Session K5: Physical Properties of Bacterial Cytoplasm

Heterogeneous dynamics in the bacterial cytoplasm

The bacterial cytoplasm is a highly crowded environment, with estimates of excluded volume reaching 40 -- 60{\%}. A diverse array of highly polydisperse components is responsible for crowding and ranges from metabolites to compact proteins to large nucleic acid polymers and macromolecular assemblies. In contrast to eukaryotic cells, bacterial cells lack membrane delimited intracellular components. To measure the material properties of the \textit{Eschericia coli} cytoplasm, I tracked foreign particles of different sizes in various cellular states. I found particle motion to be dependent on cellular metabolism at physiological pH and exhibited dynamical properties associated with glass-like materials, including particle caging and un-caging and heterogeneous dynamics in single cells. When particle mobility was compared across populations of cells, an enormous amount of heterogeneity was measured that increased over time. This inter-cellular heterogeneity could be separated into contributions from cellular components and differences in effective cytoplasmic viscosity between cells. These findings have implications for bacterial physiology and motivate experiments and simulations in colloid-polymer mixtures with high polydispersity.   

Computational models of the cytoplasm of bacterial cells

In vivo experiments of bacteria have shown that free ribosomes and ribosomes bound to mRNA possess dramatically different self diffusion coefficients. In addition, the diffusion coefficient of ribosomes is strongly affected by its interaction with DNA in the nucleoid. We have developed molecular dynamics simulations of mRNA-ribosome complexes (polysomes) in bacteria to investigate the mechanisms responsible for their slow dynamics. In preliminary studies, we showed that polysomes diffuse slower than spheres with an equivalent mass. The introduction of DNA in the simulation gives rise to a further decrease in the diffusion coefficient of polysomes. We quantify the number of entanglements between polysomes to explain the strong decrease in their diffusion coefficient. Static packings of polysomes also indicate that tension in the polysome chain can affect the jamming onset and the packing fraction at which the diffusion coefficient vanishes.   

Physico-chemical conditions for self-replication

Self-replication is a process by which an object creates a near identical copy of itself. It is the process by which asexual reproduction happens and, arguably, it was the most important process that led to the formation of living objects from inanimate matter. However, it is not clear why some systems, characterized by the fundamental interactions and the reaction rates between their constituents, can grow via self-replication, while others do not. In this talk, we aim to answer this question through the computational investigation of a simple chemical system, where we can independently control the interaction energies of the fundamental building blocks, called “atoms,” and the reaction rates between the “molecules” formed by them. Our simulation suggests that exponential growth, characteristic of self-replication, is observed in parts of the parameter space where (a) the self-replicator reacts very specifically with a few molecules, and (b) the reactions that deplete the self-replicator happen at time-scales longer than the replication reactions. We use this insight to create classes of self-replicators that have slightly different reaction rates from each other, and investigate how this variation in the reaction rates affects their replicative fitness.   

Stochastic Modeling of Bacteria Cell Size Control and Homeostasis

Besides recent breakthroughs, there is a gap of knowledge about the mechanisms underlying cell size control and homeostasis. In this context, recent studies support the incremental rule in rod-shaped bacteria: cells add a constant length to their size before dividing which is independent of their size at birth. This growing pattern, when coupled with the mid-cell division mechanism, leads to size convergence and homeostasis. However, some aberrantly long mutant strains of \emph{E. coli}, e.g. $\Delta$FtsW, do not typically divide at the middle. Whether cell size control and homeostasis apply to those mutant backgrounds, or the role played by biomechanical cues, remain open questions. Here we present a combination of theoretical, experimental, and computational approaches to address these questions. First, we introduce a Markov chain model that describes either wild-type (wt) strains or growth-defective strains. Second, we propose a polymer-like model to account for the mechanical inputs. Finally, we test experimentally some of our predictions by using wt and conditional mutant ($\Delta$FtsW) strains. Altogether, our preliminary studies suggest a way to unify the principles of cell size control and homeostasis of wt and growth-defective cell strains.   

The Location of the Bacterial Origin of Replication is Critical for Initial Ciproflaxcin Antibiotic Resistance

By using {\em E. coli} cells in which the unique origin of replication has been moved to a ectopic chromosome location distant from the native one, we probe how perturbation of gene order near the origin of replication impacts genome stability and survival under genomic attack. We find that when challenged with sub-inhibitory doses of ciprofloxacin, an antibiotic that generates replication fork stalling, cells with the ectopic origin show significant fitness loss. We show that genes functionally relevant to the cipro-induced stress response are largely located near the native origin, even in distantly related species. We show that while cipro induces increased copy number of genes proximal to the origin of replication as a direct consequence of replication fork stalling, gene copy number variation was reduced near the ectopic origin. Altered gene dosage in cells with an ectopic origin resulted in impaired replication fork repair and chromosome instability. We propose that gene distribution in the origin region acts as a fundamental first line of defense when the integrity of the genome is threatened and that genes proximal to the origin of replication serve as a mechanism of genetic innovation and a driving force of genome evolution in the presence of genotoxic antibiotics.   

