Bulletin of the American Physical Society
APS March Meeting 2014
Volume 59, Number 1
Monday–Friday, March 3–7, 2014; Denver, Colorado
Session D11: Focus Session: Bacterial Biophysics II |
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Sponsoring Units: DBIO Chair: Gerard Wong, University of California, Los Angeles Room: 203 |
Monday, March 3, 2014 2:30PM - 3:06PM |
D11.00001: Geometric control of bacterial cell shape Invited Speaker: Joshua Shaevitz How bacteria grow into specific, 3D shapes remains a central mystery in microbiology. We have developed an imaging and analysis pipeline to simultaneously probe the shape of cells and the localization of proteins in 3D during growth. We find evidence for feedback between the local geometry of the cell, localization of key morphological proteins, and cell growth that helps to ensure the maintenance of rod-shape in elongating \textit{Escherichia coli} cells. [Preview Abstract] |
Monday, March 3, 2014 3:06PM - 3:18PM |
D11.00002: Nanomechanical Response of Bacterial Cells to Cationic Antimicrobial Peptides Shun Lu, Grant Walters, Richard Parg, John Dutcher The effectiveness of antimicrobial compounds can be easily screened, however their mechanism of action is much more difficult to determine. Many compounds act by compromising the mechanical integrity of the bacterial cell envelope, and our study introduces an atomic force microscopy (AFM)-based creep deformation technique to evaluate changes in the time-dependent mechanical properties of \textit{Pseudomonas aeruginosa} PAO1 bacterial cells upon exposure to two different but structurally related antimicrobial peptides: polymyxin B and polymyxin B nonapeptide. We observed a distinctive signature for the loss of integrity of the bacterial cell envelope following exposure to the peptides. Measurements performed before and after exposure, as well as time-resolved measurements and those performed at different concentrations, revealed large changes to the viscoelastic parameters that are consistent with differences in the membrane permeabilizing effects of the peptides. The AFM creep deformation measurement provides new, unique insight into the kinetics and mechanism of action of antimicrobial peptides on bacteria. [Preview Abstract] |
Monday, March 3, 2014 3:18PM - 3:30PM |
D11.00003: Atomic Force Microscopy Measurements of the Mechanical Properties of Cell Walls on Living Bacterial Cells Richard Bailey, Nic Mullin, Robert Turner, Simon Foster, Jamie Hobbs Staphylococcus aureus is a major cause of infection in humans, including the Methicillin resistant strain, MRSA. However, very little is known about the mechanical properties of these cells. Our investigations use AFM to examine live S. aureus cells to quantify mechanical properties. These were explored using force spectroscopy with different trigger forces, allowing the properties to be extracted at different indentation depths. A value for the cell wall stiffness has been extracted, along with a second, higher value which is found upon indenting at higher forces. This higher value drops as the cells are exposed to high salt, sugar and detergent concentrations, implying that this measurement contains a contribution from the internal turgor pressure. We have monitored these properties as the cells progress through the cell cycle. Force maps were taken over the cells at different stages of the growth process to identify changes in the mechanics throughout the progression of growth and division. The effect of Oxacillin has also been studied, to better understand its mechanism of action. Finally mutant strains of S. aureus and a second species Bacillus subtilis have been used to link the mechanical properties of the cell walls with the chain lengths and substructures involved. [Preview Abstract] |
Monday, March 3, 2014 3:30PM - 3:42PM |
D11.00004: Evolution of the Min Protein Oscillation in \textit{E. coli} Bacteria During Cell Growth and Division Benjamin Baylis, Maximiliano Giuliani, John Dutcher Cell division is a key step in the life of a bacterium. This process is carefully controlled and regulated so that the cellular machinery is equally partitioned into two daughter cells of equal size. In \textit{E. coli}, this is accomplished, in part, by the Min protein system, in which Min proteins oscillate along the long axis of the rod-shaped cells. We have used high magnification, time-resolved fluorescence microscopy to characterize in detail the oscillation in \textit{E. coli} cells in which the MinD proteins are tagged with green fluorescent protein (GFP). We have used a microfluidic device to confine the bacteria into microchannels that allows us to track the evolution of the oscillation in cells as they grow and divide in LB growth media. In particular, we have tracked the loss of synchrony between the oscillations in the daughter cells following cell division. [Preview Abstract] |
Monday, March 3, 2014 3:42PM - 3:54PM |
