Figure 5
Mutations of residues interacting with UDP-sugars do not affect cell length and bacterial growth on minimal medium containing gluconate as sole carbon source. (A) Analysis of YvcK production by Western blotting. WT strain (168), ΔyvcK strain (SG471) and strains producing YvcK-T14A (SG520), YvcK-N218A (SG521), YvcK-Y265A (SG522) and YvcK-R301A (SG523) were grown at 37 °C in 20 ml of LB medium and collected at OD600 = 1. 16 μl of cell extracts were run on a 12.5% SDS-PAGE and transferred to nitrocellulose membrane by electroblotting. The blot membrane was read by the LAS4000 mini Imager. Full-length blot is presented in Supplementary data. (B) Bar graph of cell length averages for WT strain (168), ΔyvcK strain (SG471) and strains producing YvcK-T14A (SG520), YvcK-N218A (SG521), YvcK-Y265A (SG522) and YvcK-R301A (SG523). Bacteria were grown on LB medium until OD600 = 0.4 and analyzed by microscopy. The data were obtained from four independent experiments and the standard deviations are representated by the error bars. (C) Effect of yvcK point mutations on B. subtilis growth on gluconate as sole carbon source. WT strain (168; grey circle), ΔyvcK strain (SG471; empty grey diamond) and strains producing YvcK-T14A (SG520; cyan diamond), YvcK-N218A (SG521; YvcK pink square), YvcK-Y265A (SG522; yellow triangle) and YvcK-R301A (SG523; green square) were grown overnight on LB medium with 1% glucose. After centrifugation, cells were grown in CE-gluconate liquid medium at 37 °C supplemented with 0.5% xylose. (D) Effect of yvcK point mutations on cell morphology during growth on gluconate as sole carbon source. Cells were grown in minimal medium supplemented with gluconate at 37 °C and collected after 300 mn of growth. Their morphology was analyzed by microscopy: see micrographs of WT strain (168; grey circle), ΔyvcK strain (SG471; empty grey diamond) and strains producing YvcK-T14A (SG520; cyan diamond), YvcK-N218A (SG521; pink square), YvcK-Y265A (SG522; yellow triangle) and YvcK-R301A (SG523; green square).
