The biophysics of bacterial collective motion: Measuring responses to mechanical and genetic cues

Mechanobiology is an emerging field investigating mechanical signals as a necessary component of cellular and developmental regulation. These mechanical signals play a well-established role in the differentiation of animal cells, whereby cells with identical genes specialize their function and create distinct tissues depending on the physical properties of their environment, such as shear stiffness. These differences arise from the cell’s ability to use those incoming signals to inform which genes it expresses and what molecular machinery it builds and activates. Understanding the various missing factors that cause cells with specific genes to express an emergent phenotype is termed the genotype-to-phenotype problem, and mechanical signaling pathways present themselves as a significant piece of this puzzle. Despite the strong evidence for mechanosensing in eukaryotes, the pathways by which prokaryotes respond to mechanical stimuli are still largely unknown. Bacteria are among the simplest and yet most abundant forms of life. Many of their survival strategies depend on multicellular development and the coordinated formation of a colony into functional structures that may also feature cellular differentiation. This dissertation employs bacteria as a model system to investigate multiple biophysical questions of collective motion through novel experimental and analytical techniques. This work addresses the understudied mechanical relationship between a bacterial colony and the substrate it colonizes by asking “what is the effect of substrate stiffness on colony growth?” This is done by measuring bacterial growth on hydrogel substrates that decouple the effects of substrate stiffness from other material properties of the substrate that vary with stiffness. We report a previously unobserved effect in which bacteria colonize stiffer substrates faster than softer substrates, in opposition to previous studies done on agar, where permeability, viscoelasticity, and other material properties vary with stiffness.A second theme of this work probes the genetic inputs to the genotype-to-phenotype problem in multicellular development. The bacterial species Myxococcus xanthus producing macroscopic aggregates called fruiting bodies is used as a model organism for these studies. It has long been conjectured that genes may stand in for each other functionally, allowing for development to be more consistent and stable, but the extent of this redundancy has resisted measurement. We approach the question “how does redundancy among related genes lead to robust collective behavior?” by quantifying developmental phenotype in a large dataset of time lapse microscopy videos that show development in many mutant strains. We observe that when knocking out multiple genes that have a common origin (i.e. homologous genes), the resulting phenotypes differ from wild-type in a similar way. These phenotype clusters also differ from knockouts from other homologous gene families. These distinct phenotypic clusters provide evidence for the existence of networks of redundant genes that are larger than could previously be tested directly. Because of this robustness, the effects of individual gene mutations can be hidden or damped. We thus develop our analytical techniques further to address the question “how can subtle changes in phenotype be measured?” This involves quantifying the breadth of variation observed in wild-type development and creating a statistical technique to distinguish probabilistic distributions of phenotypic outcomes. We present a coherent method of visualizing large phenotypic datasets that include multiple metrics that we use to distinguish small developmental differences from wild-type, giving each mutant strain a phenotypic fingerprint that can be used in future studies on gene annotation and environmental impacts on phenotype

Bacteria colonies modify their shear and compressive mechanical properties in response to different growth substrates

Bacteria build multicellular communities termed biofilms, which are often encased in a self-secreted extracellular matrix that gives the community mechanical strength and protection against harsh chemicals. How bacteria assemble distinct multicellular structures in response to different environmental conditions remains incompletely understood. Here, we investigated the connection between bacteria colony mechanics and the colony growth substrate by measuring the oscillatory shear and compressive rheology of bacteria colonies grown on agar substrates. We found that bacteria colonies modify their own mechanical properties in response to shear and uniaxial compression with the increasing agar concentration of their growth substrate. These findings highlight that mechanical interactions between bacteria and their microenvironment are an important element in bacteria colony development, which can aid in developing strategies to disrupt or reduce biofilm growth.Comment: biophysics, soft matter, biofilm rheology, biofilm mechanic

