Person:

Berg, Howard

When grown in a rich medium on agar, many bacteria elongate, produce more flagella, and swim in a thin film of fluid over the agar surface in swirling packs. Cells that spread in this way are said to swarm. The agar is a solid gel, with pores smaller than the bacteria, so the swarm/agar interface is fixed. Here we show, in experiments with Escherichia coli, that the swarm/air interface also is fixed. We deposited MgO smoke particles on the top surface of an E. coli swarm near its advancing edge, where cells move in a single layer, and then followed the motion of the particles by dark-field microscopy and the motion of the underlying cells by phase-contrast microscopy. Remarkably, the smoke particles remained fixed (diffusing only a few micrometers) while the swarming cells streamed past underneath. The diffusion coefficients of the smoke particles were smaller over the virgin agar ahead of the swarm than over the swarm itself. Changes between these two modes of behavior were evident within 10–20 μm of the swarm edge, indicating an increase in depth of the fluid in advance of the swarm. The only plausible way that the swarm/air interface can be fixed is that it is covered by a surfactant monolayer pinned at its edges. When a swarm is exposed to air, such a monolayer can markedly reduce water loss. When cells invade tissue, the ability to move rapidly between closely opposed fixed surfaces is a useful trait.

When vegetative bacteria that can swim are grown in a rich medium on an agar surface, they become multinucleate, elongate, synthesize large numbers of flagella, produce wetting agents, and move across the surface in coordinated packs: they swarm. We examined the motion of swarming Escherichia coli, comparing the motion of individual cells to their motion during swimming. Swarming cells' speeds are comparable to bulk swimming speeds, but very broadly distributed. Their speeds and orientations are correlated over a short distance (several cell lengths), but this correlation is not isotropic. We observe the swirling that is conspicuous in many swarming systems, probably due to increasingly long-lived correlations among cells that associate into groups. The normal run-tumble behavior seen in swimming chemotaxis is largely suppressed, instead, cells are continually reoriented by random jostling by their neighbors, randomizing their directions in a few tenths of a second. At the edge of the swarm, cells often pause, then swim back toward the center of the swarm or along its edge. Local alignment among cells, a necessary condition of many flocking theories, is accomplished by cell body collisions and/or short-range hydrodynamic interactions.

In Escherichia coli, the behavior of the flagellar rotary motor near zero load can be studied by scattering light from nanogold spheres attached to proximal hooks of cells lacking flagellar filaments. We used this method to monitor changes in speed when cells were subjected to changes in temperature or shifted from a medium made with (H{2}O) to one made with (D{2}O). In (H{2}O), the speed increased with temperature in a near-exponential manner, with an activation enthalpy of (52 \pm 4) kJ/mol ((12.0 \pm 1.0) kcal/mol). In (D{2}O), the speed increased in a similar manner, with an activation enthalpy of (50 \pm 4) kJ/mol. The speed in (H{2}O) was higher than that in (D{2}O) by a factor of (1.53 \pm 0.14). We performed comparison studies of variations in temperature and solvent isotope, using motors operating at high loads. The variations were small, consistent with previous observations. The implications of these results for proton translocation are discussed.

Bacterial flagellar filaments grow at their distal ends, from flagellin that travels through a central channel (\sim 2 nm) in diameter. The flagellin is extruded from the cytoplasm by a pump powered by a proton motive force (PMF). We measured filament growth in cells near the mid-exponential-phase with flagellin bearing a specific cysteine-for-serine substitution, allowing filaments to be labeled with sulfhydryl-specific fluorescent dyes. We labeled filaments first with a green maleimide dye and then, following an additional period of growth, with a red maleimide dye. The contour lengths of the green and red segments were measured. The average lengths of red segments ((2.3 \mu m)) were the same regardless of the lengths of the green segments from which they grew (ranging from less than 1 to more than (9 \mu m) in length). Thus, flagellar filaments do not grow at a rate that decreases exponentially with length, as formerly supposed. If flagellar filaments were broken by viscous shear, the broken filaments continued to grow. Identical results were obtained whether flagellin was expressed from fliC on the chromosome under the control of its native promoter or on a plasmid under the control of the arabinose promoter.

Flagellated bacteria can swim within a thin film of fluid that coats a solid surface, such as agar; this is a means for colony expansion known as swarming. We found that micron-sized bubbles make excellent tracers for the motion of this fluid. The microbubbles form explosively when small aliquots of an aqueous suspension of droplets of a water-insoluble surfactant (Span 83) are placed on the agar ahead of a swarm, as the water is absorbed by the agar and the droplets are exposed to air. Using these bubbles, we discovered an extensive stream (or river) of swarm fluid flowing clockwise along the leading edge of an Escherichia coli swarm, at rates of order (10 \mu m/s), about three times faster than the swarm expansion. The flow is generated by the action of counterclockwise rotating flagella of cells stuck to the substratum, which drives fluid clockwise around isolated cells (when viewed from above), counterclockwise between cells in dilute arrays, and clockwise in front of cells at the swarm edge. The river provides an avenue for long-range communication in the swarming colony, ideally suited for secretory vesicles that diffuse poorly. These findings broaden our understanding of swarming dynamics and have implications for the engineering of bacterial-driven microfluidic devices.

