Research Projects
Systematic Dissection of the Biochemical Basis for the Mechanical Properties of the Outer Membrane
Figure 1. A) Gram-negative bacterial cell. l: envelope length, P: turgor pressure. B) Osmolarity of growth medium versus time during an osmotic-force extension experiment. C) Cell envelope length during the experiment. D) Mechanical strain versus shock magnitude during the experiment. E) Rank-ordered cell envelope stiffness of outer membrane mutants. F) E. coli cells color- coded with fluorescent dyes.
Motivated to understand how the outer membrane bears forces at the molecular level I aimed to determine what molecules and moieties within the outer membrane were critical for its load-bearing capacity. However, there were two experimental bottlenecks that I had to resolve. First, previous methods relied on subjecting cells to a single osmotic shock of a given magnitude, thereby altering the turgor pressure within cells, and measuring the resulting deformation of the cell envelope (Fig. 1A), which limited our ability to reveal the constitutive mechanical properties of the cell envelope (e.g. linear vs. non-linear). To overcome this, I developed and validated a new microfluidics-based “osmotic force-extension assay” in which cells were subjected to a series of osmotic shocks (Fig. 1B) and the induced mechanical strains of the cell envelope (Fig. 1C) from each shock were measured using single-cell microscopy; an empirical measure of envelope stiffness is given by the inverse of the slope of the linear regression between strain and shock magnitude (Fig. 1D). Second, most single-cell mechanics measurements are inherently low throughput, therefore, I invented a broadly useful method to color-code tens of bacterial strains with combinations of fluorophores with non-overlapping emission wavelengths, which could then be pooled into the same microfluidics-microscopy experiment (Fig. F).
Specifically, I wanted to test whether lipopolysaccharide charge, length, and protein abundance are critical for outer membrane stiffness and strength. Therefore, I constructed and collected a library of mutant bacterial strains that had alterations to lipopolysaccharide charge, length, and outer membrane protein abundance. I combined my color-coding method to pool mutants with my osmotic force extension assay to measure the cell-envelope stiffness. These mutants displayed a wide, graded range in outer membrane stiffness (Fig. 1E).
The Biomechanical Mechanism of Outer Membrane Vesiculation
Figure 2. A) Simulation of vesiculation based on mechanical model. E: total chemomechanical energy, Es: stretching energy, Eb: bending energy, Eadh: adhesion energy, Vperi: volume of periplasm Pperi: periplasmic pressure. B) E. coli Δpal cells expressing periplasm-targeted mCherry. Arrow indicates a vesicle forming.
All Gram-negative bacteria release small vesicles (20-200 nm) derived from their outer membrane, which enhance fitness by aiding in nutrient acquisition, biofilm formation, and antibiotic stress response. These vesicles are, highly abundant existing in an approximate 1:1 ratio with bacterial cells in the ocean. Despite their ubiquity and numerous functions, the mechanism of vesiculation is very poorly understood. Genetic screens have identified nearly 200 genes which result in hyper- or hypovesiculation phenotypes, obscuring the identification of the pathways which regulate vesiculation. Indeed, vesiculation is an inherently biophysical process, and few methods exist for the quantification of such processes at the single-cell level.
To address this, I am developing a mechanistic, theoretical model for vesiculation. As proof of concept, I computationally solved a preliminary version of this model by minimizing the free energy of the system (Fig. 2A). To precisely measure outer membrane vesiculation dynamics dependence on protein level and outer membrane stiffness, I will use a microscopy assay that I developed for measuring the dynamics of vesiculation at the single-vesicle level (Fig. 2B). This interdisciplinary project will allow strong quantitative predictions with our theoretical model that are borne out through rigorous experimentation.
Adhesion Forces in Bacterial Predator-Prey and Prey-Prey Systems
Image of GFP-expressing E. coli adhered to a cantilever
At SUNY New Paltz, I joined the lab of Megan Ferguson, where I investigated the biochemical and physical mechanisms of Bdellovibrio bacteriovorus predation and cell-cell adherence. B. bacteriovorus are small predatory Gram-negative bacteria found in the human gut that prey on other Gram-negative bacteria. B. bacteriovorus predation has interested many microbiologists because this bacterium has been demonstrated to prey on almost every Gram-negative species it is co-cultured with, indicating its potential utility to fight infection rather than using traditional antibiotics. In order to “eat” another bacterial cell, a B. bacteriovorus cell first has to adhere to it. However, it was unclear if adherence was a purely passive process. Indeed, I hypothesized that the magnitude of the adhesion force would depend on active predation by B. bacteriovorus rather than cell-cell contact alone. However, no methods existed for measuring B. bacteriovorus adhesion forces. Thus, to test this hypothesis precisely, my strategy was to use an atomic force microscope (AFM) to measure the adhesion force as a function of the time that the predator and prey were in contact, which is correlated with the probability of predation. I thus developed and optimized an innovative strategy to do so in which I functionalized AFM cantilevers with a cell- adhesive chemical called Cell-Tak, then these cantilevers were submerged in a culture of GFP- expressing E. coli (the prey bacterium). I used a similar protocol to coat glass slides with B. bacteriovorus. I then used AFM to engage the predator-coated slide with the prey-coated cantilever and measured the resulting adhesion forces between the two. I found that initial adhesion forces (after seconds) were greater for control homotypic E. coli-E. coli interactions then for E. coli-B. bacteriovorus ones. However, if I prolonged the interaction time to 5 minutes, E. coli-B. bacteriovorus interactions were stronger. These data supported my hypothesis that B. bacteriovorus increases adhesion upon predation.