Biofilm assays

Cells from a single colony isolated on LB plates were grown to mid-logarithmic phase in a 3-ml LB broth culture (4 hours at 37°C with shaking). Then, a drop was spotted on solid MSgg medium. For engulfment experiments, colonies were inoculated next to a disc placed at a distance of 0.3 or 0.5 cm. Plates were incubated at 30°C for the time period indicated in the legend for each figure. For floating biofilms (pellicles), cells from mid-logarithmic phase were diluted 1:100 into 3 ml of liquid MSgg and grown in six-well plates, at 30°C, without shaking, for 24 hours.

Images of brightfield and GFP signal intensity for colonies were obtained with a Stereo Discovery V20 microscope with Objective Plan Apo S 0.5× FWD 134 mm or Apo S 1.0× FWD 60 mm (Zeiss) attached to an Axiocam camera. For microscopy, cells were resuspended in phosphate-buffered saline (PBS) and washed twice, and images were taken with an Axioplan 2 microscope (Zeiss) equipped with a high-resolution microscopy Axiocam camera, at ×100 magnification. Data were captured and analyzed using ZENpro AxioVision suite software (Zeiss, Oberkochenm, Germany).