Studying the Dynamics of Flagella in Multicellular Communities of Escherichia coli by Using Biarsenical Dyes

Abstract

This paper describes a new approach for labeling intact flagella using the biarsenical dyes FlAsH and ReAsH and imaging their spatial and temporal dynamics on live Escherichia coli cells in swarming communities of bacteria by using epifluorescence microscopy. Using this approach, we observed that (i) bundles of flagella on swarmer cells remain cohesive during frequent collisions with neighboring cells, (ii) flagella on nonmotile swarmer cells at the leading edge of the colony protrude in the direction of the uncolonized agar surface and are actively rotated in a thin layer of fluid that extends outward from the colony, and (iii) flagella form transient interactions with the flagella of other swarmer cells that are in close proximity. This approach opens a window for observing the dynamics of cells in communities that are relevant to ecology, industry, and biomedicine.Swimming cells of Escherichia coli are propelled through liquids using flagella that are arranged peritrichously (e.g., uniformly distributed). Each flagellum is rotated by a motor at a rate of ϳ100 Hz using the proton motive force across the cell wall. The balance of torque across the cell results in the counter rotation of the cell body at a frequency of ϳ10 Hz, which biases the movement of cells suspended in fluids and in close contact with surfaces (6,19,24,33,37). The biophysical details of the function and dynamics of the flagella of individual E. coli cells suspended in fluids are well understood (6). In contrast, the role and dynamics of these organelles in cells that are in multicellular communities, where the majority of bacteria arguably reside, are just beginning to emerge (8,9,16,36).Extracellular organelles including flagella, pili, and curli fibers are involved in cell motility and the attachment of cells to surfaces, critical steps in the early formation of multicellular structures (13,22,39,48). In some communities, the dynamic movement of these organelles plays a central role in population-wide behavior. For example, the coordinated movement of individual bacteria in communities, referred to as "swarms," produces cohesive motion over length scales of hundreds of micrometers and provides a mechanism for the migration of colonies across surfaces (20,27,30,41,46,54). Swarming is a phenotype that plays a role in pathogenesis and makes it possible for bacterial colonies to transcend the confines of diffusion-limited growth. Swarming is a mechanism that cells use to replicate, expand rapidly across surfaces, and colonize niches that would be inaccessible to static multicellular structures (3,23,47,52).Swarms of E. coli cells consist of a heterogeneous population of cells with a morphology that ranges from a mononucleate, vegetative state, in which the cells are 2 to 3 m long and have ϳ3 to 7 flagella, to a morphology that is multinucleate, in which the cells are 5 to 20 m long and the density of flagella is ϳ2 to 3 flagella more per unit of surface area than vegetative cells (28). The most "differentiated" cells (e.g., those that are the...

Visualization of Flagella during Bacterial Swarming

“…Cells paused, but their flagella continued to spin and thus pump fluid over the agar in front of the swarm. A similar observation was made by Copeland et al (9). By measuring diffusion coefficients of small particles suspended in the surfactant monolayer on top of the swarm, we found that the fluid in front of the swarm became more shallow as one moved away from the swarm edge, over distances ranging from 10 to 20 m (44).…”

“…The hallmark of this maneuver, which is evident without flagellar visualization, is that the cell suddenly swims backwards at the same speed at which it was swimming forwards, without changing the orientation of its cell body. It is possible that this maneuver was not seen by Copeland et al (9) because their cells were swimming in a glassagar-glass sandwich and might have been oxygen deprived, while our cells were swimming between glass and a thin film of PDMS, which is oxygen permeable.…”

Bacterial Acrobatics on a Surface: Swirling Packs, Collisions, and Reversals during Swarming

“…Cells at the outermost edge of a swarm become jammed; however, the Berg and Weibel laboratories found that the flagella keep working and can be seen extending over the uncolonized agar (2,18). The stalled cells eventually manage to reverse back into the swarm, where they are actively motile.…”

“…Individual bacteria can be tracked, and fluorescently labeled flagella allow direct observation of the flagellar movement during swarming. Turner and colleagues used conjugated Alexa Fluor dyes to visualize flagella (18), while Copeland et al also recently labeled the flagella of swarming cells in another way, by using biarsenical dyes (2). Using these techniques, some old questions are put to rest-as highlighted below-and some new ones can now be addressed.…”

Water reservoir maintained by cell growth fuels the spreading of a bacterial swarm

“…Much has been learned about the genetics and biochemistry of bacterial swarming as well as its relevance to biofilm formation and pathogenic infections (1)(2)(3). More recent advances have been made at the single-cell level (6)(7)(8)(9). However, relatively little is known about the thin layer of fluid that supports flagellar motility and allows swarm cells to maintain a distinct physiological state (10,11).…”