Introduction

Outer membrane vesicles (OMVs) are extracellular vesicles produced by Gram-negative bacteria, ranging from 20–300 nm in diameter1,2,3. OMV composition includes numerous outer membrane and periplasmic components such as proteins, lipopolysaccharide (LPS), enzymes, and in the case of pathogens, toxins4. OMVs may also contain DNA5,6 or RNA7. Consequently, OMV formation and release exhibit many unique functions including acting as a protein secretion system8, delivering virulence factors by pathogenic bacteria9,10,11, and signaling between bacteria12,13 and cross-kingdom with eukaryotic cells9,13,14. OMVs are released during all stages of cell growth in liquid and solid culture15. OMVs are released in biofilms and are a part of the extracellular matrix in naturally-occurring and laboratory-grown biofilms16. OMVs may mediate biofilm formation17. For several Helicobacter pylori strains, OMV production correlates with the ability to form and maintain the biofilm18. The quorum-sensing molecule 2-heptyl-3-hydroxy-4-quinolone, used for communication during biofilm formation by Pseudomonas aeruginosa, is present within P. aeruginosa-produced OMVs19 and is required for OMV formation by this bacterium20.

Although OMVs are continually released, OMV production increases in response to stress21,22, possibly as a mechanism to quickly release misfolded periplasmic proteins22,23. OMV production under stress also appears to have defensive roles. OMVs are decoys for bacteriophages and other membrane-binding antimicrobial agents thus distancing their potential to impact the live cell24. OMVs may contain specific enzymes and other compounds to increase bacterial survival1,25,26. For example, exposure to reactive oxygen species (ROS), such as H2O2, increases OMV release in several bacteria21,27,28. However, H. pylori release catalase-containing OMVs in response to ROS stress thus protecting cells from oxidative damage28. Another example is packaging β-lactamase into OMVs by Moraxella catarrhalis to inactivate β-lactam antibiotics29.

Virulence and signaling through OMVs released by human pathogens11,30 and plant pathogens31,32 have been investigated for managing infections. In contrast, factors stimulating OMV release and OMV roles for plant health-promoting bacteria are generally unknown. The beneficial relationship between the plant and its associated microbiome requires cross-kingdom signaling that is mediated through many small metabolites, including hormones and quorum sensing molecules33,34. OMV release presents one potential method for the secretion and delivery of these signals.

The potential packaging of nucleic acids and LPS into OMVs may be important as signals to the plant which then activates plant defenses. LPS is a microbe-associated molecular pattern (MAMP) and DNA fragments are damage-associated molecular patterns (DAMPs) recognized by specific plant cell receptors to trigger innate immunity35. OMVs contain large quantities of LPS36, likely due to the high surface area of these nano-vesicles. OMVs released by P. aeruginosa may contain plasmid DNA12, coding chromosomal DNA5, or noncoding extracellular DNA (eDNA)37.

Here, OMVs of a plant health-promoting bacterium, Pseudomonas chlororaphis O6 (PcO6), were studied. PcO6 is a Gram-negative, rod-shaped bacterium originally isolated from roots of commercial dryland wheat grown in Cache Valley, UT, USA38. PcO6 is representative of many beneficial soil bacteria, boosting plant health through multiple protective pathways. PcO6 is an aggressive root colonizer and forms robust biofilms on plant roots39,40 and abiotic surfaces41. Cell growth within the biofilm is nurtured through catabolism of the metabolites in the root exudates41. In return, the bacterium protects the host by producing phenazines and other antibacterial and antifungal compounds as well as triggering systemic resistance42. PcO6 protects plants from drought, in part by producing a volatile, butanediol, that triggers partial stomatal closure43. Biofilm formation may also promote crop drought tolerance because the biofilm matrix maintains moisture around the plant roots44,45.

Potential OMVs are observed in atomic force microscopy images of PcO6 cells46,47, but OMV composition and their role in PcO6 signaling, biofilm architecture, and stress responses are currently unknown. Two relevant stressors to the rhizosphere, the space around plant roots, and their effects on PcO6 and subsequent OMV production were examined in this study. The first stressor was CuO nanoparticles (NPs). Numerous engineered metal and metal oxide NPs are explored for agricultural applications including delivering macronutrient and micronutrient to crops48,49, controlling pests and pathogens49, and protecting crops against abiotic stresses50 such as drought40,49,51,52. In some cases, NPs enhance microbial synthesis of products that improve plant health53.

