are powered by flagellar rotation, and their course can be altered by alternating the direction of rotation between clockwise and counterclockwise. Flagella rotate through the action of an intricate nano machine, a protein complex known as a flagellar motor.
Just what are these “motors”?
Flagellar motors work in the same way as the motors seen in everyday life but are more complex than most, with tens of thousands of subunits of over thirty different proteins (Figure 3). Located at the base of each flagellum, they anchor them to the bacteria and provide a mechanism for rotation. These motors are self-assembled, as discussed in an article by Elio a few years ago. In a temporally regulated process, the bacterium takes the protein building blocks, its LEGOsTM of life, and ensures that they are placed in the proper order. Building a flagellar motor is indeed a costly and lengthy process. Due to the amount of proteins needed to make functional flagella, and the complex regulation of gene operons involved, it takes more than one cell generation to assemble a fully functional flagellum, as discussed here. But clearly, these motors are advantageous and well worth the investment for the bacteria.
When ions (or just protons, depending on the species) flow across the cytoplasmic membrane, conformational change occurs in one of the subunits, MotA, which acts as a force-generating element and gets its more general name from a similar structure from an engine, the piston. For those like me, who don’t know much about cars, the piston moves and works to transfer force and impart motion. Rotation occurs from an interaction between the stator complex, which is comprised of pistons (four of the previously mentioned MotA as well as two of MotB) and the rotor. When MotA changes conformation, it causes an electrostatic interaction with the rotor (which is a disk-like protein known as FliG) and turns it, generating a rotational force, torque, used to propel the bacterium.
Is the flagellar motor regulated by anything?
Bacterial motility needs to be regulated to keep it advantageous enough to be worth the large cost it takes to build the flagella. Case in point is the interaction between the major components of the flagellum, the stator complex (the stationary component) and rotor. The interaction of the two is regulated in three ways to control rotation:
- The power from the motor can be increased by increasing the number of stator subunits bound to the rotor
- ‘Clutch proteins’ bind and disengage the rotor from stators, cutting power to the flagellum.
- ‘Brake proteins’ prevent rotation by increasing resistance between the rotor and stators.
As you can see, the car theme for naming these regulatory mechanisms continues. These mechanisms are of major interest to researchers as they help us to understand how they motors work. To this end, the lab of Dan Kearns looked into a regulatory molecule and unveiled some of the mystery behind it, as described in a recent paper.
This study focused on DgrA from Bacillus subtilis, a regulator that inhibits motility in general, and completely abolishes swarming motility. This is seen in particular when DgrA is overexpressed and bound to c-di-GMP (hence the name – DgrA is short for “diguanylate receptor A”). But DgrA was first identified and studied in Caulobacter crescentus. The messenger c-di-GMP was known to have a role in antagonistically controlling motility, so the researchers wished to purify and identify proteins which bind c-di-GMP – one of which was DgrA. In studying this protein, they discovered that it does indeed bind to c-di-GMP, that it reduces bacterial motility, and that mutants of DgrA that could not bind to c-di-GMP were unable to affect motility.
Curious as to whether this inhibited motility was due to a decreased number of flagella or impaired flagella assembly, the researchers examined these factors using electron microscopy and immunoblot analysis. They found that dgrA overexpression did not decrease the amounts of flagella or interrupt their assembly. One finding of note was the decreased concentration of the flagellar protein, FliL. In C. crescentus, fliL is required for flagellar rotation, but FliL is not assembled into the flagellum itself. FliL interacts with the stator proteins MotA and MotB and functions by strengthening stator interactions as well as increasing stator retention at the rotor, which is required to drive rotation of the flagellum. This led the authors to conclude that DgrA inhibits motility by interfering with the motor function, rather than interfering with the actual assembly of the motor, with FliL being implicated to have some role in this inhibition.
From DgrA to MotI
In their experiments, the Kearns lab confirmed that DgrA abolished powered flagellar rotation without altering the expression of flagellar genes or blocking flagellar assembly. During their studies, swarming motility was inhibited until spontaneous mutations suppressed the inhibition. The mutations were rare and occurred in the stator protein, MotA. This led the authors to conclude that DgrA inhibits flagellar rotation at the mechanistic level of MotA, leading to the renaming of DgrA as MotI – “motility inhibitor.”
