Small Metabolites Mediated Interspecific Competition

Equal amounts (10 μl of bacterial suspension with OD₆₀₀ of 1.0) of P. aeruginosa and K. pneumoniae were cocultured in disposable sterile 12-well polystyrene plate equipped with an inner well-containing a 0.4 μm permeable polycarbonate membrane (Corning, NY, United States) in the bottom of the inner well (Supplementary Figure 1). The two species were separated by the wall of the inner well, but could exchange the small extracellular metabolites through the membrane layer. The outer space of the well was supplied with 3.0 ml of LB broth and the inner space was supplied with 0.75 ml of LB broth. Coculture of 1:1 well-mixed P. aeruginosa and K. pneumoniae and pure cultures of the two species in normal 12-well polystyrene plate without membrane containing 4.0 ml of LB broth were set as control. All the capped plates were sealed by sealing films and statically cultured at 37°C for 3 days.