Figure 5

Impact of bgsB on the synthesis and anchoring of LTA in the cell wall and on hydrophobicity of E. faecalis cells. A The total amount of butanol-extracted LTA from cell-wall extracts as determined by ELISA. For the quantification of LTA tethered to the cell wall, bacteria were grown overnight and adjusted to the same OD₆₀₀. Cell walls were disrupted by shaking with glass beads, and LTA was mobilized by stirring bacterial cells with butanol/water. ELISA plates were incubated with various concentrations of the respective water phase of the extraction, and LTA was detected using a polyclonal rabbit anti-LTA antibody. Data points represent means ± SEM, *** P < 0.001, Tukey's multiple comparison test. B Cell-surface hydrophobicity of E. faecalis strains determined by adherence of bacterial cells to a mixture of dodecane and aqueous phase. Bars represent the percentage of bacteria remaining in the organic phase after partitioning of the solvent system. Data represent the means ± SEM, **P < 0.01, *P < 0.05, Tukey's multiple comparison test. C Western blot detection of LTA from 12030 wild type and deletion mutants. LTA was extracted from disrupted bacterial cells after shaking with glass beads by boiling in SDS. LTA was loaded onto 4-12% SDS PAGE gels and detected using a polyclonal rabbit antibody against enterococcal LTA. D Estimation of LTA shed into the culture medium. After overnight culture, bacterial density was adjusted to the same OD₆₀₀, and bacteria were removed by centrifugation. 100 μl of supernatant was blotted onto PVDF membrane. Bound LTA was detected using the same antibody used in the ELISA. Dilution steps of culture supernatant are indicated in the legend.