Campygen compact system

Bacterial strains used are shown in Table S1 and were cultured on solid media as described before^{39 (link)}. For the evolution study, liquid cultures of each bacterial strain were grown until stationary phase in BHI broth (37 °C, 250 rpm; Table S1). S. anginosus and G. haemolysans cultures were incubated in microaerophilic conditions (CampyGen Compact system, Thermo Fisher Scientific, USA).
For phenotypic testing and sequencing of evolved populations originating from the biofilm evolution experiment, an aliquot of the frozen biofilm (stored as glycerol stock at −80 °C) was plated on LB agar and incubated overnight at 37 °C. The entire growth on the agar plate was then divided into two Microbank cryovials with beads (Pro-Lab Diagnostics) and frozen at −80 °C. A single bead was then used to inoculate 5 ml LB broth (incubated at 37 °C, 250 rpm, 16 hours) to serve as inoculum for phenotypic tests and DNA extraction. These cultures are designated “evolved populations” throughout the text for the sake of clarity.

Bacterial liquid cultures were grown to stationary phase as described above, and diluted to 5 ​× ​10⁷ ​CFU/mL (based on OD_590nm) in BHI ​+ ​LYS medium. Diluted cultures were transferred to 96-well plates (100 μL/well) and incubated statically for 48 ​h ​at 37 ​°C in an EnVision Multilabel Plate Reader (PerkinElmer, USA), and OD_590nm was measured every 30 ​min. To generate microaerophilic conditions, the outer wells of the plate were filled with ascorbic acid from CampyGen Compact System (Thermofisher Scientific, USA), and the plate was sealed with silicone.