Session A11: Focus Session: Bacterial Biophysics I

Bacterial surface adaptation

Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including \textit{Pseudomonas aeruginosa and Vibrio cholerae}.   

Filaments in curved flow: Rapid formation of \textit{Staphylococcus aureus} biofilm streamers

Biofilms are surface-associated conglomerates of bacteria that are highly resistant to antibiotics. These bacterial communities can cause chronic infections in humans by colonizing, for example, medical implants, heart valves, or lungs. \textit{Staphylococcus aureus,} a notorious human pathogen, causes some of the most common biofilm-related infections. Despite the clinical importance of \textit{S. aureus} biofilms, it remains mostly unknown how physical effects, in particular flow, and surface structure influence biofilm dynamics. Here we use model microfluidic systems to investigate how environmental factors, such as surface geometry, surface chemistry, and fluid flow affect biofilm development in \textit{S. aureus. }We discovered that \textit{S. aureus} rapidly forms flow-induced, filamentous biofilm streamers, and furthermore if surfaces are coated with human blood plasma, streamers appear within minutes and clog the channels more rapidly than if the channels are uncoated. To understand how biofilm streamer filaments reorient in curved flow to bridge the distances between corners, we developed a mathematical model based on resistive force theory and slender filaments. Understanding physical aspects of biofilm formation in \textit{S. aureus} may lead to new approaches for interrupting biofilm formation of this pathogen.   

A bacterial swimming strategy with two alternating speeds of propagation

We used microfluidics together with high-speed video microscopy to acquire large data sets of swimming trajectories of \textit{Pseudomonas putida}, a bacterium with multiple polar flagella known for its ability to degrade aromatic hydrocarbons. The motion of cells in the bulk fluid is dominated by periods of persistent displacement along a straight line (runs) and sharp reorientation events (turns). The distribution of turning angles is bimodal with a dominating peak around 180 degrees and a minor peak around zero degrees. During the majority of turns, the cell reverses its swimming direction and the corresponding trajectories resemble a zig-zag pattern. Our analysis revealed that upon a reversal, the cell systematically changes its swimming speed by a factor of two on average. Based on the experimentally observed values for rotational diffusion and average runtime we developed a run-reverse random walk model with two distinct swimming speeds, which successfully recovers the mean square displacement and in an extended version also the observed negative dip in the directional autocorrelation. Our model demonstrates that by alternating between two swimming speeds, the cell explores its environment more efficiently than a cell swimming at a constant intermediate speed.   

Bacterial navigation in chemical and nonchemical environments

Navigation of cells to the optimal environmental niches is critical for their survival and growth. E. coli cells, for example, can detect various chemicals and move up or down those chemical gradients (i.e., chemotaxis). Using the same signaling machinery, they can also sense other external factors such as pH and temperature and navigate from both sides toward some intermediate levels of those stimuli. This mode of precision sensing is more sophisticated than the (unidirectional) chemotaxis strategy and requires distinctive molecular mechanisms. To understand different bacterial taxis behaviors, we develop a theoretical model which incorporates microscopic signaling events in individual cells into macroscopic population dynamics. We find that the equilibrium population distribution is governed by an effective potential, the landscape of which depends on the external environment (chemical stimuli, pH, and temperature). We uncover the key conditions for various taxis behaviors and directly connects the cellular taxis performances with the underlying molecular parameters. This approach is used to examine and predict how background attractants and downstream temperature effects influence the performance and stability of thermotaxis, which can be tested in future experiments.   

Mechanical Evolution of Bacterial Films at Oil-Water Interfaces

Bacteria can assemble at the interface between oil and water to form films that strongly affect the mechanical properties of the interface. In comparison with biofilms on solid substrates, such biofilm formation at fluid-fluid interfaces has been the subject of relatively little study. The microstructure of the films, which can include not only packings of bacteria but macromolecular surfactants secreted by the bacteria and the remains of dead bacteria, resembles a quasi-two-dimensional colloidal suspension in a polymer solution. We have characterized the mechanical response of bacterial films at oil-aqueous interfaces during their formation via passive microrheology and pendant drop imaging. With increasing age, the films undergo a transition from a viscous to an elastic interfacial shear rheology and eventually acquire a bending rigidity. These findings will be discussed in terms of viscoelstic models and in the context of the active nature of the bacteria in the films and in the adjoining aqueous suspension.   

Role of surface properties in bacterial attachment

Bacterial biofilms foul a wide range of engineered surfaces, from pipelines to membranes to biomedical implants, and lead to deleterious costs for industry and for human health. Designing strategies to reduce bacterial fouling requires fundamental understanding of mechanisms by which bacteria attach to surfaces. We investigate the attachment of \textit{Escherichia coli} on silanized glass surfaces during flow through a linear channel at flow rates of 0.1--1 mL/min using confocal microscopy. We deposit self-assembled monolayers of organosilanes on glass and track the position and orientation of bacteria deposited on these surfaces during flow using high-throughput image processing algorithms. Here, we report differences in deposition rate and surface-tethered motion of cells as a function of surface charge and surface energy, suggesting that attachment of bacteria on these engineered surfaces is dominated by different physical mechanisms.   

