Fig. S4.

Spatial distributions of matrix components. (A) Biofilm cluster was simultaneously stained with Cy3-conjugated anti-FLAG antibodies and Texas red-conjugated wheat germ agglutinin (WGA) to illustrate the spatial distribution of RbmC and Bap1 (yellow, Top) and compare them with the spatial distribution of Vps (red, Middle). The overlaid images (Bottom). The distributions were followed at the surface (Left) and away from the surface (Right). Away from the surface, both Bap1 and RbmC colocalize to the same extent with Vps, forming an envelope around the biofilm cluster. At the surface, however, the Bap1 signal is more pronounced under the early biofilm-forming cells, whereas the RbmC signal continues to colocalize with Vps. Hence, we suggest that RbmC and Bap1 can each achieve cell-to-surface adhesion independently, but via different mechanisms. Whereas Bap1 directly acts as an adhesive between the cells and the surface, RbmC helps Vps to form an envelope that clamps the entire cluster to the substrate. The partially redundant functions of RbmC and Bap1 are consistent with the high similarity between their sequences (26). The direct adhesion mechanism makes Bap1 more effective than RbmC in mediating cell-to-surface adhesion, as shown by the attachment assay in Fig. 2H. (Scale bar: 10 μm.) (B) RbmA links cells together both in the parent Rg strain and in the Δbap1ΔrbmC mutant. (Left) Strains constitutively expressing mTFP1 from an ectopic chromosomal location. (Center) Distribution of RbmA-3× FLAG stained with Cy3-conjugated anti-FLAG antibodies. (Right) Distribution of Vps stained with Texas red-conjugated WGA. In the biofilm clusters of the Rg parent strain, RbmA permeates throughout the entire cluster and can be imaged on the surfaces of all cells, whereas Vps primarily forms envelopes around cell clusters. In the Δbap1ΔrbmC double mutant, the RbmA signal is still present, but the Vps signal is below the detection limit. This observation suggests that one important function of RbmC and Bap1 is to prevent Vps from leaking away into the medium. (Scale bars: 10 μm.)