Session Y42: Physics of Bacteria and Viruses

Membrane-mediated interaction between retroviral capsids

A retrovirus is an RNA virus that is replicated through a unique strategy of reverse transcription. Unlike regular enveloped viruses which are assembled inside the host cells, the assembly of retroviral capsids happens right on the cell membrane. During the assembly process, the partially formed capsids deform the membrane, giving rise to an elastic energy. When two such partial capsids approach each other, this elastic energy changes. Or in other words, the two partial capsids interact with each other via the membrane. This membrane mediated interaction between partial capsids plays an important role in the kinetics of the assembly process. In this work, this membrane mediated interaction is calculated both analytically and numerically. It is worth noting that the diferential equation determining the membrane shape in general nonlinear and cannot be solved analytically,except in the linear region of small deformations. And it is exactly the nonlinear regime that is important for the assembly kinetics of retroviruses as it provides a large energy barrier. The theory developed here is applicable to more generic cases of membrane mediated interactions between two membrane-embedded proteins.   

Discrete fracture patterns of virus shells reveal mechanical building blocks

Viral shells are self-assembled protein nanocontainers with remarkable material properties. They combine simplicity of construction with toughness and complex functionality. To date we know little about how virus structure determines assembly pathways and shell mechanics. We have used atomic force microscopy to study structural failure of the shells of the bacteriophage $\Phi $29. We observed rigidity patterns following the symmetry of the capsid proteins and under prolonged force exertion, we see fractures along well-defined lines of the 2D crystal lattice. We found the mechanically most stable building block of the shells was a trimer. Our approach of ``reverse engineering'' the virus shells thus made it possible to identify stable structural intermediates. Such stable intermediates point to a hierarchy of interactions among equal building blocks correlated with distinct next-neighbor interactions. The results also demonstrate that concepts from macroscopic materials science, such as fracture, can be usefully employed in molecular engineering.   

Pili-driven surface motility of Myxococcus xanthus

Myxococcus xanthus is a common, rod-shaped soil-dwelling bacterium with complex motility characteristics. In groups, M. xanthus bacteria can move via social ``S'' motility, in which the Type IV Pili (TFP) attach to secreted exopolysaccharides (EPS). We examine this motility mechanism using high-framerate video acquisition, taking data on individual bacteria at 400 frames per second; using particle tracking algorithms, we algorithmically reconstruct the bacterial trajectories. The motion of a single bacterium as it is pulled by its TFP through the EPS layer on the surface is not smooth, but instead displays distinct plateaus and slips, with a wide range of plateau and slip lengths. The distribution of slips exhibits power law scaling, consistent with a crackling noise model; crackling noise has previously been used to model nonbiological systems such as earthquake dynamics and Barkhausen noise. We show quantitative agreement between mean field friction models and observed bacterial dynamics. We demonstrate that the crackling noise behavior of M. xanthus is strongly dependent on the presence of EPS, but is unaffected by the chemotactic behavior of the bacterium; we also demonstrate velocity coupling between pairs of bacteria in the early stages of social motility.   

Pili-taxis: Clustering of \textit{Neisseria gonorrhoeae} bacteria

The first step of colonization of \textit{Neisseria gonorrhoeae} bacteria, the etiological agent of gonorrhea, is the attachment to human epithelial cells. The attachment of \textit{N. gonorrhoeae} bacteria to surfaces or other cells is primarily mediated by filamentous appendages, called type IV pili (Tfp). Cycles of elongation and retraction of Tfp are responsible for a common bacterial motility called twitching motility which allows the bacteria to crawl over surfaces. Experimentally, \textit{N. gonorrhoeae} cells initially dispersed over a surface agglomerate into round microcolonies within hours. It is so far not known whether this clustering is driven entirely by the Tfp dynamics or if chemotactic interactions are needed. Thus, we investigate whether the agglomeration may stem solely from the pili-mediated attraction between cells. By developing a statistical model for pili-taxis, we try to explain the experimental measurements of the time evolution of the mean cluster size, number of clusters, and area fraction covered by the cells.   

The Psl economy in early P. aeruginosa biofilm development

Psl from P. aeruginosa (PAO1) is a mannose- and galactose-rich exopolysaccharide (EPS). It has been shown that Psl plays an important role in bacterial surface adhesion. Here, we examine role of Psl in controlling motility and microcolony formation during early biofilm development, by translating video microscopy movies into searchable databases of bacterial trajectories. We use a massively-parallel cell tracking algorithm to extract the full motility history of every cell in a large community. We find that at early stages of growth, P. aeruginosa motility is guided by Psl and self-organize in a manner analogous to a capitalist economic system, resulting in a power law bacterial distribution where a small number of bacteria are extremely ``rich'' in communally produced Psl. By comparing overproducers and underproducers of Psl, we find that local Psl levels determine post-division cell fates: High local Psl levels drive the formation of sessile microcolonies that grow exponentially.   

