Session A42: Physics of Biofilms

Mechanical Principles of Biofilm Formation

Biofilms are surface-associated bacterial communities embedded in an extracellular matrix. Biofilms are a major problem in the context of chronic infections, because biofilm dwelling cells have increased antibiotic resistance compared to their planktonic counterparts. In this talk, I will discuss about our recently progress in using Vibrio cholerae as a model biofilm former to reveal the mechanical and biophyisical principles behind biofilm formation. I will first present a new methodology to image living, growing bacterial biofilms at single-cell resolution, and demonstrate how cell-cell adhesion and cell-surface adhesion balance each other to cause V. cholerae to form an ordered, three-dimensional biofilm cluster. Next, I will demonstrate how matrix production enables biofilm cells to establish an osmotic pressure differential between the biofilm and the external environment, and use this osmotic pressure to facilitate biofilm expansion. Finally, I will present various mechanical instabilities that take place when biofilms grow on soft substrates, and how such instabilities shape the morphology development of bacterial colonies.   

Bacteria Sense Mechanical Stress to Know When to Start Forming a Biofilm

Biofilms are communities of sessile microbes that have different patterns of gene expression from their genetically-identical, free-swimming counterparts. Biofilms can initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For Pseudomonas aeruginosa, it has long been known that intracellular levels of the signal cyclic-di-GMP increase upon surface adhesion and that increased cyclic-di-GMP levels are required to begin biofilm development. However, what cue is sensed to notify bacteria that they are attached to the surface has not been known. Here, we show that mechanical shear acts as a cue for surface adhesion and activates cyclic-di-GMP signaling. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We identify specific protein structures as important elements in the mechanosensory process. An analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides describes our data well.

It is well-known that eukaryotic cells can sense and respond to many types of mechanical inputs. In contrast, very little is known about how prokaryotes may respond to mechanical inputs. Here, we show that bacteria can sense shear and can respond by initiating biofilms. This is an important advance in fundamental microbiology and mechanobiology. Biofilms are difficult to prevent using extant approaches. Our knowledge points the way to a hitherto-undeveloped type of antibiofilm surface that thwarts mechanosensing and thereby prevents bacteria from "knowing" that they are attached to a surface, activating cyclic-di-GMP signaling, and forming a biofilm.   

Surface sensing, motility appendages, and hydrodynamics in bacterial interactions with surfaces

Bacterial biofilms are integrated communities of cells that adhere to surfaces and are fundamental to the ecology and biology of bacteria. The accommodation of a free-swimming cell to a solid surface is more complex than simply modulation of cell adhesion. We investigate the interplay between motility appendages, molecular motors, hydrodynamics, and exopolysaccharide production near the surface environment using state of the art tools from different fields that are not usually combined, including theoretical physics, community tracking with single cell resolution, genetics, and microbiology. We show that surface sensing, multi-generational signaling, and downstream motility consequences can be understood within a Turing-inspired stochastic model, and that bacteria can have a surprisingly long memory of >10 hrs.

  

The importance of changing color: roles for redox-active pigments in sustaining biofilm metabolism

Bacteria are colorful, both literally and figuratively. And some change color depending on what is present in their environment. Take the case of Pseudomonas aeruginosa, whose cultures toggle between blue-green and clear. The name “aeruginosa” derives from the Latin word for copper rust, which is of the same blue-green hue. While microbiologists and clinicians have long used color to identify the organism, why it is colored in the first place—and why its color changes with aeration—is a question that not many have considered. We now know that phenazines, a class of redox-active pigments, are responsible not only for the blue-green color of P. aeruginosa in the presence of oxygen, but also for different colors displayed by other Pseudomonas species. In the early 20^th century, Ernst Friedheim and colleagues postulated that phenazines are “accessory respiratory pigments” that sustain bacterial “respiration” based on their ability to stimulate oxygen consumption. Their work was carried out before respiratory pathways were fully understood and well before the importance of microbial biofilms in nature and disease was widely recognized. In the interval between Friedheim’s pioneering studies and our recent work, attention shifted to exploring the roles of phenazines as virulence factors. Phenazines came to be known as "secondary metabolites", molecules produced at late stages of microbial growth in laboratory cultures whose function was thought to be to protect Pseudomonas from competitors. While the antibiotic activity of phenazines has been elegantly shown in a variety of contexts, labeling phenazines as “secondary metabolites” suggests that they are not essential to the growth or survival of their producers. In this talk, I will champion Friedheim’s original hypothesis and extend it. Specifically, I will discuss our deepening understanding of how phenazines are vital to the metabolic survival of cells in biofilms that are oxidant-limited.   

Electrical cell-to-cell signaling in bacterial communities

I will share our recent results building on our discovery of ion channel-mediated electrical signaling within bacterial biofilm communities. By affecting the membrane potential of bacteria, this electrical cell-to-cell signaling appears to play a critical role in the emergence of collective behavior and regulation of cellular activity as a function of space and time. I will discuss the intriguing physical properties associated with this new form of bacterial communication.