Guanglai Li

Surface contact stimulates the just‐in‐time deployment of bacterial adhesins

The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide‐mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili‐dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just‐in‐time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non‐productive self‐adherence, and preventing curing of the adhesive.

Amplified effect of Brownian motion in bacterial near-surface swimming

Brownian motion influences bacterial swimming by randomizing displacement and direction. Here, we report that the influence of Brownian motion is amplified when it is coupled to hydrodynamic interaction. We examine swimming trajectories of the singly flagellated bacterium Caulobacter crescentus near a glass surface with total internal reflection fluorescence microscopy and observe large fluctuations over time in the distance of the cell from the solid surface caused by Brownian motion. The observation is compared with computer simulation based on analysis of relevant physical factors, including electrostatics, van der Waals force, hydrodynamics, and Brownian motion. The simulation reproduces the experimental findings and reveals contribution from fluctuations of the cell orientation beyond the resolution of present observation. Coupled with hydrodynamic interaction between the bacterium and the boundary surface, the fluctuations in distance and orientation subsequently lead to variation of the swimming speed and local radius of curvature of swimming trajectory. These results shed light on the fundamental roles of Brownian motion in microbial motility, nutrient uptake, and adhesion.

The elastic properties of the caulobacter crescentus adhesive holdfast are dependent on oligomers of N-acetylglucosamine.

The aquatic bacterium Caulobacter crescentus attaches to solid surfaces through an adhesive holdfast located at the tip of its polar stalk, a thin cylindrical extension of the cell membrane. In this paper, the elastic properties of the C. crescentus stalk and holdfast assembly were studied by using video light microscopy. In particular, the contribution of oligomers of N-acetylglucosamine (GlcNAc) to the elasticity of holdfast was examined by lysozyme digestion. C. crescentus cells attached to a surface undergo Brownian motion while confined effectively in a harmonic potential. Mathematical analysis of such motion enabled us to determine the force constant of the stalk-holdfast assembly, which quantifies its elastic properties. The measured force constant exhibits no dependence on stalk length, consistent with the theoretical estimate showing that the stalk can be treated as a rigid rod with respect to fluctuations of the attached cells. Therefore, the force constant of the stalk-holdfast assembly can be attributed to the elasticity of the holdfast. Motions of cells in a rosette were found to be correlated, consistent with the elastic characteristics of the holdfast. Atomic force microscopy analysis indicates that the height of a dried (in air) holdfast is approximately one-third of that of a wet (in water) holdfast, consistent with the gel-like nature of the holdfast. Lysozyme, which cleaves oligomers of GlcNAc, reduced the force constant to less than 10% of its original value, consistent with the polysaccharide gel-like nature of the holdfast. These results also indicate that GlcNAc polymers play an important role in the strength of the holdfast.

Molecular Adsorption Steers Bacterial Swimming at the Air/Water Interface

Microbes inhabiting Earth have adapted to diverse environments of water, air, soil, and often at the interfaces of multiple media. In this study, we focus on the behavior of Caulobacter crescentus, a singly flagellated bacterium, at the air/water interface. Forward swimming C. crescentus swarmer cells tend to get physically trapped at the surface when swimming in nutrient-rich growth medium but not in minimal salt motility medium. Trapped cells move in tight, clockwise circles when viewed from the air with slightly reduced speed. Trace amounts of Triton X100, a nonionic surfactant, release the trapped cells from these circular trajectories. We show, by tracing the motion of positively charged colloidal beads near the interface that organic molecules in the growth medium adsorb at the interface, creating a high viscosity film. Consequently, the air/water interface no longer acts as a free surface and forward swimming cells become hydrodynamically trapped. Added surfactants efficiently partition to the surface, replacing the viscous layer of molecules and reestablishing free surface behavior. These findings help explain recent similar studies on Escherichia coli, showing trajectories of variable handedness depending on media chemistry. The consistent behavior of these two distinct microbial species provides insights on how microbes have evolved to cope with challenging interfacial environments.

Flagellin Redundancy in Caulobacter crescentus and Its Implications for Flagellar Filament Assembly

Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in ΔfljK ΔfljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.

Holdfast spreading and thickening during Caulobacter crescentus attachment to surfaces

BackgroundAdhesion to surfaces facilitates many crucial functions of microbes in their natural habitats. Thus understanding the mechanism of microbial adhesion is of broad interest to the microbiology research community.ResultsWe report a study by fluorescence imaging and atomic force microscopy on the growth in size and thickness of the holdfast of synchronized Caulobacter crescentus cells as they attach to a glass surface. We found that the holdfast undergoes a two-stage process of spreading and thickening during its morphogenesis. The holdfast first forms a thin plate on the surface. The diameter of the holdfast plate reaches its final average value of 360 nm by the cell age of ~ 30 min, while its thickness further increases until the age of ~ 60 min. Our AFM analysis indicates that the holdfast is typically thicker in the middle, with gradual falloff in thickness towards the outer edge.ConclusionsWe propose that the newly secreted holdfast substance is fluid-like. It has strong affinity to the surface and cures to form a plate-like holdfast capable of supporting strong and permanent adhesion.