Microrheology study of human mucins varying in \textit{Helicobacter pylori} binding affinity

\textit{Helicobacter pylori} is the pathogen that colonizes the human stomach and causes gastric ulcers and cancer. One of the key mechanisms by which \textit{H. pylori} establishes an infection on the gastric mucosa is by expressing adhesins that facilitate the binding of the bacterium to the host epithelial cell. We present the motility and microrheology study of a clinical isolate strain of \textit{H. pylori}, J99, and its mutant with and without particular adhesins that bind to mucins with specific alterations in their glycans coat. Our microrheology experiments show that mucin viscosity depends on the glycans coat and decreases in the presence of bacteria. We found no significant changes in bacterial motility between J99 wild type and mutant in culture broth. Unlike previous observations made with other \textit{H. pylori} strains, we did not see reversals in J99 strains. Bacteria tracking measurements are underway to examine the motility in these altered mucin solutions.   

Subinhibitory concentrations of cell wall synthesis inhibitors promote biofilm formation of Enterococcus faecalis

Enterococcus faecalis are commonly associated with hospital acquired infections, because they readily form biofilms on instruments and medical devices. Biofilms are inherently more resistant to killing by antibiotics compared to planktonic bacteria, in part because of their heterogeneous spatial structure. Surprisingly, however, subminimal inhibitory concentrations (sub-MICs) of some antibiotics can actually promote biofilm formation. Unfortunately, much is still unknown about how low drug doses affect the composition and spatial structure of the biofilm. In this work, we investigate the effects of sub-MICs of ampicillin on the formation of E. faecalis biofilms. First, we quantified biofilm mass using crystal violet staining in polystyrene microtiter plates. We found that total biofilm mass is increased over a narrow range of ampicillin concentrations before ultimately declining at higher concentrations. Second, we show that sub-MICs of ampicillin can increase mass of E. faecalis biofilms while simultaneously increasing extracellular DNA/RNA and changing total number of viable cells under confocal microscopy. Further, we use RNA-seq to identify genes differentially expressed under sub-MICs of ampicillin. Finally, we show a mathematical model to explain this phenomenon.   

Cellular cAMP uptake as trigger for electrotaxis

Cells have the ability to detect continuous current electric fields and respond to them with a directed migratory movement. Dictyostelium discoideum cells, a key model organism for the study of eukaryotic chemotaxis, orient and migrate toward the cathode under the influence of an electric field. The underlying sensing mechanism and whether it is shared by the chemotactic response pathway remains unknown. By investigating the migration in the electric field of cell strains unable to migrate chemotactically (Amib-null) and with defective cAMP relay (ACA-null) we show that the starvation-induced transcription of a set of genes involved in the early developmental stage is not necessary for electrotaxis. However, the analysis of electrotaxis of vegetative cells as well as shortly starved cells shows that cells need to be stimulated with cAMP in order for them to migrate electrotactically. Indeed 30 minutes stimulation with cAMP pulses is enough to let cells orienting with the electric field although during this time the expression of receptors and the beginning of the development has not happened yet. We believe that the reason for this observed phenomenon lies on the endocytosis of the external cAMP which triggers electrotaxis as long as endocytosis and exocytosis are not balanced.   

Investigating the Varying Effects of Weak Electromagnetic Fields on Common Bacteria

Electromagnetic fields have been previously found to alter the growth rate of Escherichia coli. These prior results can be compared and contrasted according to the chosen experimental conditions, such as the magnitude or oscillatory frequency of the fields. In light of these comparisons, we examine recent experimental results with particular emphasis on the effects arising from the use of different electromagnetic fields.   

Comparison of magnetic field effects on the growth of Staphylococcus Aureus and Staphylococcus Epidermidis

The effects of magnetic fields were investigated on two species of bacteria: Staphylococcus Aureus and Staphylococcus Epidermidis. Both cultures were grown independently in agar plates and nutrient broth with exposure to various conditions of static and oscillating magnetic fields. The effects were characterized by growth rate measurements via changes in optical density (OD) over incubation periods of 24-28 hours. Significant effects on the growth rates of both species were observed in the case of the time-varying magnetic field.   

Study of Bacterial Response to Antibiotics in Low Magnetic Fields~

Effect of low magnetic fields on bacterial growth has been well established. Current study shows how different magnetic fields effect the bacterial response to antibiotics shows that the bacterial infections treatment and disease cure is changed in the presence of weak fields. This study has focused on understanding how different types of low magnetic fields change the response the bacterium to antibiotics in a liquid medium. This low magnetic field coupled with the introduction of antibiotics to the growth medium shows a drop in the growth curve. The most significant effect of low magnetic fields was seen with the uniform electromagnetic field ~as compared to the similar strength of constant static magnetic field produced by a bar magnets.~   

Biophysics of Euglena phototaxis

Phototactic microorganisms usually respond to light stimuli via phototaxis to optimize the process of photosynthesis and avoid photodamage by excessive amount of light. Unicellular phototactic microorganisms such as Euglena gracilis only possesses a single photoreceptor, which highly limits its access to the light in three-dimensional world. However, experiments demonstrated that Euglena responds to light stimuli sensitively and exhibits phototaxis quickly, and it's not well understood how it performs so efficiently. We propose a mathematical model of Euglena's phototaxis that couples the dynamics of Euglena and its phototactic response. This model shows that Euglena exhibits wobbling path under weak ambient light, which is consistent to experimental observation. We show that this wobbling motion can enhance the sensitivity of photoreceptor to signals of small light intensity and provide an efficient mechanism for Euglena to sample light in different directions. We further investigate the optimization of Euglena's phototaxis using different performance metrics, including reorientation time, energy consumption, and swimming efficiency. We characterize the tradeoff among these performance metrics and the best strategy for phototaxis.