Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

University of Notre Dame

In very low density situations where a single myxobacterial cell is isolated from direct contact with other cells, the slime capsule interaction with the substrate or slime tracks on the substrate produce a viscous drag that results in a smooth gliding motion. Viscoelastic interactions of myxobacteria cells in a low-density domain close to the edge of a swarm are studied using a combination of a cell-based three-dimensional computational model and cell-tracking experiments. The model takes into account the flexible nature of Myxococcus xanthus as well as the effects of adhesion between cells arising from the interaction of the capsular polysaccharide covering two cells in contact with each other. New image and dynamic cell curvature analysis algorithms are used to track and measure the change in cell shapes that occur as flexible cells undergo significant bending during collisions resulting in direct calibration of the model parameters. Like aspect-ratio and directional reversals, the flexibility of cells and the adhesive cell-cell and cell-substrate interactions of M. xanthus play an important role in smooth gliding and more efficient swarming.

Study of elastic collisions of Myxococcus xanthus in swarms.

Blebs are balloon-shaped membrane protrusions that form during many physiological processes. Using computer simulation of a particle-based model for self-assembled lipid bilayers coupled to an elastic meshwork, we investigated the phase behavior and kinetics of blebbing. We found that blebs form for large values of the ratio between the areas of the bilayer and the cytoskeleton. We also found that blebbing can be induced when the cytoskeleton is subject to a localized ablation or a uniform compression. The results obtained are qualitatively in agreement with the experimental evidence and the model opens up the possibility to study the kinetics of bleb formation in detail.

Computer simulation of cytoskeleton-induced blebbing in lipid membranes.

Automatically detecting and tracking the motion of Myxococcus xanthus bacteria provide essential information for studying bacterial cell motility mechanisms and collective behaviors. However, this problem is difficult due to the low contrast of microscopy images, cell clustering and colliding behaviors, etc. To overcome these difficulties, our approach starts with a level set based pre-segmentation of cell clusters, followed by an enhancement of the rod-like cell features and detection of individual bacterium within each cluster. A novel method based on "spikes" of the outer medial axis is applied to divide touching (colliding) cells. The tracking of cell motion is accomplished by a non-crossing bipartite graph matching scheme that matches not only individual cells but also the neighboring structures around each cell. Our approach was evaluated on image sequences of moving M. xanthus bacteria close to the edge of their swarms, achieving high accuracy on the test data sets.

Detecting and tracking motion of Myxococcus xanthus bacteria in swarms.

The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.

Interconnected cavernous structure of bacterial fruiting bodies.

In this paper we develop a continuum theory of clustering in ensembles of self-propelled inelastically colliding rods with applications to collective dynamics of common gliding bacteria Myxococcus Xanthus. A multiphase hydrodynamic model that couples densities of oriented and isotropic phases is described. This model is used for the analysis of an instability that leads to spontaneous formation of directionally moving dense clusters within initially dilute isotropic "gas" of myxobacteria. Numerical simulations of this model confirm the existence of stationary dense moving clusters and also elucidate the properties of their collisions. The results are shown to be in a qualitative agreement with experiments.

Continuum modeling of clustering of myxobacteria.

Links between cell division and other cellular processes are poorly understood. It is difficult to simultaneously examine division and function in most cell types. Most of the research probing aspects of cell division has experimented with stationary or immobilized cells or distinctly asymmetrical cells. Here we took an alternative approach by examining cell division events within motile groups of cells growing on solid medium by time-lapse microscopy. A total of 558 cell divisions were identified among approximately 12,000 cells. We found an interconnection of division, motility, and polarity in the bacterium Myxococcus xanthus. For every division event, motile cells stop moving to divide. Progeny cells of binary fission subsequently move in opposing directions. This behavior involves M. xanthus Frz proteins that regulate M. xanthus motility reversals but is independent of type IV pilus "S motility." The inheritance of opposing polarity is correlated with the distribution of the G protein RomR within these dividing cells. The constriction at the point of division limits the intracellular distribution of RomR. Thus, the asymmetric distribution of RomR at the parent cell poles becomes mirrored at new poles initiated at the site of division.

Cell division resets polarity and motility for the bacterium Myxococcus xanthus.

Pseudomonas aeruginosa is a ubiquitous bacterium that survives in many environments, including as an acute and chronic pathogen in humans. Substantial evidence shows that P. aeruginosa behavior is affected by its motility, and appendages known as flagella and type IV pili (TFP) are known to confer such motility. The role these appendages play when not facilitating motility or attachment, however, is unclear. Here we discern a passive intercellular role of TFP during flagellar-mediated swarming of P. aeruginosa that does not require TFP extension or retraction. We studied swarming at the cellular level using a combination of laboratory experiments and computational simulations to explain the resultant patterns of cells imaged from in vitro swarms. Namely, we used a computational model to simulate swarming and to probe for individual cell behavior that cannot currently be otherwise measured. Our simulations showed that TFP of swarming P. aeruginosa should be distributed all over the cell and that TFP-TFP interactions between cells should be a dominant mechanism that promotes cell-cell interaction, limits lone cell movement, and slows swarm expansion. This predicted physical mechanism involving TFP was confirmed in vitro using pairwise mixtures of strains with and without TFP where cells without TFP separate from cells with TFP. While TFP slow swarm expansion, we show in vitro that TFP help alter collective motion to avoid toxic compounds such as the antibiotic carbenicillin. Thus, TFP physically affect P. aeruginosa swarming by actively promoting cell-cell association and directional collective motion within motile groups to aid their survival.

Type IV pili interactions promote intercellular association and moderate swarming of Pseudomonas aeruginosa.

Tracking the motion of Myxococcus xanthus is a crucial step for fundamental bacteria studies. Large number of bacterial cells involved, limited image resolution, and various cell behaviors (e.g., division) make tracking a highly challenging problem. A common strategy is to segment the cells first and associate detected cells into moving trajectories. However, known detection association algorithms that run in polynomial time are either ineffective to deal with particular cell behaviors or sensitive to segmentation errors. In this paper, we propose a polynomial time hierarchical approach for associating segmented cells, using a new Earth Mover's Distance (EMD) based matching model. Our method is able to track cell motion when cells may divide, leave/enter the image window, and the segmentation results may incur false alarm, detection lost, and falsely merged/split detections. We demonstrate it on tracking M. xanthus. Applied to error-prone segmented cells, our algorithm exhibits higher track purity and produces more complete trajectories, comparing to several state-of-the-art detection association algorithms.

Cameron W. Harvey

Department of Applied and Computational Mathematics and Statistics

University of Notre Dame

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Cameron W. Harvey

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Applied and Computational Mathematics and Statistics

University of Notre Dame

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Department of Applied and Computational Mathematics and Statistics