In bacteria, a host of enzymes regulates the reproducible and robust construction of the cell wall, whose mechanical integrity is crucial for viability under osmotic stress. Antibiotics that target these enzymes disrupt cell wall construction, ultimately leading to mechanical failure of the cell. Our work explores the physical mechanisms of cell growth and death, as a guide to understanding antibiotic mechanisms that disrupt mechanical properties of the cell. We use a combination of cell wall fluorescent labeling, high resolution time-lapse microscopy, and computational image processing to characterize where, and with what dynamics, cell wall and outer membrane growth occurs. Analysis of cell-surface marker fluorescence indicates that the cytoskeleton is present at sites of active growth and that chemical depolymerization of the cytoskeleton causes homogeneous, unstructured growth and eventual cell death by rupture. When combined with cell-shape analysis, our data strongly suggest that dynamic localization of the bacterial cytoskeleton is part of a curvature sensing and growth feedback mechanism that orchestrates heterogeneous growth to maintain cell shape and regulate mechanical stress. Using surface-specific protein labeling, we show that outer membrane growth also occurs in a similar heterogeneous manner. Quantitative tracking of growth is an effective method for characterizing cell wall mechanical failure resulting from antibiotic treatment. These techniques pave the way for studying the detailed dynamics of growth-associated proteins and their disturbance by antibiotics.
NOTE: IT'S ON WEDNESDAY NOT THURSDAY!