Todd hewitt broth

Genotypes used in this study are described in Supplementary Table S1. Unless otherwise stated, encapsulated S. pneumoniae were cultured statically at 35°C in 5% CO₂. Culturing on solid media used Todd-Hewitt broth supplemented with 0.5% yeast extract and 1.5% agar (Sigma-Aldrich). Media were supplemented antibiotics for selection of mutated genotypes: rifampicin (Fisher Scientific) at 4 μg ml⁻¹; kanamycin (Sigma-Aldrich) at 400 μg ml⁻¹, or chloramphenicol (Sigma-Aldrich) at 4 μg ml⁻¹. Phase contrast microscopy of colonies used a Leica DFC3000 G microscope.
Unless otherwise specified, culturing in liquid media used 10 ml of a 2:3 ratio mixture of Todd-Hewitt broth (Sigma-Aldrich) with 0.5% yeast extract (Sigma-Aldrich), and Brain-Heart Infusion media (Sigma-Aldrich); this is referred to as ‘mixed’ media. Transformation experiments with S. pneumoniae R6 derivatives used a chemically-defined medium, consisting of disodium β-glycerophosphate (20 g l⁻¹; Sigma-Aldrich), sodium pyruvate (0.1 g l⁻¹; Fluorochem), choline (0.001 g l⁻¹; Alfa Aesar), cysteine (0.4 g l⁻¹; Tokyo Chemical Industry UK), glucose (3.8 mM; Sigma-Aldrich) and galactose (12 mM; Sigma-Aldrich). Carbon source supplements were added to liquid media at a final concentration of 30 mM, unless otherwise specified.

B. pertussis B1917 (62 (link)) came from the RIVM collection. For counter selection purposes, B1917 was electroporated with the pFUS2-BctA1 suicide plasmid to acquire gentamicin resistance as described (63 (link)), thereby yielding B1917GR. After electroporation, gentamicin-resistant derivatives were checked by PCR to verify the site of insertion of the pFUS2-BctA1 suicide vector in the bacterial genome. Bacteria were cultured at 37°C on Bordet-Gengou (BG) agar (Difco), supplemented with 1% glycerol, 10% defibrinated sheep blood, and 10 μg/mL gentamycin, as described (64 (link)). After 40 hours of growth, the bacteria were harvested by scraping the plates and resuspended in PBS at the density of 5 × 10⁵ CFU/mL for infection of neonatal mice, or 5 × 10⁷ CFU/mL for infection of adult mice. Whole–B. pertussis cell lysates were prepared and used for antibody determination as described (65 (link)). S. pneumoniae serotype 1 (clinical isolate E1586) was obtained from the National Reference Laboratory, Ministry of Health, Montevideo, Uruguay, and was grown statically at 37°C with 5% CO₂ for 4–6 hours in Todd-Hewitt yeast broth, consisting Todd-Hewitt broth (Sigma-Aldrich) and 0.5% yeast extract (Becton Dickinson) as described (66 (link)). K. pneumoniae (ATCC 43816) was grown at 37°C in tryptic soy broth (Sigma-Aldrich) until mid-log phase.