In PcO6-colonized wheat, CuO NPs upregulate genes associated with drought tolerance52 and increase lignification in wheat sclerenchyma, a strengthening tissue of the plant40. Thus NP-induced gene expression can contribute to crop stress tolerance52,54. NPs also change gene expression in various beneficial and antagonistic microorganisms54. Sublethal doses of CuO NPs increase PcO6 cell size without impeding biofilm formation41, decrease the production of the Fe-scavenging siderophore pyoverdine55, and increase the production of the plant growth regulator indole-3-acetic acid56. Hydrogen peroxide (H2O2) was examined as a second bacterial stressor as ROS are generated as a stress response by plant root cells57, including roots exposed to sublethal CuO NP challenges58.

To characterize OMVs, as well as any compositional changes due to these abiotic stressors, Raman spectroscopy was chosen as the primary analytical technique. Compared to other spectroscopy methods, Raman spectroscopy is well suited for biological samples as it requires minimal sample preparation, is non-destructive, and yields a linear correlation of compound concentration to signal strength59. Raman spectroscopy is regularly used for cellular identification and characterization, even at a single-cell level60,61,62. Changes in biomolecular profiles according to the growth stage can be detected by Raman spectroscopy in mycobacterial cells63. Raman spectroscopy has been used to characterize extracellular vesicles from eukaryotic cells64,65,66, but no current literature reports Raman spectroscopy characterization of OMVs.

Raman spectroscopy was used to explore the chemical signatures of biofilm PcO6 cells under baseline conditions and after exposure to sublethal doses of CuO NP or H2O2. OMVs were isolated and characterized from both control and stressed cells. Spectra were compared using linear discriminant analysis (LDA). This machine learning technique creates an algorithm to sort input data sets according to treatments, then uses the algorithm to predict the treatments of the input data sets. The actual treatment and predicted treatment are then compared to determine consistencies and variations between spectra. Biochemical assays detecting protein, LPS, and DNA concentrations were performed to measure their presence in OMVs and to further examine changes in OMV composition.

Results

Physical characterization of outer membrane vesicles

OMV production by PcO6 biofilm cells was confirmed in situ with imaging by atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM images of PcO6 biofilm cells, transferred from minimal medium agar plates, without stressors show OMVs as aggregates between PcO6 cells (Fig. 1a) and as linear assemblies apart from the cells (Fig. 1b). Purified control and stress-induced OMVs appear as clustered and linear aggregates in AFM images (Fig. 2). SEM analysis also reveals a propensity of purified OMVs to aggregate (Fig. 3). SEM images of PcO6 biofilms formed on hollow fiber membranes draped in minimal medium, a substrate that allows imaging of intact biofilms encased in extracellular polymeric substances, also showed OMVs as part of the biofilm matrix as well as budding from and/or adhered to cells (Fig. 3b).

Figure 1
figure 1

AFM amplitude images of cells from the edges of a PcO6 cell smear from a biofilm grown on a 2% agar minimal medium plate onto a clean glass slide. OMVs are visible in both images as (a) aggregates between PcO6 cells and (b) as linear assemblies. Arrows point to examples of these OMV aggregates.

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Figure 2
figure 2

AFM height images of purified OMVs. Z-scales are from red to yellow from 0 to 25 nm, respectively. (a) OMVs harvested from PcO6 without any stressors. (b) OMVs harvested from PcO6 under H2O2 stress (3% v/v). (c) OMVs harvested from PcO6 under CuO NP stress (30 mg Cu/L). Additional AFM images shown in Supplemental Fig. S1.

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Figure 3
figure 3

SEM images of PcO6 cells and purified OMVs. (a) SEM image showing OMV aggregation of samples purified from PcO6 biofilms grown on minimal medium plates without abiotic stressors after glutaraldehyde fixation and ethanol dehydration. (b) SEM image showing the cross-section of a PcO6 biofilm grown on a hollow fiber membrane draped in minimal medium without any added stressors. Three areas of this image are highlighted to show potential OMVs which are marked with arrows.

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Size analysis of the OMVs in the AFM images (n = 100 from four images per treatment) showed average diameters of individual OMVs in the range of 40–190 nm, 45–295 nm, and 45–140 nm for control, CuO NP-induced, and H2O2-induced OMVs (Supplemental Fig. S2). Two-way ANOVA of the data, accounting for the treatment and the image used, showed statistically significant differences (p-value < 0.05) between several images within each treatment (data not shown) leaving uncertainty for the results comparing OMVs from one treatment. Furthermore, OMV heights in AFM images were a fraction of the OMV width, only 5–15 nm (data not shown), implying that the OMVs flattened during sample desiccation and these diameters may not represent true in vivo OMV size. These size ranges and this potential vesicle flattening has been observed by other researchers imaging vesicles with AFM67.

Both AFM and SEM images revealed agglomerate structures comprised of individual OMVs with clearly defined boundaries between vesicles. This propensity for OMV aggregation was confirmed with dynamic light scattering (DLS) which revealed hydrodynamic diameters ranging from 40–300 nm (Supplemental Fig. S3). The imaging and DLS results are consistent with literature reports of OMV diameters between 10 and 300 nm11, 68.

