Session Y66: Physics of Microbes II

Geometric localization of cell wall growth and shape determining proteins in Helicobacter pylori

The bacterial kingdom exhibits a wide variety of cell shapes. Our computational imaging framework allows us to extract 3D shapes of individual bacterial cells from fluorescence microscopy images. From these shapes we calculate geometric parameters such as local curvature, surface area, and the enrichment of fluorescent signals. The helical-rod shape of Helicobacter pylori, a human stomach pathogen, is important for its pathogenesis and is an ideal model system for studying complex bacterial cell shapes. In H. pylori, we have been investigating the geometric localization of the proteins and metabolic probes of cell wall growth. To propagate its helical shape, a cell must grow its longer, outer helical axis more than its inner one. Fluorescent, metabolic probes incorporated into the cell wall show enrichment of growth at both the outer and inner axes. Consistent with these labels, two of the proteins associated with cell wall homeostasis show geometric localization. MreB is enriched at regions of small and negative Gaussian curvature and CcmA is enriched at Gaussians curvatures of about 5 μm^-2, similar to the curvature of the outer axis. These data are consistent with a model of helical-rod like growth that takes the simple rod and elaborates it by defining a outer helical axis.   

Mechanical Forces to Trigger Morphological Changes in Motile Bacteria

Bacterial swarming is an important mechanism for flagellated bacteria to effectively cover large distances on soft surfaces, such as tissues. Swarming is likely initiated when the flagella sense higher viscous loads such as those experienced near solid boundaries. However, the underlying assumption that soft and porous surfaces cause significantly high loads on the flagella remains untested. Here, we developed an approach to estimate the hydrodynamic drag as a function of separation from soft interfaces. In one type of testing, optical trapping experiments were employed to estimate the diffusivities of tiny objects close to soft interfaces, using a novel flow-geometry. The method was validated by comparing results for transverse diffusion coefficients for single spherical particles near a no-slip solid boundary with predictions from Faxen’s law. The data were in good agreement with published models. In a second type of testing, the surface-induced load on the flagellar motor was interpreted from the rates of swarming in two different strains that developed differential hydrodynamic thrust. These results are anticipated to help estimate the magnitude of mechanical forces necessary to initiate the transition of a bacterial cell to the swarmer state.   

Bacterial Motors and Surface Sensing

Surface-sensing precedes the establishment of many types of bacterial colonies on surfaces. Evidence suggests that surface-sensing triggers transcriptional regulation as well as post-translational signaling that promote a variety of interesting cell phenotypes. The molecular mechanisms continue to remain unclear. I will discuss how motile cells discriminate between random forces arising due to Brownian motion and those arising due to surface contact. Our recent measurements that highlight the role of the flagella (otherwise known to be involved in motility) in surface-sensing and signaling will be discussed. Single molecule experiments that explain how flagella tune the sensitivities of modules involved in well-known signaling pathways will be presented. Data indicate an indirect link between the intracellular events and the external hydrodynamic drag acting on individual flagellum, and also hint at the regulation of certain surface-dependent phenotypes by these sensitive modules. I will conclude with a brief note on how mechanically-stimulated regulatory events influence a host of important processes such as bacterial competence, which regulates the uptake of extracellular DNA.   

Determining the Metabolic Activity and Flow Boundary Conditions of Bacteria from Diffusion Measurements

When live bacteria are adhered onto a microcantilever, the nanomechanical displacements of the microcantilever increase noticeably, suggesting that bacteria fluctuations drive the microcantilever to a higher amplitude than thermal fluctuations. Here, we have performed similar measurements by adhering various bacteria onto the surface of 8-μm-diameter microbeads. After bacteria adhesion, we image the Brownian motion of these single microbeads suspended in liquid by high-resolution optical microscopy. We then calculate the mean square displacements and diffusion constants from the trajectories of the microbeads. Interestingly, microbeads with non-motile bacteria show an unexpected diffusion profile: in a given interval, microbeads with dead bacteria on them tend to diffuse farther compared to those with live bacteria. We will discuss possible sources of this unexpected behavior, including modifications to the no-slip boundary condition on the surface of bacteria.   

Passive and active response of bacteria under mechanical compression.

The ability to maintain a positive turgor pressure, by means of higher osmolarity of the cell interior than the exterior, is a requirement for proper metabolism in walled microbial cells. Turgor pressure is sensitive to changes in external osmotic conditions, and is drastically increased upon osmotic downshock, together with cell volume.
Bacteria prevent lysis caused by excessive osmotic pressure through mechanosensitive (MS) channels: membrane proteins that release solutes (ions) in response to mechanical stress. The exact mechanism of channel gating in the natural setting, however, has been elusive due to the lack of experimental methods appropriate for the small dimensions of prokaryotes.
We here present experimental data on the gating of MS channels of E. coli subjected to compressive force under iso-osmotic conditions. We indent living cells with micron-sized beads attached to the cantilever of an atomic force microscope (AFM) and characterize the mechanical response. We show that turgor pressure can be monitored through the measured response and quantify its value and fluctuations for individual single cells before and after MS channel gating.
  

