Streptococcus pyogenes strain D471 was propagated on solid media in plates containing Todd-Hewitt broth supplemented with yeast extract (0.2%) (THY) and agar (1.4%) or in liquid THY as described by Gera et al [27 ]. S. mutans wild type (Xc) and mutants were grown in Todd-Hewitt broth with 1% yeast extract. All cultures were grown without antibiotics and without aeration at 37°C. E. coli genotypes DH5α (NEB, cat. No. C2988J), DH10α (Thermo Fisher, cat No. 18297010), BL21 (NEB, cat No. C2530H) and Origami 2 (Millipore Sigma, cat. No. 71346) were used for routine plasmid propagation or protein expression and grown in Lysogeny Broth (LB) medium supplemented with either 50 μg/ml kanamycin, 100 μg/ml ampicillin or 35 μg/ml chloramphenicol as needed.
Bacterial strains E. coli CS2775 were transformed with pRGP1 plasmid [28 (link)], gacABCDEFG [23 (link)] to produce pRha or empty plasmid control (pHD0131). The bacterial cells were grown overnight in LB containing erythromycin (150 μg/ml) at 37°C and used next day for whole cell Western blots and FACS and microscopy analysis.
Bacterial strains E. coli CS2775 were transformed with pRGP1 plasmid [28 (link)], gacABCDEFG [23 (link)] to produce pRha or empty plasmid control (pHD0131). The bacterial cells were grown overnight in LB containing erythromycin (150 μg/ml) at 37°C and used next day for whole cell Western blots and FACS and microscopy analysis.