Chemical characterization of PcO6 cells with Raman spectroscopy

Raman spectroscopy was coupled with LDA to detect differences in the chemical profiles of control and stressed PcO6 cells. Raman spectra of whole biofilm PcO6 cells (n = 24 from 6 replicates), normalized to the highest peak of 2935 cm−1 (C–H bonds and lipid C–H3 bonds), from each treatment are shown in Fig. 4. The 2935 cm−1 peak was selected for normalization as it correlates to C–H bonds from all biological molecules and lipid C–H3 bonds. Both of these bonds are plentiful in phospholipids that are present in OMV membranes, making this peak ideal for normalization.

Figure 4
figure 4

Averaged Raman spectra of intact PcO6 cells (6 replicates with 4 spectra each for a total of n = 24). (a) Averaged Raman spectra of PcO6 cells without any applied stressor from 600 to 3200 cm−1. No visible differences are seen in the relative peak intensities above 1800 cm−1 of PcO6 cells of each treatment so spectra of CuO NP and H2O2 treated cells are not shown. (b) Averaged Raman spectra of PcO6 cells treated with H2O2 stress, CuO NP stress, and a negative control with no added stressor from 600 to 1800 cm−1. In this region, differences are seen in relative peak intensity indicating differences in relative concentrations of various compounds. In both graphs, notable peak assignments are shown with an arrow and labeled with both the peak number and peak assignment. See Supplemental Table S1 for a list of all peak assignments and respective sources.

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Peak assignments were consistent with proteins containing amino acids such as phenylalanine and tyrosine as well as unsaturated lipids and polysaccharides. The signatures of bases adenine, uracil, and guanine indicate the presence of nucleic acids. A full list of peak assignments is shown in Supplemental Table S1. There were no peaks unique for any treatment nor any peaks that disappeared in any one treatment. However, peak intensities differed between treatments with the spectra of control PcO6 giving the highest relative intensities, followed by H2O2-treated PcO6, then CuO NP-treated PcO6. The following peaks showed the greatest decreases in peak intensity for stressed cells relative to the control: (a) 880 cm−1: tryptophan and lipid N+(CH3)3 bonds; (b) 1220–1280 cm−1: amide III; (c) 1330–1340 cm−1: adenine, guanine, tyrosine, and tryptophan; (d) 1365 cm−1: lipid C–H bonds; (e) 1450 cm−1: protein and lipid C–H bonds; and f) 1660 cm−1: unsaturated lipids.

Examining the majority of the Raman spectra (750–1700 and 2670–3100 cm−1) with LDA, it is apparent that PcO6 cells exhibit distinct chemical profiles according to the applied stressor (LDA plot shown in Figure 5 and LDA confusion matrix are shown in Table 1). Though several spectra are misclassified from each treatment, the algorithm had an 84.7% accuracy. LDA was also performed on narrower regions of the Raman spectra (Supplemental Fig. S4) to correlate how restricted input data sets influenced LDA grouping and accuracy. LDA results of spectra subsets trended towards increased distance between data groupings. Accuracy of the LDA assignment varied between spectral regions.

Figure 5
figure 5

LDA plot of Raman spectra from PcO6 cells. Data were truncated to 750–1700 cm−1 and 2670–3100 cm−1, where most peaks are located. The shape of the points of the plot indicate the true treatment of each spectrum whereas the color of the point indicates the predicted treatment according to LDA. Misclassified spectra are circled in purple.

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Table 1 Confusion matrix showing LDA results of Raman spectra from PcO6 cells.
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The LDA plot of the 2670–3100 cm−1 showed loose grouping of the treatments and the lowest accuracy of the examined regions was 82% (Supplemental Fig. S4b). This region of the spectra corresponds with several C–H and C–H2 signatures which would be unlikely to change across experimental groups. This region also contains the 2935 cm−1 peak (C–H bonds) which was the highest peak in all spectra and used to normalize the spectra. By contrast, LDA of lower wavenumbers tended to show tighter and more distinct groupings and higher accuracy. This is especially notable in the 1400–1500 cm−1 region (Supplemental Fig. S4f) and the 1500–1700 cm−1 region (Supplemental Fig. S4g) which both gave 94.4% accuracies. All treatments are distinctly grouped in these LDA plots, especially the H2O2 treatment in the 1400–1500 cm−1 region and the CuO NP treatment in the 1500–1700 cm−1 region. The 1400–1500 cm−1 region contains several protein signatures. The 1500–1700 cm−1 region contains various amino acid, nucleic acid, amide, and lipid signatures (see Supplemental Table S1 for a list of all peak assignments).