Single filament electrophoresis of F-actin and filamentous virus fd

We have developed an electrophoretic cell suitable for single-molecule electrophoresis. The setup works for fluorescently labeled macromolecules by direct recording of their motion under an external electric field. The electrophoretic mobility of rodlike, polydisperse actin filaments (F-actin) were measured, as well as its dependence on the orientation of the filaments. A dipping effect is observed and quantitatively accounted for by the difference in hydrodynamic drag between motions along and perpendicular to the long axis of a filament. When averaged over all orientations, the mobility of F-actin in 50 mM KCl and 2 mM MgCl(2) is determined to be -(8.5+/-0.7) x 10(-5) cm(2)(V s). This method is also used to compare the mobility of F-actin and fd virus in a mixture of them. A reliable ratio of 1.26 is measured for fd virus to F-actin. The influence of the orientation dependent drag on electrophoretic mobility is discussed and a strategy for reliable measurement is proposed.

Altered motility of Caulobacter Crescentus in viscous and viscoelastic media

BackgroundMotility of flagellated bacteria depends crucially on their organelles such as flagella and pili, as well as physical properties of the external medium, such as viscosity and matrix elasticity. We studied the motility of wild-type and two mutant strains of Caulobacter crescentus swarmer cells in two different types of media: a viscous and hyperosmotic glycerol-growth medium mixture and a viscoelastic growth medium, containing polyethylene glycol or polyethylene oxide of different defined sizes.ResultsFor all three strains in the medium containing glycerol, we found linear drops in percentage of motile cells and decreases in speed of those that remained motile to be inversely proportional to viscosity. The majority of immobilized cells lost viability, evidenced by their membrane leakage. In the viscoelastic media, we found less loss of motility and attenuated decrease of swimming speed at shear viscosity values comparable to the viscous medium. In both types of media, we found more severe loss in percentage of motile cells of wild-type than the mutants without pili, indicating that the interference of pili with flagellated motility is aggravated by increased viscosity. However, we found no difference in swimming speed among all three strains under all test conditions for the cells that remained motile. Finally, the viscoelastic medium caused no significant change in intervals between flagellar motor switches unless the motor stalled.ConclusionHyperosmotic effect causes loss of motility and cell death. Addition of polymers into the cell medium also causes loss of motility due to increased shear viscosity, but the majority of immobilized bacteria remain viable. Both viscous and viscoelastic media alter the motility of flagellated bacteria without affecting the internal regulation of their motor switching behavior.

Flagellar Motor Switching in Caulobacter Crescentus Obeys First Passage Time Statistics.

A Caulobacter crescentus swarmer cell is propelled by a helical flagellum, which is rotated by a motor at its base. The motor alternates between rotating in clockwise and counterclockwise directions and spends variable intervals of time in each state. We measure the distributions of these intervals for cells either free swimming or tethered to a glass slide. A peak time of around one second is observed in the distributions for both motor directions with counterclockwise intervals more sharply peaked and clockwise intervals displaying a larger tail at long times. We show that distributions of rotation intervals fit first passage time statistics for a biased random walker and the dynamic binding of CheY-P to FliM motor subunits accounts for this behavior. Our results also suggest that the presence of multiple CheY proteins in C. crescentus may be responsible for differences between its switching behavior and that of the extensively studied E. coli.

Tracking Bacterial Swimming Near a Solid or Air Surface

Bacterial motility near a boundary has many implications in surface contamination, biofilm formation, and infection. We study the near surface swimming behavior of microbes using a monotrichous bacterium called Caulobacter crescentus. C. crescentus is a waterborne bacterium that progresses through a two-stage life cycle, alternating between a stationary stalked cell and a motile swarmer cell. The swarmer cell uses its flagellum to propel itself through fluid. We use a non-chemotactic, forward swimming mutant strain lacking pili, SB3860, to focus on near surface motility. Using defocused particle tracking to relate the radius of a dark field image of a swarmer cell to its height away from the microscopes focal plane, we track its motion in three dimensions as a function of time. With this approach, we measure the density distribution from the surface, the speed in relation to height, and the trajectories of bacterial motion. In investigating the behavior of the cells at two different interfaces (water-solid and water-air), we have discovered significant differences in speed, density distribution, and the trajectory shape. At the glass-water interface, a much higher density of cells is found within a micron from the surface, and the average speed reaches a maximum within a few micrometers. At the water-air boundary, however, there is a much wider spread of cell distribution, and the average swimming speed close to surface is significantly lower. Furthermore, most cells at the water-air interface swim in circular trajectories, whereas at the solid-water interface, most trajectories appear straight as the forward swimming cells tend to leave the surface within a fraction of a second. These intriguing properties may be relevant to a variety of microbial functions at the interface, such as accumulation, biofilm formation, and adhesion.