One of the first observations of microbial life came from Antonie van Leeuwenhoek, a 17th century Dutchman who laid the foundations for bacteriology and protozoology. For years, he carefully observed the microscopic structures and dynamic movements of microbes, which he termed “animalcules”. According to his description, some of these animalcules probably had flagella, the structure we have now come to associate with microbial motility. But how can this tiny whip-like appendage manage to propel an entire microbe in such a wide array of environments? Even now, nearly 400 years after the discovery of bacterial motility, scientists still are still asking this question.
The movement of the flagella propel microbes in discontinuous pattern of straight runs and directional changes known as tumbles Source
Agrobacterium tumefaciens is a soil-borne pathogen and causative agent of crown gall disease in plants. This disease stimulates rapid growth of plant cells and can lead to some pretty interesting looking tumors. These bacteria are members of the Rhizobiaceae, a family of proteobacteria that are marked with a unique ability to associate with and affect plant development. Motility can play a critical role in this ability and without flagella to propel them, these bacteria would probably have a much harder time trying to infect new hosts.
Oak tree infected by A. tumefaciens showing the formation of a crown gall tumor. Source
A. tumefaciens is able to detect compounds secreted from wounded plants and respond by moving towards and associating with the plant, a process called chemotaxis. After arriving at its new host, A. tumefaciens can transfer a tumor-inducing plasmid to host cells which, when expressed, triggers enormous tumorous growths. The resulting tree tumor produces opines, an energy rich source of carbon and nitrogen for A. tumefaciens. This enables A. tumefaciens to thrive in its newly infected host. Crown gall disease can sometimes decrease an infected host’s growth and yield, making them an unwelcome pathogen for farmers. However, they can also be quite valued for avid hikers like my family and I. For years, my siblings and I have been collecting galls off of trees in nearby forests. Needless to say, our walls are now almost entirely covered with them, allowing us to marvel at their truly magnificent swirls and twirls.
One of our favorite galls (left) and a close up on another’s twisted wood (right).
However, regardless of if you are actively seeking out these tree tumors or not, you probably run into them from time to time on various trees and woody plants. But what makes them so great at infecting so many new hosts? By now, I hope it is starting to become clear that the flagella is a key player. This is because it allows for an indispensable pathogenic function: motility.
A flagellum is made of helical filaments, a basal body that rotates the flagellum, and a hook, joining the two together. A. tumefaciens has many flagella, almost resembling a “tuft” of hair. This tuft helps drive the bacterium forward in a movement typically called a run. The flagella rotate unidirectionally and clockwise until a disruption occurs from discordance in flagellar rotation rates. This causes a tumble, allowing the bacterium to change directions. The helical shape of the flagellar filaments is also an important element that allows the bacterium to propel itself in this pattern of runs and tumbles.
Images of the flagellum showing the three main parts: the basal body, the hook, and the filament. Source
Bacterial filaments can be composed of one kind of flagellin protein, which is the case for the well studied E. coli, or can be made of many different flagellin proteins, which is the case for A. tumefaciens. A. tumefaciens has four different flagellins: FlaA, FlaB, FlaC, and FlaD. The function and coordination of these flagellins have yet to be fully eluted and many questions remain about how the individual proteins function. Do the flagellins exhibit a certain level of redundancy, like what is seen in studies of C. crescentus, a crescent-shaped bacterium containing six different flagellins? Is there a single flagellin that is primarily responsible for this bacteria’s motility, which is true for other members of the Rhizobiaceae family? And lastly, are there sequence-specific regions of the flagellins that empart functional motility? Mohari et al. were determined to answer these questions and many more in their recent study, “Multiple Flagellin Proteins Have Distinct and Synergistic Roles in Agrobacterium tumefaciens Motility”.
