Figure 1
C16-HSL are associated with MVs in P. denitrificans Pd1222. (a) TEM image of MVs isolated from Pd1222. (b) C. violaceum VIR24 assay for the detection of C16-HSL. MVs isolated from Pd1222 wild-type or a pdnI mutant were analyzed. The purple pigment is indicative of the presence of C16-HSL. (c) C16-HSL inhibits Pd1222 aggregation. Arrows indicate cell aggregates of the pdnI mutant attached to the tube surface. A total of 5 μM C16-HSL or an equivalent amount of C16-HSL associated with MVs was added. The same amount of MVs derived from the wild-type or the pdnI mutant was added. (d) MV addition to a Pd1222 AHL reporter strain. A total of 5 μM C16-HSL or an equivalent amount of C16-HSL associated with MVs was added to a culture of P. denitrificans Pd1222ΔpdnI/pPLlas. Gfp expression in this strain is dependent on C16-HSL. n=3; mean±s.d. Unpaired t-test with Welch’s correction. ns, not significant; ***P<0.001. (e) Quantification of C16-HSL and their localization. C16-HSL was quantified from each fractions with or without ethyl acetate extractions before measurement with UHPLC-qToF-MS. n=3; mean±s.e. (f) MV induction by MMC. MMC was added at an OD600 of 0.5, following incubation for 5 h. MV production was measured by staining with the membrane-specific dye FM4–64. n=3; mean±s.d. Significant differences with the control were determined by two-way ANOVA with Dunnett's multiple comparisons post test. ns, not significant; ****P<0.0001. (g) C16-HSL concentration in the supernatant. MVs were induced by adding MMC as mentioned before. AHL concentration in the supernatant was measured using C. violaceum VIR24/pPROBE-vioA. Relative values are shown. n=3; mean±s.d. Significant differences were determined by two-way ANOVA with Dunnett's multiple comparisons post test. ns, not significant; ****P<0.0001.
