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emi12095-sup-0001-si.tif4404K

Fig. S1. The fibre-like Psl strands connecting multiple-cell aggregates/microcolonies at biofilm initiation. Shown are biofilms of PAO1 and WFPA801 after 22 h of growth under flow condition and stained with HHA-FITC (green, Psl matrix) and FM4-64 (red, bacteria membrane stain). Black arrows indicate the Psl fibres present in the area with a few bacteria. Scale bar, 5 μm.

emi12095-sup-0002-si.tif4364K

Fig. S2. The formation of the Psl-fibre matrix is not required for the production of Pel and alginate. Shown are biofilms of MJK8 (A), MJK8/Δpel (B), MJK8/Δpsl (C), PAO1 (D), WFPA1 (E) and WFPA802 (F) after 22 h of growth under flow condition and stained with HHA-FITC (green, Psl matrix) and FM4-64 (red, bacteria membrane stain). The lower right image in each biofilm panel is the merge of corresponding red and green images. Grey images are DIC image of biofilms. Bar, 2 μm for MJK8 and MJK8/Δpel, 10 μm for MJK8/Δpsl, 2 μm for WFPA1 and 5 μm for PAO1.

emi12095-sup-0003-si.tif982K

Fig. S3. The radial pattern Psl-fibre matrix was located at the stalk area of MSBS. Shown was a selected optical CLSM section of a ΔfliC biofilm. The blue line on the rectangle image shows the section located at the stalk of MSBS, which is depicted in the large square. Arrows indicate the MSBS (square) and the location of the radial Psl-fibre matrix within the MSBS (rectangle image). Red: Psl matrix; Green: Bacteria.

emi12095-sup-0004-si.tif5389K

Fig. S4. The Psl matrix in the pellicles of PAO1, ΔpilA, and ΔfliMΔpilA strains. Shown are selected CLSM images located in the middle of pellicles. The pellicles were stained by FITC-HHA for Psl matrix (green) after 44 h of growth under a static growth condition. Grey image was the corresponding DIC image of biofilm. Scale bar, 5 μm.

emi12095-sup-0005-si.tif662K

Fig. S5. Comparison of Psl released into PBS buffer and Jensen's media from mid-log phase bacteria of T4P mutants and relA spoT deletion mutant during 4 h of incubation at RT. Released Psl and bacterial surface-bound Psl were detected by immunoblotting with anti-Psl serum.

emi12095-sup-0006-si.tif1036K

Fig. S6. Concentrated Psl polysaccharide (green) and eDNA (red) were presented in the centre of a radial pattern Psl fibre matrix. Shown are selected CLSM images located in the middle of pellicles. The pellicle of ΔfliC strain was stained by FITC-HHA for Psl matrix (green) and PI for eDNA/dead cells after 44 h of growth under a static growth condition. Grey image was the corresponding DIC image of biofilm. Arrows indicate the colocalized Psl and eDNA in the centre of the radial pattern Psl fibre matrix. Scale bar, 5 μm.

emi12095-sup-0007-si.tif500K

Fig. S7. The twitching zone of PAO1 and pilA mutant on the Jensen's media and PBS agar plate. The hole in the middle is the inoculation hole. The red colour is the result of tetrazolium red stain. The diameter of twitching zone was measured by addition of distance a and b as depicted in the middle image. Scale bar, 7 mm. * indicates statistically significant difference between measurements (P < 0.007, t-test). TZ, Twitching Zone.

emi12095-sup-0008-si.avi9134K

Video S1. How the radial pattern Psl-fibre matrix maintain the biofilm structure of a ΔfliC strain. Shown was a serial optical CLSM section of a ΔfliC biofilm from substratum to the top of biofilm. Red: Psl matrix; green: bacteria in the biofilm.

emi12095-sup-0009-si.tif1012KSupporting Information

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