Roles of Pel and Psl in very early biofilm development

Biofilms are dynamic, multicellular communities of unicellular organisms. Biofilms cause many chronic infections; an important case is the opportunistic human pathogen \textit{Pseudomonas aeruginosa}. Bacteria in biofilms produce an extracellular matrix that binds bacteria to each other and to a surface. The two primary extracellular matrix components produced by \textit{P. aeruginosa} are the polysaccharides Pel and Psl. Here we examine the roles of Pel and Psl in the very early stages of biofilm development, just after initial surface attachment. We use high-throughput automated tracking and analysis to compare wild-type bacteria with mutants incapable of producing Pel, Psl, or both. We examine motion on a surface as well as inter-bacterial interactions. These results quantify the unique roles played by Pel and Psl and show an unexpected relationship between Pel expression and adhesion to a surface.   

Quorum activation at a distance: spatiotemporal patterns of gene regulation from diffusion of an autoinducer signal

Bacteria in colonies coordinate gene regulation through the exchange of diffusible signal molecules known as autoinducers (AI). This ``quorum signaling'' often occurs in physically heterogeneous and spatially extended environments such as biofilms. Under these conditions the space and time scales for diffusion of the signal limit the range and timing of effective gene regulation. We expect that spatial and temporal patterns of gene expression will reflect physical environmental constraints as well as nonlinear transcriptional activation and feedback within the gene regulatory system. We have combined experiments and modeling to investigate how these spatiotemporal patterns develop. We embed engineered plasmid/GFP quorum sensor strains or wild type strains in a long narrow agar lane, and then introduce AI signal at one terminus of the lane. Diffusion of the AI initiates reporter expression along the length of the lane, extending to macroscopic distances of mm-cm. Resulting patterns are captured quantitatively by a mathematical model that incorporates logistic growth of the population, diffusion of AI, and nonlinear transcriptional activation. Our results show that a diffusing quorum signal can coordinate gene expression over distances of order 1cm on time scales of order 10 hrs.   

Biofilm Formation in Microscopic Double Emulsion Droplets

In natural, medical, and industrial settings, there exist surface-associated communities of bacteria known as biofilms. These highly structured films are composed of bacterial cells embedded within self-produced extracellular matrix, usually composed of exopolysaccharides, proteins, and nucleic acids; this matrix serves to protect the bacterial community from antibiotics and environmental stressors. Here, we form biofilms encapsulated within monodisperse, microscopically-sized double emulsion droplets using microfluidics. The bacteria self-organize at the inner liquid-liquid droplet interfaces, multiply, and differentiate into extracellular matrix-producing cells, forming manifold three-dimensional shell-within-a-shell structures of biofilms, templated upon the inner core of spherical liquid droplets. By using microfluidics to encapsulate bacterial cells, we have the ability to view individual cells multiplying in microscopically-sized droplets, which allows for high-throughput analysis in studying the genetic program leading to biofilm development, or cell signaling that induces differentiation.   

Atomic Force Microscope Investigations of Bacterial Biofilms Treated with Gas Discharge Plasmas

We present investigations of bacterial biofilms before and after treatment with gas discharge plasmas. Gas discharge plasmas represent a way to inactivate bacteria under conditions where conventional disinfection methods are often ineffective. These conditions involve biofilm communities, where bacteria grow embedded in an exopolysaccharide matrix, and cooperative interactions between cells make organisms less susceptible to standard inactivation methods. In this study, biofilms formed by the opportunistic bacterium \textit{Pseudomonas aeruginosa} were imaged before and after plasma treatment using an atomic force microscope (AFM). Through AFM images and micromechanical measurements we observed bacterial morphological damage and reduced AFM tip-sample surface adhesion following plasma treatment.   

High speed dynamic characterization of an \textit{E. coli} population using advanced optical methods (DDM and DFM)

The motility of microbes/bacteria in a complex environment, especially the average motility of the whole group of microorganisms, is directly related to behavior such as virulence, biofilm formation, etc. It is challenging to use traditional tracking methods to quantify the average motility of a large population. It is even more challenging when the environment is constantly changing. Two optical methods were developed to solve the problem: differential dynamic microscopy (DDM) and dark field flickering microscopy (DFM). The key features of bacteria motility were quantified automatically: average swimming speed, motile fraction, diffusion coefficient, cell body rotation speed and flagellar bundle rotation speed. This method is able to measure $\sim $10$^{4}$ cells simultaneously. With the help of a high speed camera, the timescale of the dynamic measurement can be in a wide range from 10$^{-4}$ s to 10$^{5}$ s. Using this tool, temperature effects on \textit{E. coli} motility were studied. Potential biomedically-relevant applications will also be discussed.   

