Proliferation of Listeria monocytogenes L-form cells by formation of internal and external vesicles

L-forms are cell wall-deficient bacteria that divide through unusual mechanisms, involving dynamic perturbations of the cellular shape and generation of vesicles, independently of the cell-division protein FtsZ. Here we describe FtsZ-independent mechanisms, involving internal and external vesicles, by which Listeria monocytogenes L-forms proliferate. Using micromanipulation of single cells and vesicles, we show that small vesicles are formed by invagination within larger intracellular vesicles, receive cytoplasmic content, and represent viable progeny. In addition, the L-forms can reproduce by pearling, that is, generation of extracellular vesicles that remain transiently linked to their mother cell via elastic membranous tubes. Using photobleaching and fluorescence recovery, we demonstrate cytoplasmic continuity and transfer through these membranous tubes. Our findings indicate that L-forms’ polyploidy and extended interconnectivity through membranous tubes contribute to the generation of viable progeny independently of dedicated division machinery, and further support L-forms as models for studies of potential multiplication mechanisms of hypothetical primitive cells.