Theoretical investigation of stochastic clearance of bacteria

Understanding mechanisms of bacterial eradication is critically important for overcoming the failures of antibiotic treatments. Current studies suggest that the clearance of large bacterial populations proceeds deterministically, while for smaller populations the stochastic effects become more relevant. We developed a theoretical approach to investigate the bacterial population dynamics under the effect of antibiotic drugs using a method of first-passage processes. It allows us to explicitly evaluate the most relevant characteristics of the bacterial clearance dynamics such as extinction probabilities and extinction times. In addition, we investigate the effect of fluctuations in the population growth rates on dynamics of bacterial eradication. It is found that fluctuations for some range of parameters increase the extinction probability and the extinction times, indicating slowing the bacterial clearance. We speculate that this might be a first step in developing of antibiotic resistance.   

Mechanotransduction in bacteria: how Pseudomonas aeruginosa actively probes and responds to substrate mechanics

A growing body of evidence suggests that bacteria respond to mechanical cues such as shear flow or surface material properties, yet clear evidence that such mechanical features of the environment are integrated in a cell’s active decision making is lacking. Here, we demonstrate that the pathogen Pseudomonas aeruginosa employs arm-like, retractable appendages called pili to actively deform a substrate and probe its mechanical properties. We use a transcriptional fluorescence reporter to show that Pseudomonas tunes the expression of virulence factors based on substrate stiffness in the range of 0.1 – 1000 kPa. Using optical tweezers, traction force microscopy, and molecular modeling of the active motor components, we probe the mechanism of pilus-based mechanosensation. These results provide unique insights into the molecular functioning and control of pilus extension and retraction and how this is linked to twitching motility. We find evidence of an active bacterial mechanotransduction pathway that connects substrate mechanical properties to a genetic response via active surface deformation, similar to the mechanogenetic regulation found in stem cell differentiation.   

Strength and longevity of non-genetic memory in sister bacterial cells.

Phenotypic heterogeneity is a vital aspect of isogenic bacterial population related to the survival of the species. The focus of this study is to understand the origin of non-genetic phenotypic heterogeneity by observing the inheritance pattern of different cellular traits in the lineage of single bacterial cells using microfluidics and time-lapse microscopy. We aim to reveal the cellular factors responsible for this variability and estimate the timescale over which new phenotypes emerge. We have developed a unique microfluidic device that allows us to trap and follow E. coli sister cells for hundreds of generations in the same environment, and thus separating cellular effects from environmental ones. Our results reveal strong correlation of several measurable properties like cell size, cell-cycle duration and growth rate between E. coli sister cells for many generations while non-related cells growing in the same environment show almost zero correlation over the same period. These results allow us to quantitatively determine the strength and longevity of non-genetic memory in sister bacterial cells.   

Mechanics and dynamics of filaments on curved membranes

Protein filaments that bind to curved membranes exhibit rich behavior, as shown in previous studies of bacterial morphogenesis. Here we model the direct binding of protein filaments to membranes and show that it is typically energetically favorable for filaments to orient along the direction of largest curvature. We then model the dynamics of an ensemble of filaments, each translocating along the direction of largest curvature and subject to noise. We show that their macroscopic properties, such as localization, can vary significantly depending on membrane geometry. Finally, we discuss the implications of our study to bacterial morphogenesis and regulation of bacterial rod shape. Our work shows, from a physical perspective, how cellular-scale dynamics which dictates growth may arise from nanometer-scale properties.   

Modes of Bacterial Aging and Death

Currently, “biological aging” is well defined in human beings and multicellular organisms, and is one of the largest risk factors for most diseases. However, in single cell organisms there has been a sequence of various definitions. My research focuses on understanding the cellular processes responsible for senescence and death in E. coli bacteria as well as identifying cellular characteristics of aging. We employed an experimental setup consisting of a microfluidic device designed to trap single-cells while continuously supplying them with nutrients, allowing us to acquire images of the trapped cells. Analysis of these images provides long-term single-cell measurements of cell-size and protein content, which are then used to uncover aging effects and determine modes of cell death. Our data acquired thus far reveal two different phenotypes of cell death: 1. Part of the cells maintain their bacterial chromosome and enter a non-dividing phase. 2. A subset of the population loses its chromosome and so all cellular functions are obstructed. Our results indicate aging in all strains, which is marked by the increase in death rate with time. In addition, by comparing wild type and an ATP synthase E. coli mutant, we found different average lifetimes prior to undergoing these transitions.   

