Abstract
Members of the Corynebacterineae, including Corynebacterium and Mycobacterium, have an atypical cell envelope characterized by an additional mycomembrane outside of the peptidoglycan layer. How this multilayered cell envelope is assembled remains unclear. Here, we tracked the assembly dynamics of different envelope layers in Corynebacterium glutamicum and Mycobacterium smegmatis by using metabolic labeling and found that the septal cell envelope is assembled sequentially in both species. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. This diffusion barrier is resolved through perforations in the peripheral peptidoglycan, thus leading to the confluency of the mycomembrane before daughter cell separation (V snapping). Furthermore, the same junctional peptidoglycan also serves as a mechanical link holding the daughter cells together and undergoes mechanical fracture during V snapping. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.