Abou-Haydar, Y. | bioRxiv Science

bioRxiv2025

Extracellular vesicle production and membrane uptake promote repair and antibiotic tolerance in E. coli

Bacterial extracellular vesicles (EVs) are nanosized lipid structures released in response to environmental stressors, such as phages and antibiotics. Despite their critical role in bacterial adaptability, the mechanisms by which EVs interact with membranes under stress remain poorly understood, due to challenges in visualizing these dynamic processes in live bacteria. Here, we use high-resolution fluorescence microscopy, flow cytometry, and cryo-electron microscopy to investigate EV production and uptake in Escherichia coli exposed to sub-minimum inhibitory concentration doses of polymyxin B (Pmb), a membrane-active antimicrobial peptide. Using fluorescently labeled Pmb and EVs, we track Pmb insertion and removal from membranes, EV production and uptake, and their effects on cell growth. Our findings demonstrate that EV production rapidly sequesters Pmb in the medium and facilitates its removal from bacterial membranes. For the first time, we demonstrated that EVs act as membrane plugs by adhering to or fusing with Pmb-damaged membranes. These dynamic processes work together to reduce the antibiotic load from the membranes, turn off the RcsA-mediated membrane stress response, and enable cells to resume growth. Although EVs do not provide resistance to Pmb, they enhance the survival and tolerance of bacterial populations. This study uncovers the dual role of EVs in Pmb sequestration and membrane repair, providing new insights into antibiotic tolerance mechanisms and paving the way for innovative approaches to combat antimicrobial resistance.

microbiology↗

bioRxiv2024

Incomplete lytic cycle of a widespread Bacteroides bacteriophage leads to the formation of defective viral particles

Advances in metagenomics have led to the identification of new intestinal temperate bacteriophages. However, their experimental characterization remains challenging due to a limited understanding of their lysogenic-lytic cycle and the common lack of plaque formation in vitro. In this study we investigated the hankyphage, a widespread transposable phage of prominent Bacteroides symbionts. Hankyphages spontaneously produced virions in laboratory conditions even in the absence of inducer, but virions did not show any evidence of infectivity. To increase virion production and raise the chances of observing infection events, we identified a master repressor of the hankyphage lytic cycle, RepCHP, whose silencing amplified hankyphage gene expression, enhanced replicative transposition and virion production. However, attempts to infect or lysogenize new host cells with different capsular types remained unsuccessful. Transmission electron microscopy and capsid DNA sequencing revealed an abnormal virion morphology and incomplete DNA packaging of the hankyphage, suggesting that it cannot complete its assembly in laboratory conditions for reasons that are yet to be identified. Still, metavirome and phylogenetic analyses were suggestive of hankyphage horizontal transmission. We could also detect the activity of diversity-generating retroelements (DGRs) that mutagenize the hankyphage minor tail fiber, and likely contribute to its broad host range. This study sheds light on the life cycle of this this abundant intestinal bacteriophage and highlights important gaps in our understanding of the factors required for the completion of its life cycle. Elucidating this puzzle will be critical to gain a better understanding of the hankyphage biology and ecological role.

microbiology↗