June 24, 2013
A group of researchers led by Daisuke Nakane, of the Graduate School of Biomedical Sciences, Division of Microbiology and Oral Infection, and research fellow of the Japan Society for the Promotion of Science (now an assistant professor in Gakushuin University's Faculty of Science) and Professor Koji Nakayama demonstrated the mechanism used in the gliding motion of a certain strain of bacteria. Their research was published in the electronic version of the Proc. Natl. Acad. Sci. USA (1).
Bacteria in the phylum Bacteroidetes are found in a wide range of environments, such as in the sea, in soil, and in the human body. Many of these have the ability to move over surfaces via what is called gliding motility. Gliding motility fundamentally differs from the known motility mechanisms used by other bacteria, such as the use of flagella or pili, but its actual mechanism was a complete mystery.
A gene cluster unique to the Bacteroidetes phylum is involved in this motion. Approximately 20 genes have been identified, with multiple proteins located along the bacterial membrane being used for gliding. One interesting discovery was that Porphyromonas gingivalis, a periodontal pathogen that is similar to these gene clusters, is involved in the secretion of pathogenic protease (2).
This indicates that the Bacteroidetes bacteria contains a new mechanism linking gliding motility and protein secretion, much like the relationship between flagella and Type III secretion systems. Flavobacterium johnsoniae, known for having a high degree of motor activity, can glide along a glass surface at a speed of approximately 2μm/s.
When beads were attached to SprB, a cell-surface protein involved in motility, they were observed to move at a speed of approximately 2μm/s across the cell surface. SprB is a massive 700 kDa protein, and based on its amino-acid sequence and localization, it is believed to function as adhesion when gliding. However, it was not known whether this bead movement was caused by direct motion by the SprB protein. The research team performed direct fluorescent labeling of this cell-surface protein, investigating its dynamics.
SprB was labeled with antibodies in order to directly visualize its behavior during bacterial movement. Immunofluorescence microscopy of chemically fixed cells detected 20 to 30 signals on the cells' surfaces. In order to confirm whether these signals were moving on the surface of the cells, immunofluorescence microscopy was performed without chemically fixing cells. When antibodies were added to bacteria moving over glass, the capabilities of the cells to bind to and move across the glass were weakened, dependent on the concentration.
This suggested that SprB protein is a cell-surface protein directly involved in gliding motility. When the antibody concentration was diluted 100-fold, the cells retained approximately 60% of their binding and gliding capabilities, and no impact was observed on SprB localization. One interesting observation was that the signals moved around the circumference of the cells.
In order to investigate this protein movement and its relation to gliding motility, antiserum was added to the cells to stop their gliding motility. Bacteria generally use ATP or proton motive force (PMF) to move. When CCCP, a PMF inhibitor, was added, protein movement stopped within 3 seconds, and the cells stopped moving. This effect was reversible, and within 6 seconds of removing the CCCP, both protein and cell movement resumed. This phenomenon has not been observed with other inhibitors. These results suggest that movement of SprB is dependent on PMF, and that it is essential for gliding motility.
The SprB signals exhibited translational movement around the long axes of the cells. This type of bacteria is thin, only around 500nm wide, so both the sides near the glass and the reverse sides were visible. The researchers selected cells moving straight forward, and, treating the direction of movement of the bacteria as positive, measured the speed of the SprB along the long axis. Histograms showed two peaks, with moving at 3.4±1.1μm/s and half moving at -0.5±0.5μm/s. That is, half of the SprB moved slowly towards the rear, while half moved rapidly towards the front.
This indicates that the SprB could bond both tightly and lightly to the glass. Taking the velocity of cells (1.9±0.6μm/s) moving in a straight line into consideration, signals migrating in opposite directions appeared to move at similar speeds, roughly 2μm/s. Furthermore, at the poles the SprB changed direction, moving toward the front when reaching the rear pole, and moving toward the rear when reaching the front pole. Taken together, these findings suggest that the SprB exhibits translational movement at a constant velocity along the long axes of the cells, switching direction at the poles.
Tracing the paths of the SprB signals showed that they exhibited a zig-zagging, wavelike motion. A kymograph was made representing the spatial positions of the SprB on the top and bottom of the cells, using the colors blue and red, respectively. This showed that the orbits changed from red to blue, and back to red again. In other words, when the SprB moved translationally along the long axis, they also moved from one side to the other along the short axis.
More detailed investigation was performed using total internal reflection fluorescence microscopy, looking only at the side of the cells nearest the glass. During translational movement, the SprB only moved from one side to the other by moving left, never to the right. In other words, the SprB moved over the cell surface along a closed loop wound the left.
Observation with a cryo-electron microscopy found that the surface of the cells had a filament structure. In order to investigate whether this structure was produced by the SprB, structural observation was performed using an electron microscope. Observation of the surface structure of an SprB-deficient strain found no filament structure. To directly confirm the relationship, SprB was isolated from F. johnsoniae cells. Two surfactants and ammonium sulfate precipitation were used to produce an SprB-rich cell fraction.
Examination of this fraction found 150nm-long, straight filament structures. The filaments reacted specifically with SprB antibodies, and not with other antibodies. In other words, it was discovered that the SprB protruded from the cell surface in 150nm-long protein filament structures.
These findings suggest the following model. SprB, in 150nm-long protein filament structures, use proton motive force as their source of energy, moving helically counterclockwise from pole to pole in a loop. The adhesion of the SprB to the surface being moved across creates translational movement along the long axis.
(1) Nakane D, et al. (2013) Proc Natl Acad Sci U S A, Published online before print June 18, 2013, doi: 10.1073/pnas.1219753110.
(2) Sato K, et al. (2010) Proc Natl Acad Sci U S A, 107(1):276-281.
* The research findings above were published in the electronic version of PNAS, at the website below.