The researchers began with a mutational analysis. They found that FlaA, only one of A. tumefaciens’s four flagellin proteins, is required for proper motility. Mutants lacking FlaA exhibited straight flagellar filaments and were quite deficient in motility. Mutants lacking one or more of the other flagellins appeared to make relatively normal flagella and have proper motility. Therefore, from now on FlaA was the researchers' primary focus because motility seemed to be most affected by the lack of this protein. Mohari et al. further found that although FlaA is required for motility, it’s not sufficient; some additional secondary flagellins (FlaB, FlaC, or FlaD) are needed to produce normal motility.
Now with some of their more straight forward observations out of the way, it really starts to get interesting. When ΔflaA mutants (Δ indicates deletion of the gene) that retained flaB were left to incubate for a longer amount of time, suppressor mutations resulted. This means that these mutants gained additional mutations which enabled them to revert back to their original phenotype. Therefore, the ΔflaA mutants which should have been deficient in swimming were now somehow able to swim almost normally!
The authors called the mutants that regained their swimming abilities flagellin mutation suppressor mutants, which is a mouth full, I know. For simplicity's sake, I will refer to these as the fms mutants. The suppressor mutations in these fms mutants were often found in the coding region of flaB or in its ribosome binding site (RBS). Mutations in FlaB’s coding region could drastically alter the resulting protein’s function by changing various amino acids. Remember, a protein’s function is linked to its very specific structure, so even small structural changes can have big functional implications. Alternatively, mutations in the RBS could also cause phenotypic changes by altering the protein’s expression levels. Mutations in the RBS can change ribosomes’ ability to dock and begin translation. It seems likely that both these mutations could be working together to allow the fms mutants to regain their swimming ability.
The researchers did many more experiments to eventually figure out the exact mechanism. One of the fms mutants they focused on was fms-6, a ΔflaACD mutant with a suppressor mutation altering the 129th amino acid in FlaB (FlaBY129N). This mutant also had a mutation in the RBS of FlaB (SD*-FlaB). They started their experiment by fluorescently labeling FlaB. When viewed via fluorescence microscopy, they found that FlaB appeared almost identical to that of wild type FlaA (Figure 3)! FlaB now appeared helical. This means that somehow, the suppressor mutations in this fms mutant allowed FlaB to take on the structure and function of FlaA. In other words, FlaB can almost completely compensate for the lack of FlaA!
Figure 3: Fluorescently labeled flagellar proteins of A. tumefaciens. Shown are wild type FlaA (A), wild type FlaB (B), and labeled FlaB in fms-6 mutant (C). The structure of FlaB appears most similar to wild type FlaA.
Mohari et al. experimented more with this mutant to determine how the typically straight FlaB could somehow now appear helical. They found that the FlaBY129N mutation was independently working to allow for helical flagella formation and that the SD*-FlaB mutation only helped increase FlaB production. Furthermore, they observed that when a certain amino acid called asparagine is present at position 129, in either FlaA or FlaB, a helical structure results. This amino acid is normally present only in FlaA, explaining why FlaA is naturally helical and FlaB isn’t. It’s pretty cool how changing just one amino acid can drastically change the entire structure of the protein.
This brings us to the culmination of the data presented in Mohari et al.’s 2018 article. Overall, they found that secondary flagellins, FlaB, FlaC and FlaD combine with the primary flagellin, FlaA to contribute to motility. Furthermore, a single key amino acid in FlaA seems to be behind its helical structure. When FlaB is mutated to contain this amino acid, it too takes on a helical shape. This reflects how very subtle differences can have major implications for both the structure and function of flagellins.
A. tumefaciens has generated remarkable mechanisms for colonizing and manipulating its host. Future studies will no doubt reveal additional ways in which these bacteria manage to infect a wide array of plants and even how various environmental conditions could affect their motility. It’s truly amazing how the intricate movements of bacteria continue to inform our understanding of the microscopic world. I’m sure Leeuwenhoek would be thrilled to know his interests have been taken up by millions, all striving together to determine the workings of these tiny “animalcules”.
About the author:
Abbie Collette ‘21 is a biochemistry major and psychology minor from Maine. She works in the McMenimen lab at Mount Holyoke College studying small heat shock proteins. She enjoys running, playing the cello, and taking care of her many pets.
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