Cell Wall Nonlinear Elasticity and Growth Dynamics: How Do Bacterial Cells Regulate Pressure and Growth?

Abstract

In my thesis, I study intact and bulging <italic>Escherichia coli</italic> cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. I find strong evidence of power--law stress--stiffening in the <italic>E. coli</italic> cell wall, with an exponent of 1.22&plusmn;0.12, such that the wall is significantly stiffer in intact cells (E = 23&plusmn;8 MPa and 49&plusmn;20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells <italic>E. coli</italic> is 29&plusmn;3 kPa. The nonlinearity in cell elasticity serves as a plausible mechanism to balance the mechanical protection and tension measurement sensitivity of the cell envelope. I also study the growth dynamics of the <italic>Bacillus subtilis</italic> cell wall to help understand the mechanism of the spatiotemporal order of inserting new cell wall material. High density fluorescent markers are used to label the entire cell surface to capture the morphological changes of the cell surface at sub-cellular to diffraction-limited spatial resolution and sub-minute temporal resolution. This approach reveals that rod-shaped chaining <italic>B. subtilis</italic> cells grow and twist in a highly heterogeneous fashion both spatially and temporally. Regions of high growth and twisting activity have a typical length scale of 5 &mu;m, and last for 10-40 minutes.

Motivated by the quantification of the cell wall growth dynamics, two microscopy and image analysis techniques are developed and applied to broader applications beyond resolving bacterial growth. To resolve densely distributed quantum dots, we present a fast and efficient image analysis algorithm, namely Spatial Covariance Reconstruction (SCORE) microscopy that takes into account the blinking statistics of the fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging, which is at least an order of magnitude faster than single-particle localization based methods such as STORM and PALM. SCORE is insensitive to background and can be applied to different types of fluorescence sources, including but not limited to organic dye and quantum dot that are tested experimentally in this thesis. The second development is an extension from tracking single quantum dot to the more general cases of moving objects at high density based on active contour model. I add a repulsive interaction between open contours to the original model and treat the trajectories as extrusions in the temporal dimension. This technique is applicable to a broad range of problems and two specific tracking problems are chosen as illustrations: (i) the quantification of walking and chasing behaviors of <italic>Drosophila</italic> and (ii) the study of trajectories of gliding bacteria <italic>Myxococcus xanthus</italic> on flat surface. I demonstrate the capability of this high-through and highly automated analysis method for studying social and group behaviors in interacting organisms.