Characterization of glucose-specific catabolite repression-resistant mutants of Bacillus subtilis: identification of a novel hexose:H+ symporter

Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GIcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GIcP. The last protein exhibits greatest sequence similarity to the fucose:H⁺ symporter of Escherichia coil and the glucose/galactose:H⁺ symporter of Brucella abortus. In a wild-type B. subtilis genetic background, the glcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [¹⁴C]glucose and [¹⁴C]methyl α-glucoside uptake. In a Δpts genetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a Δpts mutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2- deoxyglucose and methyl α-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.