Comparing Raman spectra of PcO6 cells and isolated OMVs

Raman spectroscopy was used to compare the chemical profiles of PcO6 cells and isolated OMVs. Unique peaks were found in comparisons between Raman spectra of control biofilm PcO6 cells and purified OMVs from control cells (Fig. 6): 27 peaks are unique to PcO6 cells, 14 peaks are unique to OMVs, and 10 peaks are shared (a full list of peak assignments for PcO6 cells and OMVs is shown in Supplemental Table S1). Figure 6 shows these shared peak assignments, and whether relative peak intensity is higher for control PcO6 cells or resultant purified OMVs. The same trends were seen in H2O2 and CuO NP-treated PcO6 cells and purified OMVs of stressed cells (data not shown).

Figure 6
figure 6

Comparisons between averaged Raman spectra of control PcO6 cells and OMVs (n = 24 for PcO6, n = 12 for OMVs). Peaks that are shared by PcO6 and OMVs Raman spectra are marked with an arrow and labeled with the peak assignment. Text color indicates whether these peaks are higher in PcO6 cells (blue text), or isolated OMVs (orange text). See Supplemental Table S1 for a list of all peak assignments and respective sources.

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Figure 7
figure 7

Averaged Raman spectra of OMVs harvested from PcO6 colonies exposed to H2O2, CuO NPs, or no stressor at all (3 replicates with 4 spectra each for a total of n = 12) from 600 to 3200 cm-1. Differences are seen in normalized peak intensities indicating large differences in relative concentrations of compounds in the OMVs. Notable peak assignments are marked with an arrow and labeled with their respective peak number and peak assignment. For a list of all peak assignments and their sources, see Supplemental Table S1.

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The relative intensity differed between shared peaks. All but two of these shared peaks had higher relative intensity for the OMV spectra than spectra of intact PcO6 cells. Though there are few peaks shared by intact PcO6 cells and purified OMVs, several unique peaks for PcO6 and OMV Raman spectra correspond to the same chemical compounds: i.e. PcO6 peak 644 cm−1 and OMV peak 870 cm−1 both correspond with tyrosine. Raman peaks assigned to the amino acid tryptophan and the nucleic acid bases adenine, cytosine, and guanine are present in spectra PcO6 cells and purified OMVs. However, the peaks assigned to these chemistries differ between the spectra for intact biofilm cells and the isolated OMVs. Both PcO6 and OMV spectra show various lipid, carbohydrate, protein, and nucleic acid signatures although different signatures of these macromolecules appear in each: i.e. PcO6 peak 810 cm−1 indicates lipid O–P–O bonds while OMV peak 1092 cm−1 indicates lipid C–C bond (Supplemental Table S1).

Especially notable in Raman spectra of isolated OMVs was the increased intensity of nucleic acid peaks which implies that DNA and/or RNA are selectively packaged by PcO6 into and/or onto OMVs. This conclusion was confirmed by measuring OMV 260/280 UV-absorption ratios, which compares the concentration of nucleic acids to the concentration of protein at their respective absorption peaks of 260 and 280 nm. OMV 260/280 ratios did not vary according to cell stressor (Supplemental Table S2) with an overall mean of 5.91 ± 0.09. Generally, a 260/280 ratio above 1.8 is obtained with a pure DNA sample and 2.0 is obtained with a pure RNA sample. The micro-BCA assay results confirmed varying amounts of protein in OMV suspensions (Supplemental Fig. S5b). These high 260/280 ratios for OMVs could be due to other compounds within OMVs that absorb light at similar wavelengths. PcO6 is known to secrete several secondary metabolites including phenazines and siderophores42 and these results suggest that some metabolites may be packaged into OMVs.

Chemical characterization of stress-induced outer membrane vesicles with Raman spectroscopy

The chemical profiles of control OMVs were compared with H2O2- and CuO NP-induced OMVs with Raman spectroscopy and LDA. Raman spectra of OMVs purified from control and stressed cells showed consistency similar to Raman spectra of control and stressed intact PcO6 cells. Averaged Raman spectra (n = 12 from 3 replicates) from OMVs harvested from cells of all treatments are shown in Fig. 7 differences are seen in the peak intensity of control OMVs and those harvested from PcO6 exposed to CuO NPs and H2O2, most notably: (a) 1646 cm−1, glycine; (b) 1513 cm−1, adenine, cytosine, and guanine; and (c) 1348 cm−1, adenine, guanine, tyrosine, and tryptophan. For these peaks, spectra of OMVs from CuO NP-treated PcO6 had the highest intensity, followed by OMVs from H2O2-treated PcO6 then OMVs of control cells.

