Abstract Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacteriumPseudomonas chlororaphisO6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H2O2-released from roots during plant stress responses. Atomic force microscopy revealed 40–300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles ofPcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids thanPcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H2O2-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents.
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Differential Packaging Into Outer Membrane Vesicles Upon Oxidative Stress Reveals a General Mechanism for Cargo Selectivity
Selective cargo packaging into bacterial extracellular vesicles has been reported and implicated in many biological processes, however, the mechanism behind the selectivity has remained largely unexplored. In this study, proteomic analysis of outer membrane (OM) and OM vesicle (OMV) fractions from enterotoxigenic E. coli revealed significant differences in protein abundance in the OMV and OM fractions for cultures shifted to oxidative stress conditions. Analysis of sequences of proteins preferentially packaged into OMVs showed that proteins with oxidizable residues were more packaged into OMVs in comparison with those retained in the membrane. In addition, the results indicated two distinct classes of OM-associated proteins were differentially packaged into OMVs as a function of peroxide treatment. Implementing a Bayesian hierarchical model, OM lipoproteins were determined to be preferentially exported during stress whereas integral OM proteins were preferentially retained in the cell. Selectivity was determined to be independent of transcriptional regulation of the proteins upon oxidative stress and was validated using randomly selected protein candidates from the different cargo classes. Based on these data, a hypothetical functional and mechanistic basis for cargo selectivity was tested using OmpA constructs. Our study reveals a basic mechanism for cargo selectivity into OMVs that may be useful for the engineering of OMVs for future biotechnological applications.
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- Award ID(s):
- 1931309
- PAR ID:
- 10290548
- Date Published:
- Journal Name:
- Frontiers in Microbiology
- Volume:
- 12
- ISSN:
- 1664-302X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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