3 hydroxydiphenol

Capsular polysaccharide (CPS) production was quantified by the method described previously^{24 (link)}. Five hundred microliters of pneumococcal culture grown in the presence of 55 mM mannose or glucose from late exponential phase (approximately OD600 1.1 for wild type and 0.7 for the mutants) was mixed with 100 µl of 1% (v/v) Zwittergent 3–14 detergent (Sigma-Aldrich) in 100 mM citric acid (pH 2.0), and then the mixture was incubated at 50 °C for 20 min. The CPS was precipitated with 1 ml of absolute ethanol. The pellet was dissolved in 200 µl distilled water, and 1200 µl 12.5 mM borax (Sigma) in H₂SO₄ was added. The mixture was vigorously vortexed, boiled for 5 min, and cooled, and then 20 µl 0.15% 3-hydroxydiphenol (Sigma) was added. The absorbance of the mixture at 520 nm was measured, and the glucuronic acid content determined from a standard curve of glucuronic acid (Sigma).

The bacterial CPS was extracted by using a method described by Domenico (Domenico et al., 1989). Briefly, 500 μl of overnight‐cultured bacterium was mixed with 100 μl of 1% Zwittergent 3–14 (Sigma‐Aldrich, Milwaukee, WI, USA) in 100 mM citric acid (pH 2.0) and then incubated at 50°C for 20 min. After centrifugation, 250 μl of the supernatant was transferred to a new tube, and the CPS was precipitated with 1 ml of ethanol. The mixture was incubated at 4°C for 20 min. After centrifugation, the pellet was dried and dissolved in 100 μl of distilled water. The sample was appropriately diluted, and then, 1200 μl of 12.5 mM sodium tetraborate (Sigma‐Aldrich, St Louis, MO) in H₂SO₄ was added. The mixture was vigorously mixed and boiled for 5 min. After cooling, 20 μl of 0.15% 3‐hydroxydiphenol (Sigma‐Aldrich, Milwaukee, WI) was added. The tubes were shaken, and the absorbance at 520 nm was measured. Uronic acid content was determined from a standard curve of d‐glucuronic acid (Sigma‐Aldrich, Milwaukee, WI).

Quantification of capsule was performed using glucuronic acid assays, as previously described [56 (link)]. Briefly, bacteria were grown overnight in LB broth at 37°C under static growth conditions. Cultures were pelleted and resuspended in 1x PBS to an OD₆₀₀ of 1.0. A total of 500 μL of normalized culture was mixed with 100 μL of 1% Zwittergent 3–14 (Sigma) in triplicate in 100 mM citric acid and incubated at 50°C for 20 min. After centrifugation, supernatants from samples were precipitated with cold ethanol at 4°C for 20 min. Upon precipitation, samples were recentrifuged and the pellets were dissolved in 200 μL sterile water and 1200 μL of 12.5 mM tetraborate in concentrated H₂SO₄. Samples were vortexed, boiled at 95°C for 5 min, and mixed with 20 μL of 0.15% 3-hydroxydiphenol (Sigma) in 0.5% NaOH. Absorbance was measured at 520 nm using a microplate reader (Bio-Tek). The uronic acid concentration of each sample was determined using a standard curve of glucuronic acid (Sigma). The limit of detection (LOD) was previously defined [57 (link)] and reported here. Significance was determined using Mann-Whitney nonparametric tests with p<0.05. All graphs and statistics were generated using GraphPad Prism version 9.

K57 CPS, which contains uronic acid, was quantified according to previously described methods47 (link)48 (link). Briefly, extracted samples from the equivalent amounts of bacterial overnight cultures were resuspended in 0.1 mL of water and combined with 1.2 mL of 12.5 mM tetraborate in concentrated H₂SO₄. After vigorous vortexing, the mixture was boiled for 5 min. After cooling, 20 μL of 0.15% 3-hydroxydiphenol (Sigma-Aldrich, St. Louis, MO) was added. Then, the absorbance at 520 nm was measured.