LDA results of the broad portion of the spectra (750–1700 & 2670–3100 cm−1) showed a 83.3% accuracy for pure OMVs from all treatments with several misclassified spectra in each treatment (LDA plot shown in Fig. 8, LDA confusion matrix shown in Table 2). Narrower portions of the spectra were also examined with LDA (Supplemental Fig. S6). Much like the LDA examination of PcO6 spectra, LDA of different portions of the OMV spectra showed differences in the grouping on LDA plots. However, the accuracy of the algorithms did not vary greatly between the spectra portions (83–89% accuracy). Five of the six spectra that were misclassified in LDA of the broader spectra were misclassified in most or all of the narrower portions. The LDA plots of these narrower portions control spectra were far removed from the spectra of the other treatments. In contrast, the spectra of OMVs harvested from H2O2-treated and CuO NP-treated cells tended to be grouped apart as well though with a large intermingled section between them that contained many of the misclassified spectra.

Figure 8
figure 8

LDA plot of Raman spectra from purified OMVs (n = 12 for control and CuO NP-induced OMVs and n = 11 for H2O2-induced OMVs from three OMV isolations). Data were truncated to 750–1700 cm−1 and 2670–3100 cm−1, where most peaks are located. The shape of the points of the plot indicate the true treatment of each spectrum whereas the color of the point indicates the predicted treatment according to LDA. Misclassified spectra are circled in purple.

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Table 2 Confusion matrix from LDA plot of Raman spectra given by pure OMVs.
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Biochemical assays were also used to measure the LPS and protein content of OMVs. LPS content (average of 15.6 ± 2.7 endotoxin units (EU)/mL solution), which should correlate with OMV number, increased slightly in H2O2-induced OMVs (Supplemental Fig. S5a). This result matches literature sources that OMV production is increased in response to stress, including ROS stress21,27,28. Protein content (average of 23.3 ± 13.4 μg/mL solution) of OMVs was uncorrelated with cellular stressor and trial (Supplemental Fig. S5b) even when normalized to the LPS content (Supplemental Fig. S5c).

Discussion

DLS and AFM measurements showed that OMV sizes were highly polydisperse in size regardless of the cellular stressor. Differing OMV diameter according to the analysis method has been reported in the literature67, likely due to differences in OMV aggregation and/or hydration. Aggregation of OMVs observed in AFM and SEM images may be due to outer membrane surface features similar to those involved in adhesion for intact cells69. Extracellular biomolecules within the biofilm matrix, which include proteins, polysaccharides, and eDNA70, may also bind to OMVs and cause this aggregation. Similar OMV aggregation was observed in studies of OMVs produced by Neisseria lactamica by Gorringe et al.71. The researchers observed that numerous large OMV aggregates occurred at pH 7.0 with fewer aggregates occurring at pH 8.0, implying that many OMV surface proteins reached an isoelectric equilibrium at this higher pH, thus reducing the number of OMV aggregates. LPS charge may also be altered by environmental pH, influencing electrostatic and steric barriers to agglomeration.

OMV aggregation may be counterproductive to signaling and similar OMV roles, however, OMV assembly may contribute to the assembly of scaffolds to create and support the biofilm matrix. OMVs produced by biofilm Myxococcus xanthus cells form chains that tether biofilm cells together72. Xyllela fastidiosa uses OMV aggregates to mediate surface adhesion, either promoting or preventing biofilm formation by the bacterium17. PcO6 is a strong biofilm former41 and OMV release by this bacterium may promote its aggressive colonization of plant roots during adhesion and biofilm formation as patches on the plant root40.

This work established that Raman spectroscopy coupled with LDA was a reliable method for characterization of OMV content; the creation of Raman peak libraries (such as Supplemental Table S1 and references73,74,75,76) is valuable in assigning structural changes to cells under different exposures. In this study, LDA was particularly important as Raman spectra of stressed PcO6 cells did not have different peaks from those of control cells, only peak intensities differed. This same pattern occurred for OMVs isolated from stressed and control cells.

To examine what Raman bands were most heavily weighted during LDA grouping calculations, LDA was also performed on smaller spectral regions (Supplemental Fig. S4). For the spectra of PcO6 cells, it appears that LDA is primarily grouped based on peak differences in the range of 750–1700 cm−1. The 1400–1500 cm−1 and 1500–1700 cm−1 regions were especially unique for H2O2 and CuO NP treatments, respectively. LDA of these regions yielded distinct groupings with few miscalculations. For the OMV spectra, the LDA algorithm primarily converged on the 750–1700 cm−1 range as well. However, LDA of narrower spectral portions within this range did not improve LDA results, indicating that LDA groupings of OMV spectra are less reliant on single peaks and regions than LDA groupings of PcO6 cells.

