Phage-Mediated Explosive Cell Lysis Induces the Formation of a Different Type of O-IMV in Shewanella vesiculosa M7T

Nicolás Baeza; Lidia Delgado; Jaume Comas; Elena Mercade

doi:10.3389/fmicb.2021.713669

Phage-Mediated Explosive Cell Lysis Induces the Formation of a Different Type of O-IMV in Shewanella vesiculosa M7^T

Front Microbiol. 2021 Oct 8:12:713669. doi: 10.3389/fmicb.2021.713669. eCollection 2021.

Authors

Nicolás Baeza¹, Lidia Delgado², Jaume Comas³, Elena Mercade¹

Affiliations

¹ Secció de Microbiologia, Departament de Biologia, Sanitat i Medi Ambient, Universitat de Barcelona, Barcelona, Spain.
² Crio-Microscòpia Electrònica, Centres Científics i Tecnològics, Universitat de Barcelona (CCiTUB), Barcelona, Spain.
³ Citometria, Centres Científics i Tecnològics, Universitat de Barcelona (CCiTUB), Barcelona, Spain.

Abstract

Shewanella vesiculosa M7^T is a cold-adapted Antarctic bacterium that has a great capacity to secrete membrane vesicles (MVs), making it a potentially excellent model for studying the vesiculation process. S. vesiculosa M7^T undergoes a blebbing mechanism to produce different types of MVs, including outer membrane vesicles and outer-inner membrane vesicles (O-IMVs). More recently, other mechanisms have been considered that could lead to the formation of O-IMVs derived from prophage-mediated explosive cell lysis in other bacteria, but it is not clear if they are of the same type. The bacterial growth phase could also have a great impact on the type of MVs, although there are few studies on the subject. In this study, we used high-resolution flow cytometry, transmission electron microscopy, and cryo-electron microscopy (Cryo-EM) analysis to determine the amount and types of MVs S. vesiculosa M7^T secreted during different growth phases. We show that MV secretion increases during the transition from the late exponential to the stationary phase. Moreover, prophage-mediated explosive cell lysis is activated in S. vesiculosa M7^T, increasing the heterogeneity of both single- and double-layer MVs. The sequenced DNA fragments from the MVs covered the entire genome, confirming this explosive cell lysis mechanism. A different structure and biogenesis mechanisms for the explosive cell lysis-derived double-layered MVs was observed, and we propose to name them explosive O-IMVs, distinguishing them from the blebbing O-IMVs; their separation is a first step to elucidate their different functions. In our study, we used for the first time sorting by flow cytometry and Cryo-EM analyses to isolate bacterial MVs based on their nucleic acid content. Further improvements and implementation of bacterial MV separation techniques is essential to develop more in-depth knowledge of MVs.

Keywords: DNA; O-IMV; OMV; electron microscopy; explosive cell lysis; flow cytometry; gram-negative bacteria; membrane vesicles.