Figure 4

CRISPRi-mediated phenotypes associated with biofilm structure and EPS matrix. P. fluorescens SBW25 cells expressing the CRISPRi system targeting genes gacS (PFLU3777, two-component sensor kinase), rimA (PFLU0263, PDE), dipA (PFLU0458, PDE), alg44 (PFLU0988, alginase co-polymerase Alg44), gcbA (PFL0621, DGC) and bifA (PFLU4858, PDE). The control corresponds to the pPFL-gRNA plasmid with no guide RNA inserted. (A) Cells were stained with FM 1–43 and observed by confocal microscopy. 3D architecture of biofilms were reconstructed as described in Methods. Virtual shadow projections were included on the right to show thickness. (B) Biofilms grown in the presence of the Congo Red dye to reveal the structure of EPS. The ESP-stained volumes were rendered with maximum intensity projection. Inset shows a blow-up view of a clump in the EPS matrix after silencing of bifA.