Ti2 e

Overnight cultures of the indicated strains with WT or mutated rbmC or bap1 tagged with 3×FLAG at the C-terminus and constitutively expressing mNeonGreen were grown following the same procedure as described above. The initial incubation time was 10 or 30 min when biofilms were grown in the presence of asialofetuin or BSA, respectively. The wells were washed twice with M9 medium; subsequently, 100 µL of M9 medium with 0.5% glucose and 1 mg/ml asialofetuin (Sigma-Aldrich A4781) or 0.5 mg/ml BSA (Sigma-Aldrich A9647) was added to the well. BSA and asialofetuin spontaneously coat the surface under these conditions. Both conditions included 2 µg/mL anti-FLAG antibody conjugated to Cy3 (Sigma-Aldrich A9594). The lid was secured with a layer of parafilm and incubated at 30 °C for 16–24 h for asialofetuin and 40–48 h for BSA samples. Thus-prepared samples were imaged with a spinning disk confocal microscope (Nikon Ti2-E connected to Yokogawa W1) using a 100× oil immersion objective (numerical aperture = 1.35) or a 60× water immersion objective (numerical aperture = 1.20) and a 488 nm laser excitation to observe the cells and a 561 nm laser excitation to observe protein localization, with the corresponding filters. The images were captured with a sCMOS camera (Photometrics Prime BSI) at a z-step size of 0.5 µm.

Chemically synthesized peptides (Atlantic Peptides) were dissolved and stored in DMSO at 150 µM and diluted 100× into M9 media immediately before the adsorption assay. 100 µL of M9 media containing 1.5 µM of FITC-labeled peptide or FITC and 0.01% (weight percent) 5 µm silica microspheres (Polysciences 25348) was shaken in Eppendorf tubes for 30 min at room temperature. The sample was then bath sonicated for 20 min with ice before transferring to a NaOH-treated 96-well plate with a glass bottom (MatTek P96G-1.5-5-F) and allowed to settle at room temperature for 5 min before imaging. Thus-prepared samples were imaged with a spinning disk confocal microscope (Nikon Ti2-E connected to Yokogawa W1) using a 60× oil objective (numerical aperture = 1.40) and a 488 nm laser excitation or bright field. For each sample, at least three locations were imaged and captured with a sCMOS camera (Photometrics Prime BSI). Each field of view contained roughly 100–150 beads.