Rolling motion of uni-flagellated bacteria near a solid surface

Bacterial interaction with surfaces is ubiquitous in nature and is essential to many biological processes such as infection and biofilm formation. Here we study the tethering of uni-flagellated Caulobacter crescentus to a solid surface. In particular, we focus on weak reversible adhesion, which allows subsequent detachment. By applying an electrical field parallel to the surface, we observe that some initially tethered bacteria detach from the surface, but remain close to it. The detached cells perform rolling motion, a mixture of translation and rotation, different from the almost exclusive translation observed for nonmotile bacteria or swimming cells that are not previously attached. Our analysis of the rolling motion accounts for electrophoretic force, flagellar rotation, and the cell’s close proximity to the solid surface.
  

E. coli in Mazes

Bacteria often live in topologically complex environments. The path from a colony of bacteria which as exhausted its local supply of food to a source can have many branch points with false, dead-end leads. While chemotaxis can easily navigate bacteria to a food source in the presence of a food gradient, in a sufficiently complex and large maze chemotaxis will fail, leading to bacterial collapse at dead ends. Here, using nano and microfabrication, we made physical puzzles to study the collective behaviors of E. coli bacteria. We constructed mazes for bacteria to solve in order to get food and probed for their searching algorithms, with and without the presence of hydro flows.   

High Throughput 3D Tracking of Bacterial Chemotaxis in Complex Environments

Bacterial chemotaxis is an important survival mechanism by which motile bacteria bias their random walk trajectory to navigate chemical gradients. While chemotaxis has been studied extensively in E. coli, much less attention has been devoted to the diversity of bacteria with different flagellar architectures, or to motility in viscous or porous environments that more closely mimic the complexity of natural habitats. Current approaches for characterizing and comparing chemotaxis strategies are hampered by being either limited to the population scale, qualitative, low throughput, 2D, and/or dependent on theoretical models.

Here we present a simple yet powerful chemotaxis assay combining a recent high-throughput 3D tracking method with microfluidically created chemical gradients. With a typical throughput of above 3,000 individual trajectories in 5 minutes, we demonstrate that we can directly determine chemotactic drift velocities in diverse environments while simultaneously resolving 3D motility behavior, enabling unprecedented access to a mechanistic understanding and quantitative comparison of chemotactic strategies. We highlight the power of our assay by revealing the role of lateral flagella in the opportunistic pathogen V. alginolyticus for motility and chemotaxis in hydrogels.   

Bacterial Scattering from Convex Surfaces and its Effects on Motility and Chemotaxis

For many organisms across length scales, self-propulsion is a key mechanism for survival and nutrient acquisition in changing environments. The model microorganism Escherichia coli explores low Reynolds number environments executing ‘run-and-tumble’ chemotaxis in its search for resources. While many studies of chemotaxis and motility take place in open environments, typical real-world environments -- like soils, sediments, or the mammalian gut -- have physical structure across multiple length-scales which can alter or impede cellular trajectories. To understand how physical structure in the environment alters trajectories and attendant persistence lengths, we built microfluidic devices containing arrays of microscopic circular pillars and studied bacterial interactions with these structures through the lens of probabilistic scattering functions. We find that attractive interactions with the pillars and stochastic noise produce a complex set of probability distributions that characterize bacterial motion in structured environments. Thus, understanding how cells navigate chemical gradients and acquire resources in such complex environments is crucial to our understanding of microbial ecology and may enable design of devices for controlling bacterial motility.   

Run-and-tumble motility enables equatorial magnetoaerotaxis

Guided by Earth’s magnetic field, magnetotactic bacteria (MTB) seek out microoxic environments in the sediments of swamps, lakes, and oceans for survival. To accomplish this, MTB synthesize magnetic nanoparticles that mechanically orient the cells along Earth’s magnetic field, which is mostly parallel to the water column over a broad range of latitudes. In the current paradigm, magnetotaxis provides a global orientation for the cells and facilitates a one-dimensional aerotactic search along naturally occurring, parallel oxygen gradients through ‘run-and-reverse’ motility. However, a variety of MTB species thrive at the equator, in spite of the orthogonal magnetic field and oxygen gradients. Using a microfluidic device and Helmholtz coil, we independently control an oxygen gradient and an orthogonal magnetic field, enabling precise measurements of MTB motility under equatorial conditions. In contrast to the current paradigm, we show that Magnetococcus marinus (MC-1) achieves ‘run-and-tumble’ motility, which facilitates exploration and aerotaxis perpendicular to the magnetic field. These results establish a new survival mechanism for equatorial magnetoaerotaxis in bacteria.