Correlative Super-Resolution Optical and Atomic Force Microscopy Reveals Relationships Between Bacterial Cell Wall Architecture and Synthesis in Bacillus subtilis

Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. Atomic force microscopy (AFM) can provide near molecular resolution images of biological systems but typically provides limited chemical information. Conversely, while super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain. Here, we combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Using clickable D-amino acid incorporation, we fluorescently label and spatially localize a short and controlled period of peptidoglycan synthesis and correlate this information with high-resolution AFM of the resulting architecture. During division, septal synthesis occurs across its developing surface, suggesting a two-stage process with incorporation at the leading edge and with considerable in-filling behind. During growth, the elongation of the rod occurs through bands of synthesis, spaced by similar to 300 nm, and corresponds to denser regions of the internal cell wall as revealed by AFM. Combining super-resolution optics and AFM can provide insights into the synthesis processes that produce the complex architectures of bacterial structural biopolymers.

Automated summary

This study combines STORMForce and SIMForce technologies to map the synthesis of peptidoglycan, a structural macromolecule of bacterial cell walls, during growth and division in Bacillus subtilis. It reveals a two-stage process of septal synthesis during division and bands of synthesis during growth. The combination of super-resolution optics and AFM provides insights into the synthesis processes of bacterial structural biopolymers.