Fig 1.
Effects of tolC deletion on biofilm formation of A. pleuropneumoniae.
Biofilm formation was determined at various time points (0, 4, 6, 12, 24 and 36h) by crystal violet staining using static broth cultures of WT SC1516, ΔtolC and the genetically-complemented strain ΔtolC/tolC. Data are means ± standard deviations (SDs) of three independent experiments with three replicates. Asterisks indicate statistical significance (P< 0.05).
Fig 2.
Effects of TolC on initial surface adherence, autoaggregation and cell surface hydrophobicity.
(A) The surface attachment assay was determined in 96-well microtiter plates by staining with crystal violet. Attached cells in the well were quantitated by measuring the optical density at 595 nm. Data represents mean values for triplicate wells from three independent experiments. Error bars indicate SDs and the asterisks show significant differences (p< 0.05). (B) The ΔtolC cells remained mostly in suspension (middle), while the WT (left) and complemented strain (right) autoaggregated and settled to the bottom of the tubes after 6 h static incubation at room temperature. (C) The surface hydrophobicity of ΔtolC cells was significantly reduced when compared against WT. Values are expressed as means ± SE, * p < 0.05.
Fig 3.
Detachment of biofilm colonies of WT A. pleuropneumoniae and ΔtolC grown in 96-well microtiter plates.
Biofilms were cultured for 4h (A), 6h (B) and 12h (C) and treated with dispersin B, DNase I and proteinase K, respectively. The remaining biofilms were quantitated by crystal violet staining. Data represent mean values ± SDs of three independent experiments with three replicates. Results with significant differences when compared to the untreated controls or between two strains were marked with asterisks (P< 0.05).
Fig 4.
CLSM images of biofilms formed by WT and ΔtolC.
Both 4-h and 6-h static biofilms cultured in 6-well microtiter plates were stained with the SYTO-9 and propidium iodide (A-C) to label live versus dead cells, or with WGA-conjugated to Oregon green to label PGA (D).
Fig 5.
Relative expression of pgaA and cpxR in biofilms of ΔtolC versus WT.
The mRNA levels of pgaA and cpxR genes in biofilms at 4h (A), 6h (B) and 12h (C) were determined by qRT-PCR. Data presented are means of three independent experiments with triplicates. Error bars indicate SDs. Results with statistical significant are marked with asterisks (P< 0.05).
Fig 6.
Coculturing with WT rescued biofilm formation defects of ΔtolC.
Biofilm formation was performed by coculturing ΔtolC with WT in Transwell chambers. (A) Biofilms in the lower chamber were stained with crystal violet. (B) Quantitative analysis of biofilms. Data presented here are means of triplicate experiments and error bars indicate the SDs. *, statistical significance (P< 0.05).