Urea

Precipitated chromatin proteins were dissolved in 60 µL of 100 mM Tris-HCl, pH 8.5 containing 8M urea (Sigma). The protein was reduced by incubation in 5 mM tris(2- carboxyethyl)phosphine (TCEP) for 20 minutes at room temperature followed with carboxyamidomethylation of cysteines by incubation at room temperature for 30 minutes in the dark in 10 mM iodoacetamide. The sample was diluted 2-fold (to 4M of the concentration of urea) by the addition of an equal volume of 100 mM Tris-HCl, pH 8.5 and then Lys-C (Promega) was added at ∼1∶100 enzyme to substrate ratio (wt∶wt) and incubated at 37°C for 4 hours in the dark. The sample was then further diluted to the concentration of 2M urea and trypsin (Promega) was added at ∼1∶100 enzyme to substrate ratio (wt∶wt) and incubated at 37°C overnight in the dark. The resulting peptides from the digests were dissolved using 90% formic acid to a final concentration of 2% formic acid. The sample was stored at −20°C prior to LC/LC-MS/MS analysis.

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.

Cell pellets were lysed in 8 M urea (Bio-Rad Laboratories Inc., CA, United States, 161-0731), 100 mM Tris-HCl, pH 8.5 (Sigma-Aldrich, St. Louis, MO, United States, 10812846001) by sonication in 1.5 ml Micro Tubes (TPX Plastic for Sonication from Diagende Inc.) using a Bioruptor® sonication system (Diagenode Inc., New Jersey, United States, B01020001) with 30 s/30 s on/off cycles for 15 min in a water bath at 4°C. After subsequent centrifugation at 14,000 rcf for 20 min, protein concentrations were determined by Bradford protein assay (BioRad, Hercules, 5000006). A 20 µg equivalent of protein from each sample was reduced with 5 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP, Sigma-Aldrich, C4706) for 30 min at room temperature, and the resulting free cysteine thiols were alkylated with 10 mM chloroacetamide (CAA, Sigma-Aldrich, C0267) for 30 min at room temperature in the dark. Samples were diluted with 50 mM Tris HCl, pH 8.5, to a final urea concentration of 2 M for Trypsin/Lys-C based overnight protein digestion at 37°C (1:100 protease: substrate ratio, mass spectrometry grade, Promega Corporation, Madison, WI, V5072).