The Problem

How does a cell establish its shape? While spheres are favored by physical laws, cells are rarely spherical. Instead, many cells employ intricate molecular machineries and complex regulatory networks to build and maintain various shapes for diverse biological functions. Morphogenesis (the ability to build defined cell shapes) is especially important for bacteria, because bacterial cell shapes are usually defined by the peptidoglycan (PG) cell wall, an exoskeleton also essential for their survival. Since the machineries for PG synthesis constitute the best targets for antibiotics, understanding bacterial morphogenesis will provide critical information for the control of infectious diseases. To understand morphogenesis, we are investigating how bacteria form rods, the simplest non-spherical shapes.

The Approach

• An exceptional model organism. When induced by chemicals, rod-shaped vegetative cells of the Gram-negative bacterium Myxococcus xanthus thoroughly degrade their cell wall and shrink into spherical spores. As these spores germinate, rod- shaped cells rebuild cell wall without preexisting templates, which provides a rare opportunity to visualize de novo cell wall synthesis and bacterial morphogenesis. Using M. xanthus as the model organism, we visualize cell wall synthesis using fluorescent dyes, label key components in the cell wall synthesis machinery with fluorescent tags, and investigate how spherical spores build rod- shaped cell wall through germination.
• A powerful technique. Because of the diffraction limit of visible light, the resolution limit for con- ventional light microscopy is 200-250 nm. For this reason, the localization and dynamics of single molecules cannot be resolved in bacterial cells. To solve this problem, we have built a super-resolu- tion microscope for single-particle tracking pho- toactivatable localization microscopy (sptPALM) and stochastic optical reconstruction microscopy (STORM). Most importantly, we are able to track single-particle dynamics in live cells at 10-ms inter- vals (100 frames/second).

The Innovations and Discoveries

• We found that germinating spores first synthesize cell wall on spherical surfaces in an isotropic manner, then elongate into rods by growing cell wall at nonpolar regions. Spores establish different shapes by altering the distribution pattern of their cell wall synthesis machineries, which in turn, alters the growth pattern of cell wall.
• Special protein regulators survey the status of cell wall synthesis in germinating spores and trigger the switch of growth pattern through a system including a cytoskeleton and molecular motor.
• We are now able to weaken bacterial cell walls and generate different cell shapes by manipulating the above system, which could usher in novel methods for the control of bacterial infection.