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Extracellular vesicles (EVs) are nano-sized, biocolloidal proteoliposomes that have been shown to be produced by all cell types studied to date and are ubiquitous in the environment. Extensive literature on colloidal particles has demonstrated the implications of surface chemistry on transport behavior. Hence, one may anticipate that physicochemical properties of EVs, particularly surface charge-associated properties, may influence EV transport and specificity of interactions with surfaces. Here we compare the surface chemistry of EVs as expressed by zeta potential (calculated from electrophoretic mobility measurements). The zeta potentials of EVs produced by Pseudomonas fluorescens, Staphylococcus aureus, and Saccharomyces cerevisiae were largely unaffected by changes in ionic strength and electrolyte type, but were affected by changes in pH. The addition of humic acid altered the calculated zeta potential of the EVs, especially for those from S. cerevisiae. Differences in zeta potential were compared between EVs and their respective parent cell with no consistent trend emerging; however, significant differences were discovered between the different cell types and their EVs. These findings imply that, while EV surface charge (as estimated from zeta potential) is relatively insensitive to the evaluated environmental conditions, EVs from different organisms can differ regarding which conditions will cause colloidal instability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bacterial outer membrane vesicles (OMVs) are 20- to 200-nm secreted packages of lipids, small molecules, and proteins that contribute to diverse bacterial processes. In plant systems, OMVs from pathogenic and beneficial strains elicit plant immune responses that inhibit seedling growth and protect against future pathogen challenge. Previous studies of OMV-plant interactions suggest functionally important differences in the protein composition of Pseudomonas syringae and Pseudomonas fluorescens OMVs, and that their composition and activity differ as a result of medium culture conditions. Here, we show that plant apoplast-mimicking minimal medium conditions impact OMV protein content dramatically in P. syringae but not in P. fluorescens relative to complete medium conditions. Comparative, 2-way analysis of the four conditions reveals subsets of proteins that may contribute to OMV-mediated bacterial virulence and plant immune activation as well as those involved in bacterial stress tolerance or adaptation to a beneficial relationship with plants. Additional localization enrichment analysis of these subsets suggests the presence of outer-inner membrane vesicles (OIMVs). Collectively, these results reveal distinct differences in bacterial extracellular vesicle cargo and biogenesis routes from pathogenic and beneficial plant bacteria in different medium conditions and point to distinct populations of vesicles with diverse functional roles. IMPORTANCE Recent publications have shown that bacterial vesicles play important roles in interkingdom communication between bacteria and plants. Indeed, our recently published data reveal that bacterial vesicles from pathogenic and beneficial strains elicit immune responses in plants that protect against future pathogen challenge. However, the molecules underlying these striking phenomena remain unknown. Our recent work indicated that proteins packaged in vesicles are critically important for vesicle-mediated seedling growth inhibition, often considered an indirect measure of plant immune activation. In this study, we characterize the protein cargo of vesicles from Pseudomonas syringae pathovar tomato DC3000 and Pseudomonas fluorescens from two different medium conditions and show that distinct subpopulations of vesicles contribute to bacterial virulence and stress tolerance. Furthermore, we reveal differences in how beneficial and pathogenic bacterial species respond to harsh environmental conditions through vesicle packaging. Importantly, we find that protein cargo implicates outer-inner membrane vesicles in bacterial stress responses, while outer membrane vesicles are packaged for virulence.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence., (© 2021 The Authors.)

All cells produce extracellular vesicles (EVs). These biological packages contain complex mixtures of molecular cargo and have a variety of functions, including interkingdom communication. Recent discoveries highlight the roles microbial EVs may play in the environment with respect to interactions with plants as well as nutrient cycling. These studies have also identified molecules present within EVs and associated with EV surfaces that contribute to these functions. In parallel, studies of engineered nanomaterials have developed methods to track and model small particle behavior in complex systems and measure the relative importance of various surface features on transport and function. While studies of EV behavior in complex environmental conditions have not yet employed transdisciplinary approaches, it is increasingly clear that expertise from disparate fields will be critical to understand the role of EVs in these systems. Here, we outline how the convergence of biology, soil geochemistry, and colloid science can both develop and address questions surrounding the basic principles governing EV-mediated interkingdom interactions.

Bacterial outer membrane vesicles (OMVs) perform a variety of functions in bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions. • OMVs from P. syringae and P. fluorescens protect against bacteria and oomycetes • Bacterial OMVs elicit distinct sets of plant immune responses • OMV-mediated effects withstand biochemical disruption • OMVs can serve as new tools to probe the plant immune system The role that bacterial outer membrane vesicles (OMVs) play in plant-microbe interactions is poorly characterized. McMillan et al. show that OMVs elicit plant immune responses that protect against pathogens. This study also reveals a use for OMVs as tools to probe the plant immune system. [ABSTRACT FROM AUTHOR]