Capsular polysaccharides were extracted from wild-type and mutant strains grown in a 1.2-liter Bioflo 110 batch fermentor (New Brunswick Scientific; 800 ml culture volumes in MM-glucose (5 (link))). Briefly, cultures were grown to mid-log phase (A600 for three biological replicates were 0.60, 0.59, and 0.62 for wild type; 0.60, 0.63, 0.61 for ΩBT3992, and 0.73, 0.76, and 0.73 for a ΩBT3992/ΔCPS2 double mutant). Bacterial cells were collected by centrifugation at 5000 × g for 20 min and immediately frozen at −80 °C. Each cell pellet was thawed by the addition of 50 ml of warm water. Phenol (90% w/w; 50 ml) was then added, and the mixture was held at 65 °C with gentle stirring for 1 h followed by incubation at 4 °C overnight. The extraction mixture was subsequently centrifuged at 1600 × g for 20 min to separate the aqueous and phenol phases, and the aqueous phase was collected and dialyzed (1-kDa cutoff) exhaustively against deionized distilled water. Dialyzed samples were lyophilized to dryness and dissolved in 20 ml of boiling double distilled H2O followed by continuous agitation overnight at 4 °C. Samples were subjected to two consecutive rounds of centrifugation to remove insoluble particulates, the first at 7,000 × g for 20 min and the second at 150,000 × g in an ultracentrifuge for 4 h (both steps at 4 °C). Finally, the cleared sample was lyophilized and dissolved in hot (95 °C) double distilled H2O at a concentration of 10 mg ml−1.
Neutral and acidic sugars were assayed in extracellular polysaccharide samples by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after acid hydrolysis of the samples in 2 m trifluoroacetic acid (100 °C for 4 h). Thirteen individual peaks corresponding to different sugars were distinguished under the conditions used. Nine of these eluted identically to known standards, whereas four did not behave like any of the standards examined (fucose, N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, mannose, galacturonic acid, glucuronic acid, and iduronic acid). Because both of the unknown monosaccharides that were abundant in CPS2 eluted similarly to acidic sugar standards, we also ran a sample of acid-hydrolyzed alginic acid, which is composed of mannuronic and guluronic acids. The presence of N-acetylgalactosamine and N-acetylglucosamine, which were deacetylated during acid hydrolysis, was inferred from the presence of galactosamine and glucosamine, respectively.