Figure 1.
Lectin staining results of surface attached bacteria; Psl is anchored on the bacteria cell surface in a helical pattern.
(A) MOA-FITC (green) and membrane stain FM4-64 (red) double staining of WFPA801 cells. The green image depicts the staining of helical structures around the cell surface as indicated in the inset. The white arrow points out the division site. (B) An optical section of HHA-FITC stained WFPA801 cells without (1) or with (1') deconvolution. (C) HHA-FITC and MOA-TRITC (white) double staining of WFPA801 cells. (D) HHA-FITC stained PAO1 cells. In all panels, green and red signals are merged images of the FITC-lectins and FM4-64 stained cells. The gray signal represents DIC images while green with gray are merged images of the FITC-lectins with DIC images. Scale bars, 1 µm.
Figure 2.
The effect of cellulase treatment on Psl localization and biofilm formation.
(A) HHA-FITC (green) staining of surface-attached WFPA801 cells with or without cellulase treatment. (B) The biofilms of GFP-tagged PAO1 grown in flow cells in the presence or absence of cellulase. The total biomass was quantified by COMSTAT software. Values shown on the upper left corner of the corresponding image have been normalized to the result of the non-cellulase treatment sample (3.1 µm3/µm2). The arrows reveal Psl that has dissociated from the bacterial surface and adhered with the substratum. Scale bars, 1 µm for panel A and 5 µm for panel B.
Figure 3.
Psl at an early stage of biofilm development: a matrix formed by Psl holding bacterial cells in the biofilm and on the surface.
(A–D) Staining of Psl matrix in biofilms formed by strains PAO1, WFPA801, and WFPA800: images were acquired after 20 hours in Jensen's media under continuous flow conditions with 2% arabinose. Biofilms in reactors were stained for 2 hours with lectins as follows: HHA-FITC and FM4-64 stained WFPA801 biofilm. (E–H) HHA-FITC and FM4-64 stained PAO1 biofilm. (I–L) HHA-FITC and FM4-64 stained WFPA800 biofilm. (M–P) MOA-TRITC staining of GFP-tagged WFPA801 biofilm. (Q–T) MOA-TRITC staining of GFP-tagged PAO1 biofilm, grown under flow conditions in Jensen's media with cellulase. Scale bars, 5 µm.
Figure 4.
How the Psl matrix maintains the biofilm architecture.
Shown are sets of optical sectioned images acquired at different locations of the biofilm (position indicated on the top of each panel). DIC images are in gray. Green and red merged images are shown at the lower right corner for panels A and B. Bar, 5 µm for panels A and B, 10 µm for panels C and D. (A) Psl matrix (green, MOA-FITC staining, bottom left) in a multilayer biofilm (4 µm thickness) of WFPA801 (red, FM4-64 staining, upper left). (B) Psl matrix (green, MOA-FITC staining, bottom left) in a WFPA801 biofilm microcolony (FM4-64 staining, 24 µm thickness, upper left). The top-down view (square) and side view (rectangle) of 3D reconstituted images are shown, which reveals how the peripherally localized Psl matrix encases the bacteria in a mushroom-like microcolony. (C) The newly synthesized Psl matrix (red) covers the existing Psl matrix (green). The Psl matrix of WFPA801 biofilms was stained with MOA-FITC (green) at 40-h-growth and stained again by MOA-TRITC (red) at 60-h-growth. The large square image is a horizontal section at the top of microcolony. The blue line in the side view images marks the location of the section (rectangle). Green and gray merged images are at the lower right. Green and red merged images are at the right panel and the bottom middle of the middle panel.
Figure 5.
The Psl matrix of microcolonies before and after dispersion.
