Growth and Maintenance of S. pyogenes

S. pyogenes was routinely grown in Todd-Hewitt (TH; BD Biosciences) supplemented with 0.2% yeast extract (Y; Amresco) or in an improved chemically-defined medium (CDM) based on a previously described recipe [21] (link), [51] (link). Briefly, we found that the preparation of this medium could be streamlined by combining components into stock solutions that could be stored at −20°C or room temperature (as described in Protocol S1 in Supporting Information). Furthermore, we found that L-asparagine, absent from the original recipe, added to a final concentration of 100 mg L⁻¹ supported robust growth of the bacteria. Cultures were stored at −80°C in THY supplemented with 20% glycerol. When appropriate, antibiotics were used at the following concentrations: chloramphenicol (cm, 3 µg mL⁻¹), erythromycin (erm, 0.5 µg mL⁻¹), spectinomycin (spec, 100 µg mL⁻¹). E. coli cloning strains DH10β (Invitrogen), BH10c [86] (link), and XL-10 Gold (Stratagene) were maintained in Luria broth (LB) or on Luria agar with the following antibiotics used at the indicated concentrations: cm (10 µg mL⁻¹), erm (500 µg mL⁻¹), spec (100 µg mL⁻¹). E. coli expression strain C41(DE3) [87] (link) was maintained on ampicillin (amp) at 100 µg mL⁻¹.