FIG. 5.

Repression of ntdABC transcription by GlcP-mediated glucose transport. (A) Construction of glcP-interrupted strains. B. subtilis glcP-interrupted strains carrying ntdC::pMutinT3 or glcP::pMutinT3 were constructed by integrating a plasmid, pMutinT3, immediately upstream or downstream of T_ntdABC, respectively. These strains enabled us to control the expression of the glcP gene via the IPTG-dependent P_spac. A glcP-disrupted strain carrying ΔglcP::pMutinT3 also was constructed. In this strain, T nucleotide in the second codon of glcP was deleted, resulting in a −1 frameshift and a nonsense mutation at Leu-2 (TTA) (→ochre [TAA]). (B) Repression of ntdABC by induction of glcP. Total cellular RNAs were prepared from cells of strains TI167 (rpoB⁺ ntdC::pMutinT3), TI168 (rpoB⁺ glcP::pMutinT3), TI168Δ4 (rpoB⁺ ΔglcP::pMutinT3), TI167R5 (rpoB5 ntdC::pMutinT3), TI168R5 (rpoB5 glcP::pMutinT3), and TI168R5Δ4 (rpoB5 ΔglcP::pMutinT3), which were grown for 10 h in S7N medium containing excess (1%) glucose with or without 2 mM IPTG. RNA samples (10 μg each) were spotted directly onto a membrane and hybridized with the RNA probe for ntdB. (C) Repression of ntdABC by GlcP-mediated sugar transport. B. subtilis strain TI168 (rpoB⁺ glcP::pMutinT3) was grown in S7N medium containing excess (1%) glucose (Glc) until OD₆₅₀ reached 1.0, after which the cells were harvested by centrifugation and transferred into the same S7N medium (left lane), S7N medium without glucose (center lane), or S7N medium supplemented with 1% 2-deoxy-glucose (2DG) instead of glucose (right lane). Where indicated, IPTG was added to final a concentration of 2 mM just after the cells were transferred. After incubation for an additional 4 h, cells were harvested and RNA samples (4 μg each) were spotted directly onto a membrane and hybridized with the RNA probe for ntdC.