Materials science and mechanosensitivity of living matter

Living systems are composed of molecules that are synthesized by cells that use energy sources within their surroundings to create fascinating materials that have mechanical properties optimized for their biological function. Their functionality is a ubiquitous aspect of our lives. We use wood to construct furniture, bacterial colonies to modify the texture of dairy products and other foods, intestines as violin strings, bladders in bagpipes, and so on. The mechanical properties of these biological materials differ from those of other simpler synthetic elastomers, glasses, and crystals. Reproducing their mechanical properties synthetically or from first principles is still often unattainable. The challenge is that biomaterials often exist far from equilibrium, either in a kinetically arrested state or in an energy consuming active state that is not yet possible to reproduce de novo. Also, the design principles that form biological materials often result in nonlinear responses of stress to strain, or force to displacement, and theoretical models to explain these nonlinear effects are in relatively early stages of development compared to the predictive models for rubberlike elastomers or metals. In this Review, we summarize some of the most common and striking mechanical features of biological materials and make comparisons among animal, plant, fungal, and bacterial systems. We also summarize some of the mechanisms by which living systems develop forces that shape biological matter and examine newly discovered mechanisms by which cells sense and respond to the forces they generate themselves, which are resisted by their environment, or that are exerted upon them by their environment. Within this framework, we discuss examples of how physical methods are being applied to cell biology and bioengineering

A Torsion-Based Rheometer for Measuring Viscoelastic Material Properties

Rheology and the study of viscoelastic materials are an integral part of engineering and the study of biophysical systems. Tissue rheology is even used in the study of cancer and other diseases. However, the cost of a rheometer is feasible only for colleges, universities, and research laboratories. Even if a rheometer can be purchased, it is bulky and delicately calibrated, limiting its usefulness to the laboratory itself. The design presented here is less than a tenth of the cost of a professional rheometer. The design is also portable, making it the ideal solution to introduce viscoelasticity to high school students as well as for use in the field for obtaining rheological data

Mechanobiology as a tool for addressing the genotype-to- phenotype problem in microbiology

The central hypothesis of the genotype–phenotype relationship is that the phenotype of a developing organism (i.e., its set of observable attributes) depends on its genome and the environment. However, as we learn more about the genetics and biochemistry of living systems, our understanding does not fully extend to the complex multiscale nature of how cells move, interact, and organize; this gap in understanding is referred to as the genotype-to-phenotype problem. The physics of soft matter sets the background on which living organisms evolved, and the cell environment is a strong determinant of cell phenotype. This inevitably leads to challenges as the full function of many genes, and the diversity of cellular behaviors cannot be assessed without wide screens of environmental conditions. Cellular mechanobiology is an emerging field that provides methodologies to understand how cells integrate chemical and physical environmental stress and signals, and how they are transduced to control cell function. Biofilm forming bacteria represent an attractive model because they are fast growing, genetically malleable and can display sophisticated self-organizing developmental behaviors similar to those found in higher organisms. Here, we propose mechanobiology as a new area of study in prokaryotic systems and describe its potential for unveiling new links between an organism\u27s genome and phenome

Spreading rates of bacterial colonies depend on substrate stiffness and permeability

The ability of bacteria to colonize and grow on different surfaces is an essential process for biofilm development. Here, we report the use of synthetic hydrogels with tunable stiffness and porosity to assess physical effects of the substrate on biofilm development. Using time-lapse microscopy to track the growth of expanding Serratia marcescens colonies, we find that biofilm colony growth can increase with increasing substrate stiffness, unlike what is found on traditional agar substrates. Using traction force microscopy-based techniques, we find that biofilms exert transient stresses correlated over length scales much larger than a single bacterium, and that the magnitude of these forces also increases with increasing substrate stiffness. Our results are consistent with a model of biofilm development in which the interplay between osmotic pressure arising from the biofilm and the poroelastic response of the underlying substrate controls biofilm growth and morphology

Bacteria Colonies Modify Their Shear and Compressive Mechanical Properties in Response to Different Growth Substrates

Bacteria build multicellular communities termed biofilms, which are often encased in a self-secreted extracellular matrix that gives the community mechanical strength and protection against harsh chemicals. How bacteria assemble distinct multicellular structures in response to different environmental conditions remains incompletely understood. Here, we investigated the connection between bacteria colony mechanics and the colony growth substrate by measuring the oscillatory shear and compressive rheology of bacteria colonies grown on agar substrates. We found that bacteria colonies modify their own mechanical properties in response to shear and uniaxial compression in a manner that depends on the concentration of agar in their growth substrate. These findings highlight that mechanical interactions between bacteria and their microenvironments are an important element in bacteria colony development, which can aid in developing strategies to disrupt or reduce biofilm growth

BioInspired Institute 2020

An activity space for 2020 OSF Workshop at the BioInspired Institute