Endocytosis-like DNA uptake by cell wall-deficient bacteria

Abstract

Horizontal gene transfer in bacteria is widely believed to occur via conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. Here, we report that cell wall-deficient bacteria can engulf DNA and other extracellular material via an endocytosis-like process. Specifically, we show that L-forms of the filamentous actinomycete Kitasatospora viridifaciens can take up plasmid DNA, polysaccharides (dextran) and 150-nm lipid nanoparticles. The process involves invagination of the cytoplasmic membrane, leading to formation of intracellular vesicles that encapsulate extracellular material. DNA uptake is not affected by deletion of genes homologous to comEC and comEA, which are required for natural transformation in other species. However, uptake is inhibited by sodium azide or incubation at 4 °C, suggesting the process is energy-dependent. The encapsulated materials are released into the cytoplasm upon degradation of the vesicle membrane. Given that cell wall-deficient bacteria are considered a model for early life forms, our work reveals a possible mechanism for primordial cells to acquire food or genetic material before invention of the bacterial cell wall.

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

“…Cryo-Airyscan confocal microscopy (CACM) 19 is used to locate the region of interest (ROI) for 3D cryoFIB/SEM imaging as well as to determine the height, width and depth of the target volume in high pressure frozen tissue samples, however, registration of the two imaging modalities is complicated by the featureless surface of the vitrified ice layer 20 . By using a FinderTOP HPF carrier 36,37 , a square grid pattern is imprinted in the ice surface during the vitrification, which is recognizable by both light and electron imaging modalities. Subsequent correlation is achieved computationally through rotational and translational alignment of the imaging modalities.…”

“…The cryoFIB/SEM images were processed using the EM processing toolbox of Zen Connect 3.5 (blue edition, Carl Zeiss) to correct for defects such as curtaining, misalignment and local charging, analogous to our previously reported approach 37 , but with keeping the same processing settings for the whole stack. The same software was used for subsequent noise reduction and contrast enhancement.…”

Mycobacteria form viable cell wall-deficient cells that are undetectable by conventional diagnostics

“…Since the closely related vaccine strain M. bovis BCG Russia (Borsch et al, 2007) was also shown to form CWD cells in response to these antibiotics, it raises the question if wall-deficiency plays a role in pathogenesis and persistence of M. tuberculosis. The formation of such cells may have further contributed to the emergence of multi-, extensively-, and totally drug-resistant (MDR/XDR/TDR) TB strains (Levin-Reisman et al, 2017), given that such walldeficient cells are more competent for DNA uptake (Kapteijn et al, 2022). The involvement of CWD cells in persistent infections could also explain the necessity of synergistical administration of cell wall-targeting antibiotics with agents that target other essential cellular processes, such as rifampicin (WHO, 2020).…”

“…Considering the importance of the cell wall, it is surprising that under specific stressful conditions a range of bacteria can transiently escape from their enclosing cell wall to adopt a viable but CWD lifestyle (Allan et al, 2009;Claessen and van Wezel, 2014;Errington, 2013Errington, , 2017Errington et al, 2016;Kawai et al, 2015;Klieneberger, 1935;Mercier et al, 2014;Ramijan et al, 2018;Errington, 2019, Lazenby et al, 2022). These cells are competent with regard to DNA uptake (Woo et al, 2003, Kapteijn et al, 2022 and can revert to their walled state when the stressful conditions have diminished.…”

High‐Precision Synthesis of RNA‐Loaded Lipid Nanoparticles for Biomedical Applications

“…We also note that there are many other synergistic uses of LNP beyond therapeutic purposes in bioengineering fields that include medical imaging for traceable therapy, [ 364–367 ] cosmetics, [ 368 ] nutraceutical designs, [ 369 ] bioreactors, [ 370 ] bioprinting with 3D reconstructed scaffold, [ 371–373 ] life science study, [ 363 ] and intracellular trafficking. [ 374 ] For instance, bio‐printed nanofiber can be utilized to design and validate optimal RNA‐loaded LNP vaccine a priori and help in improving gene editing efficiency.…”