Having deduced that MotI may inhibit MotA, they considered two possibilities for how MotI functions, either as a molecular brake or as a molecular clutch. If MotI were a brake, flagellar rotation would be locked and rotate only 3° due to torsion on the flagellum. If, instead, it was a clutch, the flagella would be depowered but Brownian motion (the random motion of particles suspended in a fluid) would still be allowed. This would mean that while they would not be expected to make a complete rotation, the flagella could randomly move and rotate to a smaller extent than normal, as opposed to being locked specifically at 3°. In studying the angular motion of flagella (Figure 4A), they showed shown that rotation in the MotI-inhibited cells was more than 20° but less than 360°, which indicated no rotational resistance, suggesting that MotI was not acting as a break. Thus, it inhibits MotA in a different manner. This was comparable to results observed in cells expressing EpsE (a known molecular clutch protein used as a control), as well as cells with motA or motB mutations. The flagella were not locked in place, however, so there was still minimal rotation (but not complete rotations) due to Brownian motion – a similar result as that seen using the control molecular clutch protein.
This led the authors to conclude that MotI is a molecular clutch protein, which led them to develop a model for its mechanistic function (Figure 4B). Here, it can be seen that MotI (in red) binds to MotAB (in brown), which prevents MotAB from interacting with the purple FliG (the rotor that is turned by conformational change of MotA) and prevents rotation of the flagellum. By determining the 3D structure of MotI, along with studying inhibited cells using fluorescent imaging, they showed that MotI interacts directly with MotA (likely near the Glu92 residue of MotA, specifically) and forms puncta (sharp points/tips) when interacting (Figure 4C). This was shown through the use of an sfGFP fusion to MotI.
This result suggests that MotI functions by sequestering individual stator units. It led the authors to hypothesize that concentrations of MotI below the amount required to fully inhibit motility would proportionally reduce swimming speed. To test this, the authors measured swim speed at different concentrations of MotI. They found that partial clutch inhibition of the flagellar rotor does decrease swimming speed. Using an IPTG-inducible MotI strain, they measured swimming velocity at different ITPG concentrations. Without any, the bacteria swam with a velocity just over 30 μm/s. As the concentration of IPTG increased, the velocity decreased; for example, at about 10μM IPTG the velocity was reduced to around 25μm/s and at about 25μM, to around 20μm/s. This further confirmed their model of MotI inhibition of MotA.
A Peak Behind The Curtain
The essence of scientific research is to see something that isn’t quite understood or doesn’t make sense and to decide to go poke it with a stick. In doing so, researchers can hope to uncover just the slightest bit of new information which builds upon the mountain of previously uncovered knowledge. Flagellar motors are indeed marvelous microscopic machines that demonstrate a remarkable complexity behind a seemingly simple action. The Kearns lab was able to uncover a previously unknown method regulating these motors. It was previously demonstrated that MotI inhibits cellular motility, but the actual mechanism behind this was unknown. It has now been shown to act as a molecular clutch, and to inhibit flagellar rotation via interaction with individual MotA subunits. As the previous work had uncovered that FliL, which functions through interactions with MotA, has its concentration decreased when MotI is expressed, this new discovery helps to reveal why that might be. In uncovering the mechanism behind one of the regulators of these machines, the Kearns lab has helped to shed some light on some of the mystery and has aided in our understanding of this interesting topic of how a bacterium shakes its groove thing.
Mike is a recent graduate of Canisius College, having majored in Biology. He not only had the opportunity to take the microbiology class taught by STC Associate Blogger Daniel Haeusser, but he also joined Dr. Haeusser’s research lab. Since graduating, Mike has completed a Master’s degree in Science Education and is currently in a Genetics graduate program. Despite now working in a developmental genetics lab, Mike has maintained an interest in microbiology and is excited to be encountering the complexities of microbial genetics in his current studies