Culturing and Antibiotic Selection of Streptococcus

S. pyogenes and S. mutans were grown in Todd-Hewitt broth (BD) supplemented with 0.2% yeast extract (Amresco) or a chemically-defined medium (CDM; [5 (link),16 (link)]) as indicated; broth cultures were grown at 37x° C without shaking, and agar plates were cultured at 37° C with 5% CO₂. All cloning was done in E. coli strain BH10c [17 (link)] which was routinely cultured in Luria-Bertani broth (BD) at 30° C with agitation. All strains were stored at -80° C in 20% glycerol. Antibiotics were added at the following concentrations when appropriate–S. pyogenes: chloramphenicol (Cm), 3 μg mL^-1; erythromycin (Erm), 0.5 μg mL^-1; kanamycin (Km), 200 μg mL^-1; spectinomycin (Spec), 200 μg mL^-1; S. mutans: Cm 7.5 μg mL^-1; Erm, 1.5 μg mL^-1; Km, 750 μg mL^-1; Spec, 500 μg mL^-1; and E. coli: Erm, 500μg mL^-1; Spec, 150μg mL^-1.