D11.00005: Single Cell Response to Time-dependent Stimuli using a Microfluidic Bioreactor Eric M. Johnson-Chavarria, Utsav Agrawal, Melikhan Tanyeri, Thomas E. Kuhlman, Charles M. Schroeder Cellular adaptation is critical for survival under uncertain or dynamic environmental conditions. Recent studies have reported the ability of biological systems to implement low-pass filters to distinguish high frequency noise in environmental stimuli from lower frequency input signals, yet we still lack a complete understanding of this phenomenon. In this work, we report a microfluidic-based platform for single cell analysis that provides dynamic control over periodic, time-dependent culture media. Single cells are confined in free solution by the sole action of gentle fluid flow, thereby enabling non-perturbative trapping of cells for long time scales. In this way, our microfluidic-based technique provides the ability to control external stimuli with precise methods while observing non-adherent cells over long timescales. Using this approach, we observed intranucleoid diffusion of genetic repressor proteins released from a chromosomal binding array. Overall, this microfluidic approach provides a direct method for sustaining periodic environmental conditions, measuring growth rates, and detecting gene expression of single cells in free solution. [Preview Abstract] |
Monday, March 3, 2014 3:54PM - 4:06PM |
D11.00006: quenched-smFISH: Counting small RNA in Pathogenic Bacteria Douglas Shepherd, Nan Li, Sofiya Micheva-Viteva, Brian Munsky, Elizabeth Hong-Geller, James Werner Here, we present a modification to single-molecule fluorescence in situ hybridization, quenched smFISH (q-smFISH), that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. Exploiting an automated, multi-color wide-field microscope and GPU-accelerated data analysis package, we analyzed the statistics of sRNA expression in thousands of individual Yersinia pseudotuberculosis and Yersinia pestis bacteria before and during a simulated infection. Before infection, we find only a small fraction of either bacteria express the small RNAs YSR35 or YSP8. The copy numbers of these RNA are increased during simulated infection, suggesting a role in pathogenesis. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in bacterial regulatory networks. [Preview Abstract] |
Monday, March 3, 2014 4:06PM - 4:18PM |
D11.00007: Towards rationally redesigning bacterial signaling systems using information encoded in abundant sequence data Ryan Cheng, Faruck Morcos, Herbert Levine, Jose Onuchic An important challenge in biology is to distinguish the subset of residues that allow bacterial two-component signaling (TCS) proteins to preferentially interact with their correct TCS partner such that they can bind and transfer signal. Detailed knowledge of this information would allow one to search sequence-space for mutations that can systematically tune the signal transmission between TCS partners as well as re-encode a TCS protein to preferentially transfer signals to a non-partner. Motivated by the notion that this detailed information is found in sequence data, we explore the mutual sequence co-evolution between signaling partners to infer how mutations can positively or negatively alter their interaction. Using Direct Coupling Analysis (DCA) for determining evolutionarily conserved interprotein interactions, we apply a DCA-based metric to quantify mutational changes in the interaction between TCS proteins and demonstrate that it accurately correlates with experimental mutagenesis studies probing the mutational change in the \emph{in vitro} phosphotransfer. Our methodology serves as a potential framework for the rational design of TCS systems as well as a framework for the system-level study of protein-protein interactions in sequence-rich systems. [Preview Abstract] |
Monday, March 3, 2014 4:18PM - 4:30PM |
D11.00008: Single Bacteria as Turing Machines Julia Bos, Qiucen Zang, Saurabh Vyawahare, Robert Austin In Allan Turing's famous 1950 paper on Computing Machinery and Intelligence, he started with the provocative statement: ``I propose to consider the question, `Can machines think?' This should begin with definitions of the meaning of the terms `machine' and `think'.'' In our own work on exploring the way that organisms respond to stress and evolve, it seems at times as if they come to remarkably fast solutions to problems, indicating some sort of very clever computational machinery. I’ll discuss how it would appear that bacteria can indeed create a form of a Turing Machine, the first example of a computer, and how they might use this algorithm to do rapid evolution to solve a genomics problem. [Preview Abstract] |
Monday, March 3, 2014 4:30PM - 4:42PM |
D11.00009: Influence of Helical Cell Shape on Motility of \textit{Helicobacter Pylori} Joseph Hardcastle, Laura Martinez, Nina Salama, Rama Bansil Bacteria's body shape plays an important role in motility by effecting chemotaxis, swimming mechanisms, and swimming speed. ~A prime example of this is the bacteria \textit{Helicobacter Pylori; }whose helical shape has long been believed to provide an advantage in penetrating the viscous mucus layer protecting the stomach lining, its niche environment. ~To explore this we have performed bacteria tracking experiments of both wild-type bacteria along with mutants, which have a straight rod shape. A wide distribution of speeds was found. This distribution reflects both a result of temporal variation in speed and different shape morphologies in the bacterial population. Our results show that body shape plays less role in a simple fluid. However, in a more viscous solution the helical shape results in increased swimming speeds. In addition, we use experimentally obtained cell shape measurements to model the hydrodynamic influence of cell shape on swimming speed using resistive force theory. The results agree with the experiment, especially when we fold in the temporal distribution. Interestingly, our results suggest distinct wild-type subpopulations with varying number of half helices can lead to different swimming speeds. [Preview Abstract] |
