Many species of swimming bacteria have a rotary structure called a "flagellum," consisting of more than twenty different kinds of proteins. By rotating their flagellar filaments and gaining propulsion, bacteria can swim freely in water. Flagella-mediated motility is essential for bacteria to move in search for better habitats and two forms have been known to date: (i) "run and tumbling" seen in peritrichous bacteria such as Escherichia coli; and (ii) "forward run-reverse-flick" seen in Vibrio alginoliticus. Such forms of flagella-mediated motility are adopted when moving in water, but they are also adopted by pathogenic bacteria to reach our internal organs. Thus, they are widely recognized as virulence factors.

Although bacterial flagella-mediated motility is closely linked to virulence, it is also known to have a significant role in allowing several symbiotic systems between animals and bacteria to function. For example, bobtail squids cannot emit light by themselves so they incorporate Aliivibrio fischeri, a bioluminescent bacteria species, to diffuse their appearance under moonlight and avoid getting caught by predators. Motility of the bioluminescent bacteria is essential for this symbiotic relationship to work. Another example can be seen with bean bugs, recognized as agricultural pests, which are known to acquire insecticide resistance by incorporating bacteria of the Burkholderia genus. Flagellar motility of the bacteria plays an important role for this form of symbiosis to work as well.

There is an extremely narrow constricted passage filled with polysaccharide-rich mucous between a bean bug's gut and its symbiotic organ. Dr. Kikuchi and his team have previously reported that only Burkholderia symbiont with motility can pass through this constricted passage while mutant strain bacteria lacking motility cannot. Interestingly, previous studies have shown that motile non-symbionts such as E. coli are unable to pass through this constricted passage, and therefore cannot reach the symbiotic organ. A similar constricted passage also exists in bobtail squids and it is known that only bioluminescent bacteria can selectively pass through it. However, the question "Why can only symbionts get through?" remains unanswered. Upon starting this study, our team hypothesized that "symbiotic Burkholderia symbiont have a unique motility mechanism that allows them to pass through the constricted area."

Imaging flagellar filaments

Flagella-mediated motility in the Burkholderia symbiont needs to be directly captured in order to verify our hypothesis. However, a flagellar filament has a diameter of only approximately 20 nm (a 100,000th of 2 mm) and can be captured only with electron microscopy observation. By discovering that flagellar filaments can be visualized under a fluorescence microscope when the cell body is treated with fluorescent dyes, we solved this issue. In addition, with the use of an EMCCD camera, considered to be the most sensitive type of camera in the world, we were able to capture flagellar filament movement at a rate of 400 frames per second. From this observation, we found that the Burkholderia symbiont can swim at a speed of 25 ?m/s (about ten times its body length) by rotating its flagellar filament at 150 rotations per second.

The discovery of a third form of flagella-mediated motility

Only known forms of motility shown by other bacteria such as E. coli have been observed with the Burkholderia symbiont in normal liquid media. Dr. Yoshiaki Kinosita, a member of the research group, created an environment simulating the sticky internal condition of the constricted passage with methylcellulose. While normal motility forms were observed as in low-viscosity media, he was also able to successfully observe flagellar filament-wrapping swimming frequently under this condition. This flagellum-wrapping movement does not match any known forms of flagella-mediated motility and can be described as a third form of flagella-mediated motility. Motility efficiency was approximately halved and was thus lower than other forms of motility when the Burkholderia symbiont moved through viscous liquid in this motility form like a drill bit.

Flagella-mediated gliding-like motility

Why does the Burkholderia symbiont display a unique motility form even when its efficiency is low? When we placed E. coli, a non-symbiont, in the same condition, we were able to obtain interesting results. As time passed, E. coli became bound to the glass surface, unable to move. On the contrary, no Burkholderia cells were observed being captured by the glass and rendered unable to move. Upon closer observation, the Burkholderia symbiont moving with normal motility forms were captured but then they wrapped their flagella and freed themselves, becoming able to freely move around the glass surface. Furthermore, by using a total internal reflection fluorescence microscope, which allows high-resolution observation within the vicinity of the glass surface, we were able to show that there is contact between the flagellar filament and the glass surface when gliding-like motility is shown. Considering that the glass surface is uneven, it can be assumed that the flagellar filament wrapped around the cell body perfectly fits into the surface grooves, rotates, and generates propulsion, allowing for effective motility on solid surfaces. Thus, this third form of motility could be described as essential to move effectively on extracellular matrix surfaces.

Visualization of flagellum-wrapping movement in squid symbiotic bacteria

Is the flagellum-wrapping movement unique to the Burkholderia symbionts symbionts? To address this question, we looked at A. fischeri, a symbiont of bobtail squids. Like bean bugs, bobtail squids have a mechanism to selectively incorporate symbiotic bacteria, and only motile A. fischeri are known to be able to reach the symbiotic organs. When A. fischeri were treated with fluorescent dyes and observed in the same way as the Burkholderia symbiont, we found that A. fischeri also swim by wrapping their flagellar filaments around their bodies. This implies the possibility of the newly discovered third form of flagella-mediated motility being adopted across many different species of symbiotic bacteria.

This study revealed that the transition from normal forms of motility to flagellar filament-wrapping motility is achieved by reversing the direction of flagellum rotation from counterclockwise to clockwise. However, this change in rotational direction can also be seen in E. coli and other bacteria species that do not display flagellum wrapping. Therefore, the transition to the third form of motility cannot be explained by the change in rotational direction alone. At the next stage, we will address this issue through structural analysis of proteins necessary to trigger the third form of flagellar motility and exhaustive gene analysis. Furthermore, we will analyze flagella-mediated motility inside the host body and see if swimming/gliding form-switching takes place area-specifically within the bean bug gut. This study will show whether the normal motility forms or the flagellum-wrapping form is more efficient when moving inside the constricted passage. By thoroughly revealing the relationship between symbiosis and the diverse forms of motility shown by bacteria at the gene level based on our studies, it is expected that the development of novel insecticides that prevent the infection and colonization of symbionts will follow.