When cells of Escherichia coli are grown in broth and suspended at low density in a motility medium, they swim independently, exploring a homogeneous, isotropic environment. Cell trajectories and the way in which these trajectories are determined by flagellar dynamics are well understood. When cells are grown in a rich medium on agar instead, they elongate, produce more flagella, and swarm. They move in coordinated packs within a thin film of fluid, in intimate contact with one another and with two fixed surfaces, a surfactant monolayer above and an agar matrix below: they move in an inhomogeneous, anisotropic environment. Here we examine swarm-cell trajectories and ways in which these trajectories are determined by flagellar motion, visualizing the cell bodies by phase-contrast microscopy and the flagellar filaments by fluorescence microscopy. We distinguish four kinds of tracks, defining stalls, reversals, lateral movement, and forward movement. When cells are stalled at the edge of a colony, they extend their flagellar filaments outwards, moving fluid over the virgin agar; when cells reverse, changes in filament chirality play a crucial role; when cells move laterally, they are pushed sideways by adjacent cells; and when cells move forward, they are pushed by flagellar bundles in the same way as when they are swimming in bulk aqueous media. These maneuvers are described in this report.

Video techniques were used to analyze the motion of the gliding bacterium Cytophaga sp. strain U67. Cells moved singly on glass along the long axis at a speed of about 2 micrometers/s, advancing, retreating, stopping, pivoting about a pole, or flipping over. They did not flex or roll. Cells of different lengths moved at about the same speed. Cells sometimes spun continuously about a pole at a frequency of about 2 HZ, the body moving in a plane parallel to that of the glass or on the surface of a cone having either a large or a small solid angle. Polystyrene latex spheres moved to and fro on the surfaces of cells, also at a speed of about 2 micrometers/s. They moved in the same fashion whether a cell was in suspension, gliding, or at rest on the glass. Two spheres on the same cell often moved in opposite directions, passing by one another in close proximity. Small and large spheres and aggregates of spheres all moved at about the same speed. An aggregate moved down the side of a cell with a fixed orientation, even when only one sphere was in contact with the cell. Spheres occasionally left one cell and were picked up by another. Cell pretreated with small spheres did not adhere to glass. When the cells were deprived of oxygen, they stopped gliding, and the spheres stopped moving on their surfaces. The spheres became completely immobilized; they no longer moved from cell to cell or exhibited Brownian movement. Cytophaga spp. are known to have a typical gram-negative cell envelope: an inner (cytoplasmic) membrane, a thin peptidoglycan layer, and an outer (lipopolysaccharide) membrane. Our data are consistent with a model for gliding in which sites to which glass and polystyrene strongly adsorb move within the fluid outer membrane along tracks fixed to the rigid peptidoglycan framework.

Flagellated bacteria can swim across moist surfaces within a thin layer of fluid, a means for surface colonization known as swarming. This fluid spreads with the swarm, but how it does so is unclear. We used micron-sized air bubbles to study the motion of this fluid within swarms of Escherichia coli. The bubbles moved diffusively, with drift. Bubbles starting at the swarm edge drifted inward for the first 5 s and then moved outward. Bubbles starting 30 μm from the swarm edge moved inward for the first 20 s, wandered around in place for the next 40 s, and then moved outward. Bubbles starting at 200 or 300 μm from the edge moved outward or wandered around in place, respectively. So the general trend was inward near the outer edge of the swarm and outward farther inside, with flows converging on a region about 100 μm from the swarm edge. We measured cellular metabolic activities with cells expressing a short-lived GFP and cell densities with cells labeled with a membrane fluorescent dye. The fluorescence plots were similar, with peaks about 80 μm from the swarm edge and slopes that mimicked the particle drift rates. These plots suggest that net fluid flow is driven by cell growth. Fluid depth is largest in the multilayered region between approximately 30 and 200 μm from the swarm edge, where fluid agitation is more vigorous. This water reservoir travels with the swarm, fueling its spreading. Intercellular communication is not required; cells need only grow.

Mechanosensing by flagella is thought to trigger bacterial swarmer-cell differentiation, an important step in pathogenesis. How flagellar motors sense mechanical stimuli is not known. To study this problem, we suddenly increased the viscous drag on motors by a large factor, from very low loads experienced by motors driving hooks or hooks with short filament stubs, to high loads, experienced by motors driving tethered cells or 1-μm latex beads. From the initial speed (after the load change), we inferred that motors running at very low loads are driven by one or at most two force-generating units. Following the load change, motors gradually adapted by increasing their speeds in a stepwise manner (over a period of a few minutes). Motors initially spun exclusively counterclockwise, but then increased the fraction of time that they spun clockwise over a time span similar to that observed for adaptation in speed. Single-motor total internal reflection fluorescence imaging of YFP–MotB (part of a stator force-generating unit) confirmed that the response to sudden increments in load occurred by the addition of new force-generating units. We estimate that 6–11 force-generating units drive motors at high loads. Wild-type motors and motors locked in the clockwise or counterclockwise state behaved in a similar manner, as did motors in cells deleted for the motor protein gene fliL or for genes in the chemotaxis signaling pathway. Thus, it appears that stators themselves act as dynamic mechanosensors. They change their structure in response to changes in external load. How such changes might impact cellular functions other than motility remains an interesting question.