Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production

Abstract

Members of the genus Burkholderia are known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterized Burkholderia thailandensis biofilm development under flow conditions and sought to determine whether QS contributes to this process. B. thailandensis biofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by "dome" structures filled with biofilm matrix material. We showed that this process was dependent on QS. B. thailandensis has three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the three B. thailandensis QS systems, we show that QS-1 is required for proper biofilm development, since a btaR1 mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. The btaR1 mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions.

Importance: The saprophyte Burkholderia thailandensis is a close relative of the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms, B. thailandensis is an ideal model organism for investigating questions in Burkholderia physiology. In this study, we characterized B. thailandensis biofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows that B. thailandensis produces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience of B. thailandensis biofilms against changes in the nutritional environment.

FIG 1

B. thailandensis forms biofilms with dome structures. (A) Biofilms of YFP-expressing wild-type B. thailandensis were grown in a flow cell system and were imaged at the times indicated. Representative images are shown with the cross-sectional view on top and a side view on the bottom. Bar, 50 μm. (B) Close-up of the dome structure in a 96-h wild-type biofilm. On the far left is a side view. Cross-sectional views are shown on the right. Insets for two cross-sections are shown below with arrowheads marking the cells inside the domes. Bar, 50 μm.

FIG 2

QS-1 is required for proper biofilm formation. (A and B) Representative images of 96-h YFP-expressing B. thailandensis biofilms. WT, Wild type; + AHLs, exogenous addition of AHLs. The genotypes of isogenic mutants are indicated. The btaI1 btaI2 btaI3 mutant (btaI1-3) cannot produce AHLs; the btaR1, btaR2, and btaR3 mutants lack QS-1, QS-2, and QS-3, respectively. (C) Time course of biofilm development for YFP-expressing WT and btaR1 mutant strains. For the images of the 72-h and 96-h btaR1 biofilms and of the 96-h btaI1 btaI2 btaI3 biofilms but not for earlier time points, the contrast has been enhanced to increase the visibility of the biomass. Bars, 50 μm.

FIG 3

Dome formation is impaired but not absent in the ΔCPSIV mutant strain. Representative images of Syto9-stained 96-h biofilms of the wild type (WT) and of strains that cannot produce specific capsular polysaccharides (ΔCPSI, ΔCPSII, ΔCPSIII, and ΔCPSIV) are shown. Bar, 50 μm.

FIG 4

Dome structures of wild-type biofilms do not contain DNA, proteins, or lipids. Shown are representative images of 96-h YFP-expressing wild-type biofilms stained with various biomarkers: Syto62, for the visualization of DNA (A), NanoOrange, for the visualization of proteins (B), and FM4-64, for the visualization of lipids (C). Within each panel, a merged image of the biomass (yellow) and the biomarker (magenta) is shown on the left, and the biomarker alone is shown on the right. Bar, 50 μm.

FIG 5

QS-1-deficient biofilms are not stained with a fucose-binding lectin. Shown are representative images of 96-h YFP-expressing biofilms stained with the following lectins to visualize matrix polysaccharides: HHA, which stains for mannose (A); PNA, which stains for galactose (B); RCA, which stains for galactose and N-acetylgalactosamine (C); and UEA, which stains for fucose (D). Within each panel, the wild-type (WT) biofilm is shown at the top and the btaR1 mutant biofilm at the bottom. A merged image of the biomass (yellow) and the lectin (magenta) is shown on the left, and an image of the lectin alone is shown on the right. In the images of btaR1 biofilms, the contrast has been enhanced for the biomass but not the lectin to increase visibility. Bar, 50 μm.