K2hpo4

Surface hydrophobicity of GAS cells measured by the ability to adhere to n-hexadecane was performed based on a previously described protocol (Ofek et al., 1983 (link); Rosenberg et al., 1980 ). Overnight cultures of M1 5448 wt pDCerm, Δess pDCerm, and Δess pDCerm::ess were harvested by centrifugation for 10 minutes at 10,000 ^×g at room temperature, and pelleted cells were washed twice and suspended in PUM buffer (22.2 g K₂HPO₄ ⋅ 3H₂0, 7.26 g KH₂PO₄, 1.8 g urea [Promega Corporation], 0.2 g of MgSO₄ ⋅ 7H₂0 [Sigma Aldrich], per 1 L of ddH₂O). Next, 2.4 mL of the bacterial suspension was transferred into 13 ^× 100 mm borosilicate glass disposable culture tubes (Fisher Scientific) and 0.4 mL of n-hexadecane (Fisher Scientific) was added. Bacteria without addition of n-hexadecane were used as controls for spontaneous cell lysis. The OD₆₀₀ was measured from the side of the tube using a SPECTRONIC 200 Spectrophotometer. Tubes were next vortexed for 3 minutes, allowed to settle for 15 minutes, and the OD₆₀₀ of the bottom fraction was measured. Hydrophobic properties of bacterial cells are represented by the percentage of bacteria bound to the n-hexadecane, calculated using the following formula:((T0 OD600 − T15 OD600) /T0 OD600)×100, where T0 OD600 = OD₆₀₀ value before vortexing and T15 OD600 = OD₆₀₀ value after vortexing. Experiments were performed in three biological replicates.