Projects -Fluorescent flagella -Swimming without flagella -Gliding motility -Twitching motility -Fluorescent chemotaxis proteins -Chemotactic signaling studied by FRET -Motor force generation -Switching under load

Swimming cells with different kinds of flagellar bundles. Single fields are shown (de-interlaced). The waveforms of the flagellar bundles are A, normal; B, normal or curly 1; C, curly 1 (with one of the filaments on the cell at the right with a normal distal segment); and D, semi-coiled (with one filament with a normal distal segment). Fluorescent flagella We have used fluorescence microspcopy to look in detail at the flagellar filaments of swimming Escherichia coli. Dark-field microscopy has the necessary depth of field, but the cell body scatters so much light that nothing can be seen closer than about 4 µm from its center. Differential-interference-contrast microscopy also works (pioneered earlier by Karen Fahrner and Steve Block), but it has a very shallow depth of field. Linda Turner has now succeeded with fluorescent microscopy by using amino-specific Alexa Fluor dyes (succinimidyl esters) to label the flagellar filaments. The cell bodies also are fluorescent, but not inconveniently so, while the filaments are highly fluorescent. If care is taken to inhibit photodynamic oxidation (singlet oxygen) the cells are fully motile. A number of our movies show fluorescent swimming Escherichia coli cells stained with an amino-specific Alexa Fluor dye, exposed to 0.2 ms pulses of laser light (514 nm) at 60Hz and recorded with an ordinary black-and-white CCD camera. See Movies, Swimming E. coli. A cell is propelled by rotation of helical flagellar filaments, which arise at random points on its surface. The filaments are several micrometers long but only about 20 nm in diameter, each driven at its base by a reversible rotary motor at rates of order 100 Hz. A cell "runs" (moves steadily forward) when pushed by a bundle of filaments and "tumbles" (moves erratically in place with little net displacement) when the bundle comes apart. The motors turn either clockwise or counterclockwise (as seen by an observer looking at the drive shaft as it emerges from the cell wall). When a motor turns counterclockwise, its filament tends to be left handed; when it turns clockwise, its filament tends to transform to one of several right-handed forms. Illustration of the different polymorphic forms produced by counterclockwise or clockwise motor rotation. Filaments in the bundle are usually normal, i.e., left-handed helices with pitch about 2.5 µm and diameter about 0.5 µm, with the motors turning counterclockwise. During the tumble, one or more motors switch to clockwise, and their filaments leave the bundle and transform to semi-coiled, i.e., right handed helices with pitch about half of normal. Polymorphic transformations tend to occur in the sequence normal, semi-coiled, and curly 1, with changes in the direction of a run correlated with transformation to the semi-coiled form. As the cell begins to move in a new direction other structures are seen; e.g., a curly 1 filament, in which the helices are right handed (pitch and diameter each about half of normal). When the motors driving the curly 1 filaments switch back to counterclockwise, their filaments relax to normal and rejoin the bundle. Then for a time, the cell is propelled by normal filaments turning counterclockwise (still in the bundle) and one or more curly 1 filaments turning clockwise. The traditional view has been that cells run when all of the filaments spin counterclockwise and tumble when they all spin clockwise, causing the bundle to fly apart and some of the filaments to transform from normal to curly. As noted above, our fluorescence data indicate that things are more complicated. Runs can occur with filaments of any polymorphic form; although, the normal form predominates. For a cell to tumble, not every filament needs to change its direction of rotation. Different filaments can change directions at different times, or a tumble can result from the change in direction of only one. See Movies, Swimming E. coli. Reference Turner, L., Ryu, W.S., and Berg, H.C. "Real-time imaging of fluorescent flagellar filaments." J. Bacteriol. 182, 2793-2801 (2000).

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