Comparing Raman spectra of intact PcO6 cells and isolated OMVs, it is clear that certain PcO6 cell components are enriched while others are excluded in OMVs, which agrees with studies of other Gram-negative bacteria8,77. The decreased peak intensity of H2O2-treated PcO6 cells is likely because unsaturated lipids react with H2O2 to form lipid peroxides and reactive aldehydes78. The fact that the same peaks are affected in CuO NP-stressed PcO6 cells likely indicates that ROS are formed in cells as a response to CuO NP stress, a cell response that contributes to the dose-dependent bactericidal activity of CuO NPs79. The relative greater content of unsaturated lipids in OMVs harvested from these stressed cells compared to OMVs produced by control cells could relate to changes in membrane integrity of cells under stress.

Also notable is the high amount of nucleic acids in OMVs compared to whole biofilm PcO6 cells, implicating the potential role of OMVs to transport DNA and/or RNA in PcO6 biofilms. RNA in OMVs has been reported in the forms of ribosomal RNA, mRNA, and small RNAs80. OMVs released by P. aeruginosa may contain plasmid DNA12, coding chromosomal DNA5, or noncoding eDNA37. These nucleic acids extend proposed OMV roles in cell-to-cell communication to both gene and transcription levels assuming OMV contents are taken up by live cells. OMVs of P. aeruginosa contain chromosomal DNA encoding genes related to bacterial survival under stress conditions5. The eDNA is an important part of the biofilm matrix45 and is involved in adhesion to surfaces, aggregation of bacterial cells, and the exchange of genetic information70. In H. pylori, eDNA was observed on OMV surfaces, with suggested roles in OMV aggregation and cell-to-cell binding6 including within the biofilm matrix72.

The biochemical assays confirmed the presence of protein, LPS, and nucleic acids in the OMVs from PcO6 that are indicated from the Raman spectral peaks. The inability of these assays to discriminate among OMVs according to the cellular stressor is unsurprising: OMVs are chemically heterogeneous in nature6,16,68 due to the multiple potential mechanisms and pathways that lead to OMV formation11,81,82. The presence of LPS, lipoproteins, and DNA in OMVs is significant because these are among the structures known as MAMPs and DAMPs that induce plant innate resistance35. Thus, the contents of the OMVs released from PcO6 cells may be implicated in the induction of systemic resistance observed in plants with roots colonized with PcO6.

The findings of OMV production from the plant-beneficial microbe, PcO6, and the changes in their composition with NP and ROS exposure revealed features that influence plant and bacterial responses in the rhizosphere. The nature of the charge on the OMV surfaces and the eDNA content might affect biofilm formation. Proteins such as catalase found in OMVs from other bacteria28 potentially protect the bacterial cell from oxidative damage. DNA and RNA release through OMVs could affect plant gene expression. These bacterial nucleic acids, along with other confirmed MAMPs such as LPS, carried by OMVs, could induce plant resilience to stress.

In summary, research into the heterogeneous nature and multiple roles of OMVs is supported by applying sensitive techniques such as Raman spectroscopy supported by appropriate analyzation algorithms such as LDA. In this study, these methods were able to chemically characterize purified OMVs and categorize these vesicles based on two cellular stressors relevant to the soil environment of the plant health-promoting bacterium, PcO6. The ability to differentiate H2O2-induced and CuO NP-induced OMVs reveals that these stressors do not merely induce OMV production. Rather, PcO6 responds to these stressors with changes in OMV contents and composition. The construction of an OMV Raman peak assignment library presented here provides a baseline for future analyses by our group and others.

Methods

Bacterial growth conditions

PcO6 stocks were kept at − 80 °C in 15% glycerol and thawed before use. PcO6 biofilms were grown at 22 °C on minimal medium (K2HPO4—10.5 g/L, KH2PO4—4.5 g, Na*citrate*2H2O—0.5 g/L, (NH4)2SO4—1 g/L, sucrose—2 g/L, anhydrous MgSO4—0.125 g/L) 2% agar plates (15 × 100 mm) for 48 h to a confluent lawn.

PcO6 biofilms were also grown on hollow fiber membranes for SEM imaging41. Hollow fiber membranes were inoculated with 2 μL PcO6 suspended in sterile double distilled H2O (ddH2O) (resistance > 18 MΩ cm) at a concentration of 106 colony forming units (CFUs)/mL, draped across wells with liquid minimal medium, and allowed to grow at 22 °C for 28 h.

Abiotic stressor preparations

Sterile ddH2O was used as the control treatment. Commercial CuO NPs (nominal size < 100 nm, 99.95% purity) were obtained from American Elements as a nanopowder and stored protected from light. NP size distribution and agglomeration profiles were confirmed with scanning electron microscopy (FEI Quanta FEG 650) (Supplemental Fig. S7). NP elemental composition was determined by scanning electron microscopy with energy-dispersive X-ray spectroscopy using an X-Max Detector (Oxford Instruments). NP stress used CuO NPs suspended in sterile ddH2O (30 mg Cu from CuO NPs/L) through sonication (Q500, QSonica LLC) for 10 min with an alternating 10 s on/off cycle at 25% amplitude. The ROS stress was 3% H2O2 (v/v).