CdiGMP signaling at early stages of biofilm formation in \textit{Pseudomonas Aeruginosa}

Biofilm communities on surfaces constitute an important physiological state of bacteria. CdiGMP is a secondary messenger that has recently emerged as a master regulator of biofilm behavior. It has been shown that cdiGMP can affect bacterial adhesion, motility and exopolysaccharides production, which are important in regulating biofilm formation. However, at a single cell level, the details of how cdiGMP regulate bacterial behavior are largely unknown. Here we examine the dynamics of intracellular cdiGMP levels at early stages of biofilm in \textit{Pseudomonas Aeruginosa}, by using cell tracking techniques. We show that cells with different cdiGMP levels play different roles in the microcolony development at early stages of biofilm. The correlation between Psl and cdiGMP levels is also investigated.   

Theoretical and Experimental Study of Bacterial Colony Growth in 3D

Bacterial cells growing in liquid culture have been well studied and modeled. However, in nature, bacteria often grow as biofilms or colonies in physically structured habitats. A comprehensive model for population growth in such conditions has not yet been developed. Based on the well-established theory for bacterial growth in liquid culture, we develop a model for colony growth in 3D in which a homogeneous colony of cells locally consume a diffusing nutrient. We predict that colony growth is initially exponential, as in liquid culture, but quickly slows to sub-exponential after nutrient is locally depleted. This prediction is consistent with our experiments performed with E. coli in soft agar. Our model provides a baseline to which studies of complex growth process, such as such as spatially and phenotypically heterogeneous colonies, must be compared.   

EPS forces in Bacillus subtilis biofilms

Bacteria have evolved to congregate in complex communities known as biofilms. The structure that holds a biofilm together is a matrix called extracellular polymeric substance (EPS). It has been observed in previous studies that EPS up-regulation occurs when the nutrient levels fall below a threshold concentration; this increase in EPS concentration produces an osmotic pressure that forces the colony to spread outward. This osmotic pressure may drive nutrient uptake, but the stresses generated by the EPS matrix has never been measured. Here we present measurements of the forces exerted by a biofilm on its supporting substrate and on its fluid nutrients. In our experiments, we use a technique analogous to traction force microscopy to measure strain in agar nutrient substrates imposed by Bacillus subtilis biofilms. By running additional test to measure the permeability and elastic modulus of the agar, we can estimate the pressure generated by the biofilm.   

The Spatial-Temporal Evolution of the Interface Between Growing {\em E.~coli} Colonies

An \textit{Escherichia coli} colony is a popular model used to study the physical interactions of a multicellular system. However, the development of the interface between two interacting colonies has not been well studied. In this work, we tracked the development and interaction of two cellular colonies formed from single founder cells. We observed that the colony-colony interface exhibited a range of roughening, sometimes producing a linear interface (zero roughening) and other times producing a highly sinuous interface (increased roughening). Using time-lapse microscopy, we captured images of a number of interacting colonies and quantified the evolution of their interface and show that it is highly correlated with a number of factors such as colony distance, growth rate, and age. To connect the microscopic details of the spatial orientation of cells to the macroscopic roughening, we simulated growing colonies and found that the orientation of the cells at the interface plays an important role in the roughening of the interface. Initially cells are highly aligned along the interface, but as time progresses, the cell alignment becomes more anisotropic, and it is the level of anisotropy that is highly correlated with the interface roughening.   

Spatiotemporal evolution of bacterial biofilm colonies

Many bacteria on earth live in surface-attached communities known as biofilms. Gene expression in a biofilm is typically varied, resulting in a variety of phenotypes within a single film. These phenotypes play a critical role in biofilm physiology and development. We use time-resolved, wide-field fluorescence microscopy to image triple-labeled fluorescent Bacillus Subtilis colonies grown on agar to determine in a non-invasive fashion the evolving phenotypes. We infer their transition rates from the resulting spatiotemporal maps of gene expression. Moreover, we correlate these transition rates with local measurements of nutrient concentration to determine the influence of extracellular signals on gene expression.   

Spatio-temporal Kinetics of Nontypeable \textit{Haemophilus influenzae }(NTHi) Biofilms

Bacteria can form complex spatial structures known as biofilms. Biofilm formation is frequently associated with chronic infections due to the greatly enhanced antibiotic resistance of resident bacteria. However, our understanding of the role of basic processes, such as bacteria replication and resource consumption, in controlling the development and temporal change of the spatial structure remains rudimentary. Here, we examine the growth of cultured biofilms by the opportunistic pathogen NTHi. Through spatial information extracted from confocal microscopy images, we quantitatively characterize the biofilm structure as it evolves over time. We find that the equal-time height-height pair correlation function decreases with distance and scales with time for small length scales. Furthermore, both the surface roughness and the correlation length perpendicular to the surface growth direction increase with time initially and then decrease. We construct a spatially resolved agent based model beginning with the simplest possible case of a single bacteria species Fisher-Kolmogorov-Petrovsky-Piscounov equation. We show that it cannot describe the observed spatio-temporal behavior and suggest an improved two-species model that better captures the dynamics of the NTHi system.