Monday, March 3, 2014 4:42PM - 4:54PM |
D11.00010: Quantifying the Dynamics of Bacterial Colony Expansion: From Individual Cells to Collective Behavior Erin Shelton, Maximiliano Giuliani, Robert Moscaritolo, Matt Kinley, Lori Burrows, John Dutcher Type IV pili (T4P) are very thin (5-8 nm in diameter) protein filaments that can be extended and retracted by certain classes of Gram-negative bacteria including \textit{P. aeruginosa} [1]. These bacteria use T4P to move across viscous interfaces, referred to twitching motility. Twitching can occur for isolated cells or in a collective manner [2]. We have developed experimental and data analysis techniques to quantify the expansion of the bacterial colony. Using a custom-built, temperature and humidity controlled environmental chamber, we have studied the transition from individual to collective motion. We have used optical flow analysis to characterize the evolution of the expanding colonies. We have also incorporated fluorescently tagged, non-motile cells, obtained by knocking out proteins essential for twitching motility, into the colonies to observe their transport as cargo by the motile cells. By measuring the flow of the motile cells while also tracking the motion of the non-motile cargo cells, we have obtained a direct measure of the efficiency of the transport of the cargo cells. [1] Burrows, L.L. (2012) Annu. Rev. Microbiol. 66: 493--520. [2] Semmler, A.B. \textit{et al}. (1999) Microbiology 145: 2863-2873. [Preview Abstract] |
Monday, March 3, 2014 4:54PM - 5:06PM |
D11.00011: Microscale swimming through heterogeneous networks Henry Fu, Mehdi Jabbarzadeh, YunKyong Hyon Although there have been many investigations of how swimming microorganisms are affected by complex media which treat the medium as a homogeneous material represented by a continuum constitutive equation, in many cases biological environments have microstructure at similar lengthscales as microorganisms. In that case continuum approaches are not valid and the microstructure and swimmer must be treated on equal footing. For example, cervical mucus contains a network of mucin filaments with a mesh size that can vary from approximately 0.5 to 30 microns, in the same size range as sperm. I will present results which investigate a simple theoretical model of a swimmer moving near similar-size obstructions. First, spherical obstructions are used to deduce physical principles linking the swimmer flow field, forces on obstructions, and changes in swimming velocities. Then single rod-like obstructions are studied which are similar to the filaments of networks. Using these results, we deduce the effect of a network of filaments. Notably, swimming properties such as the change in swimming speed and variance of the swimming speed reflect the density and orientation correlations of the microstructure, and hence swimming properties can be used as probes of microstructure. [Preview Abstract] |
Monday, March 3, 2014 5:06PM - 5:18PM |
D11.00012: Collective Motion of Magnetotactic Bacteria Solomon Barkley, Cecile Fradin, Kari Dalnoki-Veress Magnetotactic bacteria produce magnetic crystals that align the cells with an external magnetic field. Due to the field these bacteria preferentially swim along magnetic field lines in a behaviour known as magnetotaxis. Previous work has focused on bacteria in isolation, with investigations into the degree of orientation with the magnetic field as well as the response to magnetic field reversal. However, the motion of a cell in isolation cannot be extended to a cell with many neighbours, where collisions and collective effects cannot be ignored. The increased interaction between magnetotactic bacteria at very high concentrations alters the ability of any individual cell to align with an applied magnetic field. We will present experiments on the interplay between magnetotaxis and collectivity and the effects on the spatial and temporal organization of cells. [Preview Abstract] |
Monday, March 3, 2014 5:18PM - 5:30PM |
D11.00013: The Evopopbot Chip: Ultra High-throughput Evolutionary Population Bottlenecking using Drop-Based Microfluidics Connie Chang, Assaf Rotem, Adrian Serohijos, Huidan Zhang, Ye Tao, Audrey Fischer Hesselbrock, Peter Thielen, Thomas Mehoke, Joshua Wolfe, Christiane Wobus, Andrew Feldman, Eugene Shakhnovich, David Weitz The study of how viruses propagate is important for curing disease and preventing viral outbreaks.~ In nature, viruses can compete with one another, and the most evolutionary fit virus usually takes over a population.~ Yet there exist variants in the population that can escape subjected evolutionary pressures and eventually dominate the population.~ Successful studies of viral epidemics hinges on the ability to access these variants. Here, we present the use of droplet-based microfluidics as a simple method to segregate and propagate a viral population as individual viral lineages, simultaneously performing millions of in vitroevolutionary bottlenecking experiments. We introduce a novel microfluidic device, called the ``Evopopbot Chip'', that allows for simultaneous passaging of millions of evolutionary bottlenecking events by splitting drops containing previous generations of viruses and merging with drops containing new host cells. After several generations of viral replication in the evolution chip, we discover hundreds of new viruses that are able to escape a neutralizing antibody selection pressure compared to bulk passaging. [Preview Abstract] |
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