Treatments of PcO6 biofilm cells before OMV isolation

The confluent lawns of PcO6 biofilm cells on minimal medium agar were treated by flooding with 10 mL of ddH2O, H2O2, or CuO NPs, added in 1 mL aliquots. These conditions were maintained for 60 min, during which time the cells detached from the surface and became suspended in the liquid. CFUs were counted with serial dilutions on LB 2% agar petri dishes to check the effect of stressors on cell viability. CuO NPs had no significant effect on the ability of PcO6 to generate colonies on plate medium whereas H2O2 exposure reduced colonies < 10%.

Isolating and purifying outer membrane vesicles

OMVs were isolated from PcO6 cells using a method adapted from Zhou et al.83. Twenty minimal medium plates with confluent PcO6 lawns (a total surface area of 0.628 m2) were used per treatment to maximize OMV yields for analysis. The cell suspension (10 mL/plate for a total of 200 mL/treatment) was poured from the petri dishes into 50 mL polypropylene conical tubes and centrifuged (10,000×g, 20 min) to generate a cell pellet leaving OMVs and other secreted materials in the supernatant. This step also removed CuO NPs from the solution when this stressor was used. The OMVs were concentrated from the supernatant by ammonium sulfate precipitation. Ammonium sulfate (Mallinckrodt chemicals) was added over the course of two hours (240 g/L total) at 15 min intervals to the supernatant, which was left undisturbed at 22 °C until precipitates formed (anywhere from 2–8 h). The precipitates were pelleted by centrifugation (10,000×g, 10 min), resuspended in 1 mL sterile deionized H2O, and dialyzed against ddH2O for at least 16 h. The solution was sterile filtered (0.45 μm, Ultrafree PVDF centrifugal filter units, Beckman Coulter Inc.) to remove any remaining cells or contaminants.

To purify OMVs from flagella, pili, and secondary metabolites (Supplemental Fig. S8), density gradient ultracentrifugation was performed using methods adapted from Chutkan et al.84. The crude OMV suspension was mixed with OptiPrep iodixanol gradient medium (Sigma Aldrich) to create a 45% OptiPrep solution (vol/vol). A 2 mL aliquot was loaded into the base of each ultracentrifuge tube (Ultraclear 12.5 mL centrifuge tube, Beckman Coulter) and covered sequentially with 2 mL layers of 40, 35, 30, 25, and 20% OptiPrep before centrifugation (212,000×g, 3 h, 4 °C) (Optima LE-80 K Centrifuge, Beckman). Aliquots of 1 mL were collected from the top of the gradient in a cold room to minimize diffusion. OMV-containing fractions, the top 1 mL of each tube, were pooled from each treatment, diluted at least 10 times with sterile deionized H2O, loaded into centrifuge tubes (26.3 mL Polycarbonate Bottle with Cap, Beckman Coulter), and centrifuged (40,000×g, 3 h) to pellet the OMVs. The OMV pellet was resuspended in 750 μL sterile ddH2O and sterile filtered (0.45 μm, Ultrafree PVDF centrifugal filter units). The presence of OMVs in the filtrate was confirmed with AFM imaging. Pure OMV preps were stored at 4 °C until used for Raman spectroscopy, which took place within 48 h of OMV isolation and purification. Samples were frozen (− 20 °C) until used for biochemical assays.

Atomic force microscopy (AFM)

AFM was performed on a Nanoscope III Bioscope (Digital Instruments, Inc.) in tapping mode. Budget Sensors-Tap 300AL-G cantilevers with a tip radius of curvature < 10 nm, length of 125 μm, width of 30 μm, thickness of 4 μm, and a 40 N/m force constant were employed. Images were collected at 256 × 256 resolution and 1 Hz over a range of scan sizes and scan angles. For intact biofilm PcO6 cells, a sample of the bacterial lawn on a sterile inoculation loop was smeared on a clean glass slide and immediately imaged. For OMVs, 20 μL purified OMV suspension was pipetted onto a clean glass slide, allowed to dry, and immediately imaged. OMV diameter was determined using the horizontal distance and the vertical distance in the line cut feature in the Nanoscope software (Supplemental Fig. S9). Four images were used for each treatment with a total of 100 OMVs measured per treatment.

