Session L5: Single Cells and Bacteria II

Analysis of Peristaltic Waves & their Role in Migrating Physarum Plasmodia

The true slime mold \emph{Physarum polycephalum} exhibits a vast array of sophisticated manipulations of its intracellular cytoplasm. Growing microplasmodia of \emph{physarum} have been observed to adopt an elongated tadpole shape, then contract in a rhythmic, traveling wave pattern that resembles peristaltic pumping. This contraction drives a fast flow of non-gelated cytoplasm along the cell longitudinal axis. It has been hypothesized that this flow of cytoplasm is a driving factor in generating motility of the plasmodium. In this work, we use two different mathematical models to investigate how peristaltic pumping within \emph{physarum} may be used to drive cellular motility. We compare the relative phase of flow and deformation waves predicted by both models to similar phase data collected from \emph{in vivo} experiments using \emph{physarum} plasmodia. Both models suggest that a mechanical asymmetry in the cell is required to reproduce the experimental observations. Such a mechanical asymmetry is also shown to increase the potential for cellular migration, as measured by both stress generation and migration velocity.   

Changes in the flagellar bundling time account for variations in swimming behavior of flagellated bacteria in viscous media

The motility of bacteria E.coli in viscous fluids has been widely studied, although conflicting results on the effect of viscosity on swimming speed abound. The swimming mode of wild-type E.coli is idealized as a run-and-tumble sequence in which periods of straight swimming at a constant speed are randomly interrupted by a tumble, defined as a sudden change of direction with a very low speed. Using a tracking microscope, we follow cells for extended time and find that the swimming behavior of a single cell can exhibit a variety of behaviors including run-and-tumble and ``slow-random-walk'' in which the cells move at relatively low speed without the characteristic run. Although the characteristic swimming speed varies between individuals and in different polymer solutions, we find that the skewness of the speed distribution is solely a function of viscosity, and uniquely determines the ratio of the average speed to the characteristic run speed. Using Resistive Force Theory and the cell-specific measured characteristic run speed, we show that differences in the swimming behavior observed in solutions of different viscosity are due to changes in the flagellar bundling time, which increases as the viscosity rises, due to lower rotation rate of the flagellar motor.   

The coordination between mechanical and chemical subsystems initiates locomotion of Physarum plasmodial fragments

Physarum polycephalum is a multinucleated slime mold whose endoplasm flows periodically driven by the contraction of its ectoplasm, a dense shell of F-actin cross-linked by myosin molecular motors and attached to the cell membrane. We find that physarum fragments smaller than 100 microns remain round and stay in place. However, larger fragments break symmetry leading to sustained forward locomotion, in process that is reminiscent of an interfacial instability that seems to settle around two different limit cycles (traveling waves and standing waves). We use both theory and experiments to study how coordination emerges between the different mechanical and chemical subsystems of the fragment to initiate locomotion. The role of many involved factors, such as fragment size, substratum adhesiveness, rheological properties, actin polymerization and traction stresses are investigated, and we find they agree well with our predictive model.   

Helical and rod-shaped bacteria swim in helical trajectories with little additional propulsion from helical shape

It has frequently been hypothesized that the helical body shapes of flagellated bacteria may yield some advantage in swimming ability. The helical-shaped pathogen Helicobacter pylori allows us to test these claims. Using fast time-resolution and high-magnification phase-contrast microscopy to simultaneously image and track individual bacteria we determine cell body shape as well as rotational and translational speeds. Using the method of regularized Stokeslets, we directly compare observed speeds and trajectories to numerical calculations to validate the numerical model. Although experimental observations are limited to select cases, the model allows quantification of the effects of body helicity, length, and diameter. We find that due to relatively slow body rotation rates, the helical shape makes at most a 15\% contribution to propulsive thrust. The effect of body shape on swimming speeds is instead dominated by variations in translational drag required to move the cell body. Because helical cells are one of the strongest candidates for propulsion arising from the cell body, our results imply that quite generally, swimming speeds of flagellated bacteria can only be increased a little by by body propulsion.   

Shear banding of bacterial ``superfluids'' under confinement

Flow behaviors of bacterial suspensions have drawn tremendous attentions during the past years. Although the existence of active ``superfluids'' with zero or even negative apparent shear viscosity has been demonstrated in bulk rheological measurements of bacterial suspensions, the shear-induced microscopic dynamics of such exotic ``superfluids'' have not been fully explored experimentally. Here, we study concentrated \textit{E. coli} suspensions under oscillatory rectangular shear. Using high-speed confocal microscopy, we directly image the velocity profile of bacterial suspensions, which exhibits unusual symmetric shear bands under weak shear. We quantitatively show that such symmetric shear bands arise from a local stress balance and the average of different shear configurations. Consistent with our model, we also find that the correlation length of collective bacterial swarming increases linearly with the scale of confinement induced by the two parallel shear plates. Our study reveals the unique dynamics of active fluids under shear and sheds new light on the behavior of confined bacterial suspensions.   

Microfluidic~systems for investigating host-microbe relationship

The symbiosis between the bioluminescent bacterium,~\textit{Vibrio fisheri,}~and the Hawaiian bobtail squid,~\textit{Euprymna scolopes,}~has been widely studied, and this association~is used~as a model system for studying bacterial~colonization of~ciliated~host tissues. The recruitment of~\textit{Vibrio fisheri}~to a~specialized light organ in the nascent squid is facilitated by various chemosensing and mechanosensing events.~To decipher the effects of such environmental and host-derived sensors on bacterial physiology, we use specifically designed microfluidic channels to engineer chemical and mechanical fields~similar to those~observed in the light organ of the squid.~These~\textit{in vitro~}studies are aimed at~complementing ongoing \textit{in vivo}~studies in the~system squid-vibrio system. This approach enables~us, for the first time, to isolate the effect of mechanical and chemical cues on bacterial motility in this symbiosis~and to quantify the bacterial response to these cues.   

