Mycolic acid-containing bacteria trigger distinct types of membrane vesicles through different routes.

Bacterial membrane vesicles (MVs) are attracting considerable attention in diverse fields of life science and biotechnology due to their potential for various applications. Although there has been progress in determining the mechanisms of MV formation in Gram-negative and Gram-positive bacteria, the mechanisms in mycolic acid-containing bacteria remain an unsolved question due to its complex cell envelope structure. Here, by adapting super-resolution live-cell imaging and biochemical analysis, we show that form distinct types of MVs via different routes in response to environmental conditions. DNA-damaging stress induced MV formation through prophage-triggered cell lysis, whereas envelope stress induced MV formation through mycomembrane blebbing. The MV formation routes were conserved in other mycolic acid-containing bacteria. Our results show how the complex cell envelope structure intrinsically generates various types of MVs and will advance our knowledge on how different types of MVs can be generated from a single cell organism.