Scanning electron microscopy (SEM)

SEM was performed with an FEI Quanta FEG 650 equipped with an Oxford X-Max EDS housed in the Microscopy Core Facility at Utah State University. Hollow fiber membranes with PcO6 biofilms were dipped in sterile distilled H2O, fixed in methanol for 10 min, and dried in two 10 min bath of 100% ethanol. Samples were critical point dried and sputter coated in an Au/Pd coat. Images were captured under high vacuum. Purified OMVs were pipetted onto a clean substrate, fixed with 2.5% glutaraldehyde, dehydrated using two 5 min washes of increasing ethanol concentrations (50, 70, & 95%) followed by three 15 min washes with 100% ethanol. Samples were imaged under high vacuum without coatings.

Dynamic light scattering

DLS measurements were performed on a DynaPro NanoStar (Wyatt Technology Corporation, Santa Barbara, CA) employing Dynamics Software (version 7.0.3, Wyatt Technology Corporation, Santa Barbara, CA) and a 658 nm laser. The purified OMV suspensions were diluted 1:10 in sterile deionized water, and 70 μL was transferred to DLS cuvettes. The intensity autocorrelation function (see Supplemental Fig. S10 for autocorrelation graphs) was used to calculate a hydrodynamic diameter based on the Stokes–Einstein equation using a regularization method employed in the software.

Raman spectroscopy and linear discriminant analysis (LDA)

To examine the intact cells, confluent PcO6 lawns from two minimal medium plates with or without the stress treatments were centrifuged from solution (14,000×g, 10 min), and resuspended in 1 mL sterile, deionized H2O. This process was repeated two additional times to remove stressors. After the third centrifugation, PcO6 cells were resuspended in 200 μL sterile, deionized H2O. A total of 6 PcO6 replicates were performed per treatment. The majority of OMVs, cell secretions, and other cell debris remained in the supernatant which was discarded after each centrifugation. A 10 μL aliquot of the PcO6 suspension was pipetted onto aluminum tape affixed to a clean glass microscope slide and allowed to dry. For OMVs, no additional preparation was needed for Raman spectroscopy after isolation and purification steps. A total of 3 isolations were performed with each treatment. Twenty μL purified OMV suspension was pipetted onto aluminum tape affixed to a clean glass microscope slide and allowed to dry.

Raman spectra were obtained using a Renishaw inVia Raman microscope with a 633 nm laser, 1200 g/mm grating, and 14 mW laser power. Spectra were obtained over a 30 s acquisition time with a wavenumber range of 200–3200 cm−1, with 3 accumulations per spectrum. Four spectra were captured per treatment during each replicate for a total of n = 24 for PcO6 spectra and n = 12 for OMV spectra. Spectrogryph85 was used to visualize spectra, obtain peak numbers, and average spectra. LDA of spectra was performed with R following background subtraction (WiRE 4.1), removal of cosmic rays (WiRE 4.1), and normalization of spectra (R). Data were truncated to 750–1700 cm−1 and 2670–3100 cm−1 for broad-spectrum analysis. PcO6 cell spectra were further truncated to 750–1700 cm−1, 2670–3100 cm−1, 700–950 cm−1, 950–1200 cm−1, 1200–1400 cm−1, 1400–1500 cm−1, or 1500–1700 cm−1 for additional examination with LDA. OMV spectra were further truncated to 750–1700 cm−1, 2670–3100 cm−1, 1000–1200 cm−1, 1200–1500 cm−1, 1500–1600 cm−1, or 1500–1600 cm−1 for additional examination with LDA.

Micro-bicinchoninic acid (micro-BCA) assay

OMV protein content was quantified with a Pierce micro-BCA assay kit (Thermo Scientific). Frozen OMV samples were thawed and prepared according to the manufacturer's instructions and absorbance was read at 562 nm (Synergy HT, BioTek Instruments Inc.) with appropriate bovine serum albumin standards from the manufacturer with concentrations of 0 to 200 μg protein/mL. Duplicates were run for each standard and sample.

Lipopolysaccharide (LPS) quantification

OMV LPS content was quantified with a Pierce Endotoxin Quantification Kit (Thermo Scientific). Frozen samples were thawed and prepared according to the manufacturer's instructions and absorbance was read at 405 nm (Synergy HT, BioTek Instruments Inc.) with LPS standards from Escherichia coli supplied by the manufacturer in the range of 0.1 to 1 Endotoxin Units (EU)/mL. Duplicates were run for each standard and sample.

260/280 absorption ratio

The nucleic acid to protein ratio was determined based on the 260/280 UV-absorption ratio. Frozen samples were thawed and 2 μL of each sample was pipetted into wells of a Take3 Micro-Volume plate (BioTek Instruments Inc.) and absorbance was read at 260 and 280 nm (Synergy HT, BioTek Instruments Inc.). Standards of bovine serum albumin (40 μg/mL) from a micro-BCA assay kit (Thermo Scientific) and double-stranded DNA (100 μg/mL) from a PicoGreen DNA quantification kit (Thermo Scientific) were also run. Duplicates were run for each sample and standard.