Panels A–C show a 2-day-old PAO1 biofilm stained by HHA-FITC (green) and FM4-64 (red). Bar, 10 µm. (A) The Psl matrix of a microcolony undergoing dispersion (28 µm thickness). On the left is a horizontal sectioned image of the matrix (square) close to the surface and two vertical sectioned images of the matrix (rectangle). The small squares on the right show horizontal sectioned images from the middle of the same microcolony. The white arrows point out the areas with swimming dispersing cells and the two black arrows indicate the immobile bacterial wall. The merged image reveals how the Psl matrix enmeshed the bacteria in the immobile wall and covers dispersing cells in the matrix cavity. (B) The Psl matrix of a microcolony after seeding dispersal. A horizontal sectioned image (square) of the microcolony near the top and two vertical sectioned images (rectangle) are shown. (C) A Psl matrix cavity in a microcolony (33 µm thickness) with no visible swimming cells. Shown on the right is a horizontal sectioned image of the matrix close to the surface (square) and two vertical sectioned images of the matrix (rectangle). The large white arrow points to the Psl staining in the center of a Psl matrix cavity. Two sets of images sectioned at the top of the microcolony (middle panel) or close to the surface (left panel) are shown.
Figure 6.
Cell death and lysis contributes to the Psl matrix cavity formation.
In panel A, Psl matrix was stained in green, whereas in panel B, the green fluorescent signal represents viable cells stained by SYTO9. In all panels, red fluorescence is due to propidium iodide (PI) staining of either eDNA (weak and diffuse red) or cells with a compromised cell membrane (dead cells, bright concentrated red). Bar, 5 µm for WFPA801 in panel A and 10 µm for the other images. (A) Images of 2-day-old WFPA801 and rpoN mutant biofilms stained by HHA-FITC (green) and PI (red). A top-down view of 3D reconstructed images is shown in left panel. Sets of images optically sectioned (horizontally) at the neck of the same microcolony are shown in the right panel. The corresponding merge image of Psl matrix and DNA matrix is shown in the middle panel (large square). Two corresponding vertical section images are also shown (rectangle). (B) LIVE/DEAD viability staining of PAO1 and WFPA801 biofilm microcolonies show that there are viable swimming cells in the lower center of the microcolony and dead cells/extracellular DNA that fill up all void spaces. Three microcolonies prior to dispersion and one microcolony (the upper right) post the seeding dispersal are shown. The arrow points out the swimming cells in the center of the microcolony.
Figure 7.
The P. aeruginosa cidAB and lrgAB genes control cell death and lysis, as well as the timing of seeding dispersal.
(A) A sequence alignment between P. aeruginosa CidA (putative holin) and LrgA (putative anti-holin), the predicted CidA protein structure, and a diagram of P. aeruginosa cid/lrg genetic organization. Identical residues between the two sequences and residues absent in CidA are shaded. The black rectangle represents a sequence predicted to form a trans-membrane helix. _, residues predicted to be cytoplasmic; -, residues predicted to be periplasmic. (B) A growth comparison of P. aeruginosa PAO1, ΔcidAB, and ΔlrgAB (left graph) and the corresponding complemented strains (right graph). (C) The Psl matrix (green, HHA-FITC staining) cavity in the 2-day-old biofilm of ΔlrgAB, PAO1, and the ΔcidAB mutant. A horizontal sectioned image (square) and a vertical sectioned image (rectangle) are shown. The white bar marked by letter “a” represents the height of the Psl matrix cavity and the “b” bar shows the height of the corresponding microcolony (MC).
Figure 8.
How the P. aeruginosa Psl biofilm matrix forms and develops.
(A) A schematic showing five stages of biofilm development. Created by P. Dirckx, K. Sauer, and D. Davies and used with permission of the authors [4] and the Annual Review of Microbiology, Volume 56 ©2002 by Annual Reviews (www.annualreviews.org). (B) Selected images of Psl staining (red) during each development stage of biofilm formation. Green fluorescence signal is derived from GFP-labeled P. aeruginosa. The circle in the image V-I depicts the Psl matrix cavity. (C) A proposed mechanism showing how a P. aeruginosa microcolony sacrifices a portion of cells in the center and disrupts the existing matrix to free cells for dispersion. The pink material represents the Psl matrix, the light green cells represent live bacteria, and the dark green cells represent dead bacteria undergoing autolysis.