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

Abstract

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.

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes

“…While the cell wall is considered an essential component of virtually all bacteria, most species can be manipulated under laboratory conditions to produce so-called L-forms that are able to propagate without their wall 14 – 17 . Typically, L-forms are generated by exposing walled bacteria to high levels of lysozyme combined with antibiotics that target cell wall synthesis in media containing high levels of osmolytes 18 , 19 .…”

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes

“…While the cell wall is considered an essential component of virtually all bacteria, most species can be manipulated under laboratory conditions to produce so-called L-forms that are able to propagate without their wall 14–17 Typically, L-forms are generated by exposing walled bacteria to high levels of lysozyme combined with antibiotics that target cell wall synthesis in media containing high levels of osmolytes 18, 19 Stable L-forms that can propagate indefinitely without the cell wall require two mutations that fall in separate classes 18 . The first class of mutations leads to an increase in membrane synthesis, either directly by increasing fatty acid biosynthesis, or indirectly by reducing cell wall synthesis 20 .…”

Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria

“…Based on these assumptions, we evaluated L-form cells of Listeria monocytogenes as recipients and rebooting compartments for native, purified phage gDNA. Listeria L-form cells can be generated by prolonged subcultivation in the presence of cell wall-active antibiotics in an osmoprotective environment (24,27). We used a L. monocytogenes EGDe strain variant designated "Rev2," which can rapidly be induced to grow as an L-form (designated "Rev2L") (27).…”

“…Listeria L-form cells can be generated by prolonged subcultivation in the presence of cell wall-active antibiotics in an osmoprotective environment (24,27). We used a L. monocytogenes EGDe strain variant designated "Rev2," which can rapidly be induced to grow as an L-form (designated "Rev2L") (27). To provide a proof of concept for the rebooting approach, we used gDNA of the virulent L. monocytogenes phage P35.…”