14 encapsulated and 307 14 nonencapsulated) were taken The serot

14 encapsulated and 307.14 nonencapsulated) were taken. The serotype was confirmed by Quellung reaction. Electron microscopy Bacteria were cultured as described above for the FITC-dextran exclusion assay, grown to OD600nm of 0.2–0.25 in CDM, pH 7, 5.5 mM glucose and harvested by centrifugation. Serotype was confirmed by Quellung reaction after overnight incubation at 37°C with 5% CO2 atmosphere on CSBA plates. Bacteria were cryopreserved by high-pressure freezing

as described before [52]. Acetone containing 2% osmium tetroxide, 0.1% PLX 4720 uranyl acetate, 0.2% ruthenium hexamine trichloride (RHT) and a total of 4% H2O served as medium for freeze substitution. The RHT added improves capsule resolution [53]. Electron micrographs from cross-sectional bacterial preparations were taken at a magnification of 53 000×. The FDA-approved Drug Library polysaccharide capsule thickness was measured perpendicular

to the bacterial cell wall from at least 30 randomly selected bacterial cell bodies in 15 pictures using the free software ImageJ v1.45 l (National Institutes of Health, USA, http://​imagej.​nih.​gov/​ij). One to four measurements were taken at distinct positions of a given cell body. Growth assays Strains were streaked onto CSBA plates and incubated at 37°C in 5% CO2 overnight and then subcultured in the semi-defined, nutritionally relatively rich Lacks medium [49-51] supplemented with 20 mM glucose and with the following modifications: 14.7 mM C2H3NaO2 · 3H2O, 5.41 μM CaCl2, 0.89 μM MnSO4 · H2O (all Merck, Germany) and ≥ 12 800 U catalase (Sigma, C40) per liter Lacks medium, no NaC2H3O2 and no bovine albumin. For growth assays, CDM [54] representing a nutritionally limited environment was used. Since pH may affect growth and competence, CDM was stabilized using Sørensen pentoxifylline buffer (KH2PO4, Na2HPO4 · 2H2O), pH 7 instead of double-distilled water (Additional file 1: Table S2). Half-loopfuls of colonies were used to SU5402 concentration inoculate 10 ml Lacks supplemented

with 20 mM glucose. The bacteria were grown to OD600nm of 0.5 and frozen at -80°C in aliquots in 15% glycerol. Thawed bacterial suspensions were diluted in PBS pH 7.4 and plated on CSBA to determine the number of colony forming units (CFU) per ml the next day. The serotype was confirmed by Quellung reaction. For growth assays an inoculum of 5 × 107 CFUs was used for subculture in 20 ml CDM, 5.5 mM glucose. Bacteria were grown for 10 hours at 37°C in a water bath and the OD600nm measured every 30 minutes. Growth assays were repeated on three different days. Transformation frequency To compare transformation frequencies between the two phenotypes the bacteria were cultured as described for the FITC-dextran exclusion assay and grown to OD600 = 0.15 in CDM, 5.5 mM glucose, pH 7. 0.5 ml of the culture were transferred to 9.5 ml TSB competence medium pH 8.0 prewarmed to 30°C and incubated for 15 min at 30°C.

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