Hindered bacterial mobility in porous media flow enhances dispersion

Swimming bacteria live in porous environments characterized by dynamic fluid flows, where they play a crucial role in processes ranging from the bioremediation to the spread of infections. We study bacterial transport in a quasi-two-dimensional porous microfluidic device, which is complemented by Langevin simulations. The cell trajectories reveal filamentous patterns of high cell concentration, which result from the accumulation of bacteria in the high-shear regions of the flow and their subsequent advection. Moreover, the effective diffusion coefficient of the motile bacteria is severely hindered in the transverse direction to the flow due to decorrelation of the cells’ persistent random walk by shear-induced rotation. The hindered lateral diffusion has the surprising consequence of strongly enhancing the longitudinal bacterial transport through a dispersion effect. These results demonstrate the significant role of the flow and geometry in bacterial transport through porous media with potential implications for understanding ecosystem dynamics and engineering bioreactors.   

Directed transport of active magnetotactic bacteria in porous media flow

Swimming cell migration through porous media is a topic of ecological and technical relevance for understanding sediment ecosystems and bioremediation of soil for decontamination. We focus on magnetotactic bacteria -- which align passively with Earth's magnetic field and migrate in such sediment environments -- as a model system. The transport properties of magnetotactic bacteria are measured in a 2D microfluidic porous medium as a function of the porous microstructure geometry and under a variety of environmental conditions. In a quiescent fluid and in the absence of an external, guiding magnetic field, the effective diffusion of cells' random walk is unsurprisingly hindered with decreasing porosity due to cell-surface interactions. When guided by a magnetic field, cell trajectories acquire a net direction and form lanes, a behavior that is enhanced with increasing magnetic field. When the directed motility is coupled with an opposing fluid flow through the porous medium, convective cells form and locally trap the swimming bacteria. These results, which are corroborated by Langevin Simulations are an important step toward understanding magnetotactic bacterial ecology as well as for the magnetic guidance of microrobots in complex environments.   

Biomechanics of {\it Tetrahymena} escaping from dead ends

Behaviors of swimming microorganisms in complex environments are important in understanding cells’ distribution in nature and in industries. Although cell’s swimming and spreading in an infinite fluid has been intensively investigated, that in a narrow region bounded by walls is still unclear. Thus, in this study, we used {\it Tetrahymena thermophila} as a model microorganism, and experimentally investigated its behavior between flat plates with an angle. The results showed that the cells tended to escape from the narrow region, and the swimming velocity and the radius of curvature of the trajectories decreased as they swam narrower region. We then developed a computational model of swimming {\it Tetrahymena}. The results showed that the escaping behavior could be well explained by fluid mechanics. The obtained knowledge is useful in understanding cells’ behaviors in complex environments, such as in porous media and in a granular matter.   

Hydrodynamics of bacteriophages

Bacteriophage viruses, one of the most abundant entities in our planet, lack the ability to move independently. Instead, they crowd fluid environments in anticipation of a random encounter with bacteria. Once they 'land' on their victim's surface, they eject their genetic material inside the host cell. A big fraction of phage species, however, first attach to the flagella of bacteria. Being immotile, these so-called flagellotropic phages still manage to reach the cell body for infection, and the process by which they move up the flagellum has intrigued the scientific community for over four decades. In 1973 Berg and Anderson proposed the nut-and-bolt mechanism in which, just like a nut being rotated moves along a bolt, the phage wraps itself around a flagellum possessing helical grooves (due to the helical rows of flagellin molecules) and exploits the rotation of the flagellum in order to passively travel along it. We provide here a first-principle theoretical model for this nut-and-bolt mechanism and show that it is able to predict experiment observations.   

Large behavioral variability of motile E. coli revealed in 3D spatial exploration

Bacterial motility determines the spatio-temporal structure of microbial communities, controls infection spreading and the microbiota organization in guts or in soils. Quantitative modeling of chemotaxis and statistical descriptions of active bacterial suspensions currently rely on the classical vision of a run-and-tumble strategy exploited by bacteria to explore their environment. Here we report a large behavioral variability of wild-type E. coli, revealed in their three-dimensional trajectories. We found a broad distribution of run times for individual cells, in stark contrast with the accepted vision of a single characteristic time. We relate our results to the slow fluctuations of a signaling protein which triggers the switching of the flagellar motor reversal responsible for tumbles. We demonstrate that such a large distribution of run times introduces measurement biases in most practical situations. These results reconcile a notorious conundrum between observations of run times and motor switching statistics. Our study implies that the statistical modeling of transport properties and of the chemotactic response of bacterial populations need to be profoundly revised to correctly account for the large variability of motility features.   

Formation of Linear Gradient of Antibiotics on Microfluidic Chips for High-throughput Antibiotic Susceptibility Testing

To determine the most effective antimicrobial treatments of infectious pathogen, high-throughput antibiotic susceptibility test (AST) is critically required. However, the conventional AST requires at least 16 hours to reach the minimum observable population. Therefore, we developed a microfluidic system that allows maintenance of linear antibiotic concentration and measurement of local bacterial density. Based on the Stokes-Einstein equation, the flow rate in the microchannel was optimized so that linearization was achieved within 10 minutes, taking into account the diffusion coefficient of each antibiotic in the agar gel. As a result, the minimum inhibitory concentration (MIC) of each antibiotic against \textit{P. aeruginosa} could be immediately determined 6 hours after treatment of the linear antibiotic concentration. In conclusion, our system proved the efficacy of a high-throughput AST platform through MIC comparison with Clinical and Laboratory Standards Institute (CLSI) range of antibiotics.