Bacterial Swimming at Air/Water and Oil/Water Interfaces

The microbes inhabiting the planet over billions of years have adapted to diverse physical environments of water, soil, and interfaces between water and either solid or air. Following recent studies on bacterial swimming and accumulation near solid surfaces, we turn our attention to the behavior of Caulobacter crescentus, a singly flagellated bacterium, at water/air and water/oil interfaces. The latter is motivated by relevance to microbial degradation of crude oil in light of the recent oil spill in the Gulf of Mexico. Our ongoing study suggests that Caulobacter swarmer cells tend to get physically trapped at both water/air and water/oil interfaces, accumulating at the surface to a greater degree than boundary confinement properties like that of solid surfaces would predict. At the water/air interface, swimmers move in tight circles at half the speed of swimmers in the bulk fluid. At the water/oil interface, swimming circles are even tighter with further reduced swimming speed. We report experimental data and present preliminary analysis of the findings based on low Reynolds number hydrodynamics, the known surface tension, and surface viscosity at the interface. The analysis will help determine properties of the bacterium such as their surface charge and hydrophobicity.   

Bacterial thermotaxis by modulation of the swimming speed

It has been long established that random walkers such as bacteria can migrate in inhomogeneous environments, even without actively responding to changes they sense around them, by modulating their swimming speed and/or run time. We will show that \textit{E.coli} bacteria migrate in shallow temperature gradients due to their speed dependence on temperature even without the presence of sensing receptors. Interestingly however, we find that the direction of their migration in the gradient depends on the serine concentration in the medium. This results from the fact that the bacterial swimming speed exhibits a two-state function of serine concentration and the difference between the two states increases with temperature. Our results show that the swimming speed of the bacteria increases monotonically with temperature when serine is present at high concentration, while it decreases for temperatures above 30\r{ }C at low serine concentration. This observed difference in the speed dependence on temperature is found to be due to a change in the intracellular pH of the bacteria when serine is added to their surroundings, which occurs only in the presence of the serine receptor Tsr. We will discuss some details of the mechanism underlying this effect and its consequences for the bacterial behavior.   

Role of cell bending and slime navigation in swarms of \textit{M. xanthus}

Many bacteria use motility described as swarming to colonize surfaces that allows them to optimize their access to nutrients. The swarming of the bacterium \textit{M. xanthus} on surfaces is a remarkable interplay between motility mechanisms, cell flexibility, cell-cell adhesive interactions and directional reversals.~ The properties of individual cells from different mutant strains and density regimes will be demonstrated in this talk. Then, a computational model based on subcellular elements for cell representation and implemented on graphical processing units (GPUs) will be presented. High-quality high magnification movies of bacterial motility together with biologically justified computational simulations will be used for investigation of collective motion and order in swarming populations of bacteria.~ Collective motion will be shown to include the dynamical formation of cell clusters as well as streams of cells moving over networks of cell-generated slime tracks.   

Effect of Antimicrobial Agents on MinD Protein Oscillations in \textit{E. coli} Bacterial Cells

The pole-to-pole oscillation of MinD proteins in~\textit{E. coli} cells determines the location of the division septum, and is integral to healthy cell division. It has been shown previously that the MinD oscillation period is approximately 40 s for healthy cells [1] but is strongly dependant on environmental factors such as temperature, which may place stress on the cell [2,3]. We use a strain of \textit{E. coli} in which the MinD proteins are tagged with green fluorescent protein (GFP), allowing fluorescence visualization of the MinD oscillation. We use high-resolution total internal reflection fluorescence (TIRF) microscopy and a custom, temperature controlled flow cell to observe the effect of exposure to antimicrobial agents on the MinD oscillation period and, more generally, to analyze the time variation of the spatial distribution of the MinD proteins within the cells. These measurements provide insight into the mechanism of antimicrobial action. [1] Raskin, D.M.; de Boer, P. (1999) Proc. Natl. Acad. Sci. 96: 4971-4976. [2] Touhami, A.; Jericho, M; Rutenberg, A. (2006) J. Bacteriol. 188: 7661-7667. [3] Downing, B.; Rutenberg, A.; Touhami, A.; Jericho, M. (2009) PLoS ONE 4: e7285.   

Residence and transit times of MinD in \textit{E. coli} bacterial cells

A key step in the life of a bacterial cell is its division into two daughters cells of equal size. This process is carefully controlled and regulated so that an equal partitioning of the main cell components is obtained, which is critical for the viability of the daughter cells. In \textit{E. coli} this regulation is accomplished in part by the Min protein system, that determines the localization of the division machinery. Of particular interest is the MinD protein that exhibits an oscillation between the poles in the rod shaped bacteria. The oscillation relies on a ATP mediated dimerization of the MinD protein that allows its insertion into the inner membrane at one of the poles of the cell, followed by an interaction with the MinE protein, which releases the MinD from the membrane, allowing it to travel to the other pole of the cell where the cycle is repeated. We have studied the spatio-temporal characteristics of the MinD oscillation from which we extract the average times for the two main processes that determine the oscillation period: the residence time in the membrane and the transit time to travel the length of the cell. Additionally, we explore how these two timescales are affected by stresses on the bacterial cells due to unfavorable physiological conditions.