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. 2014 Jul;93(1):183-98.
doi: 10.1111/mmi.12650. Epub 2014 Jun 4.

Extracellular Vesicles Produced by the Gram-positive Bacterium Bacillus Subtilis Are Disrupted by the Lipopeptide Surfactin

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Extracellular Vesicles Produced by the Gram-positive Bacterium Bacillus Subtilis Are Disrupted by the Lipopeptide Surfactin

Lisa Brown et al. Mol Microbiol. .
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Abstract

Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harbouring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin.

Figures

Figure 1
Figure 1. Bacillus subtilis produces extracellular vesicles
Vesicles were isolated and pelleted by ultracentrifugation from strains (A) 168, (B) 3610, and (C) heat-killed 168. Vesicles from strain 168 were visualized by (D) negative staining TEM and (E) embedded TEM. Scale bars 100 nm.
Figure 2
Figure 2. Vesicles are produced directly by the cell not by aggregation of lipids
Strain 168 cells were grown in the presence of GXM and vesicles were isolated and left natural or sonicated. (A) Schema of experimental design. Vesicles were predicted to either (i) be gold-free if they were formed by cellular processes at the cell membrane or (ii) encapsulate gold particles if they formed in the presence of GXM (e.g. spontaneous formation in supernatant containing GXM). Immunogold was used to delineate the presence of GXM in individual vesicles at ultrastructural scales. In the schema, black dots represent gold particles and red lines, GXM. (B) Representative micrograph showing immunogold staining of GXM in a vesicle preparation. Note that all gold particles occur outside of vesicles. (C) Percentage of natural and sonication-derived vesicles in the presence of GXM that contained gold particles. Sonicated vesicles contained significantly more gold than natural vesicles (4.8 and 1.8%, n=564 and n=712, p<0.01 Chi2) and demonstrated a significantly smaller (D) diameter and standard deviation (94.54 ± 1.869, n=392 and 115.4 ± 2.69, n=355, p<0.0001 unpaired t-test, n=3) than natural vesicles. Black arrows indicate vesicles and white arrow indicates GXM. Scale bars 100 nm.
Figure 3
Figure 3. B. subtilis vesicles are heterogeneous in diameter and density
(A) Vesicle diameters measured from TEM images had a mean of 174.4 nm after applying 1.27 correction factor. (B) Dynamic light scattering indicated vesicle diameters having two peaks approximately 50 and 150-200 nm. (C) Vesicle diameters and standard deviations were measured and mean densities scored of (D) least electron density (+) (214.5 ± 73.28 nm, n=85), (E) medium electron density (++) (152.2 nm ± 53.95, n=137), and (F) most electron dense (+++) (120.4 ± 51.14 nm, n=105) from strain 168 (p<0.0001, one-way ANOVA). Scale bars 100 nm.
Figure 4
Figure 4. Structures suggestive of extracellular vesicles forming on B. subtilis cells
Strain 168 cells were visualized by (A-B) embedded TEM, (C) negative staining EM, and (D) SEM. Arrows indicate protrusions suggestive of emerging vesicles; scale bars 100 nm.
Figure 5
Figure 5. Vesicle production increases and vesicle stability decreases over time
Strain 168 cells were labeled with 14C PA and vesicles were isolated. (A) Vesicle pellet radioactivity as fraction of total sample isolated at 0, 4, 12, 18, and 24 h and (B) vesicle pellet stability in PBS compared to supernatant recovered at 0, 24, 48, and 72 h (n=4). DLS of vesicles incubated in PBS at (C-F) 0, 24, 48, and 72 h, respectively.
Figure 6
Figure 6. Vesicles are enriched in specific proteins and have a negative zeta-potential
Proteomic analysis of strain 168 (A) vesicles, cell membrane, and whole cells. (B) Vesicle preparations from strains 168, 3610, and RL2663 exhibit various banding patterns on silver stained SDS-PAGE gel. (C) The zeta-potential and standard deviation of vesicles in 10 mM dextrose and 1 mM KCl is −54.2917 +/− 9.24 and −36.724 +/− 10.2, respectively.
Figure 7
Figure 7. Vesicles are found in B. subtilis biofilms
Vesicles isolated from disrupted strains 168 (i-ii) and 3610 (iii-iv) biofilms at 4 and 8 days, respectively. (A) Negatively stained electron micrographs and (B) DLS size distribution of biofilm vesicles. (C) Embedded TEM and (D) SEM micrographs of strain 3610 whole biofilms show the presence of vesicles. Black arrow indicates vesicles, white arrow indicates vesicle protrusions. Scale bars 70 nm.
Figure 8
Figure 8. Nonfunctional sfp gene results in large quantities of recovered vesicles
Cells from strains (A) 168 and (B) 3610 were labeled with 14C PA and the radioactivity of each fraction measured (cells, FT, supernatant, and vesicle pellet). (C) Strain 168 vesicle pellet contained significantly more radioactivity than that of strain 3610 (p<0.01 t-test, n=3). Vesicle pellets of B. subtilis strains 168 and AD3610 harboring (D) nonfunctional sfp, contain significantly higher radioactivity in the vesicle pellet than that of functional sfp strains 3610, RL3090, and SL3610 (p<0.01 one-way ANOVA, n=3). (E) Density gradient of 14C PA labeled vesicles indicating the location and density of vesicles from each strain. (F) Vesicle pellets from strains 168 and RL2663 contain significantly more radioactivity than that of the surfactin-producing strain 3610 (p<0.01 one-way ANOVA, n=3).
Figure 9
Figure 9. Surfactin production disrupts extracellular vesicles
Cells from strain 168 and Sterne 34F2 were labeled with 14C PA and the radioactivity of vesicle pellets compared to radioactivity of the supernatant fractions was measured after 24 h incubation to determine disruption. (A) B. subtilis vesicle pellets incubated in media or strain 168 cell-free supernatant were stable and radioactivity remained high compared to radioactivity in the supernatant, whereas the radioactivity of vesicle pellets incubated in strain 3610 cell-free supernatant was almost completely lost (p<0.05 one-way ANOVA, n=3). (B) B. subtilis vesicle pellets incubated in PBS and 10% EtOH remained stable, whereas the radioactivity of vesicle pellets compared to supernatants diminished in 10 μg ml-1 and 100 μg ml-1 of surfactin, indicating vesicle disruption (p<0.05, p<0.01, p<0.0001 one-way ANOVA, n=3). (C) B. anthracis vesicle pellets from B. anthracis were more stable in media, Sterne 34F2 strain supernatant, and strain 168 supernatant compared to pellet radioactivity when incubated in strain 3610 supernatant (p<0.05, p<0.001, p<0.0001 one-way ANOVA, n=3). B. anthracis vesicles are incredibly stable in strain 168 cell-free supernatant. (D) B. anthracis vesicle pellet radioactivity was greatly reduced when incubated in pure surfactin and was more stable in PBS and 10% EtOH (p<0.01 one-way ANOVA, n=3).

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