Characterization of Glucose-Specific Catabolite Repression-Resistant Mutants of Bacillus subtilis: Identification of a Novel Hexose:H+ Symporter

Catabolite repression (CR) is a ubiquitous phenomenon whereby the presence of a rapidly metabolizable carbon source such as glucose inhibits the expression of genes encoding proteins concerned with the utilization of alternative carbon sources (32, 33). In Escherichia coli this phenomenon is mediated by two transcriptional regulatory proteins and their cognate cytoplasmic signaling molecules, the cyclic AMP receptor protein which binds cyclic AMP to effect positive control of operon expression, and the catabolite repressor/activator (Cra) protein, which effects positive control except when a metabolite such as fructose-1-phosphate or fructose-1,6-bisphosphate is bound to it (34, 35). In low-GC-content gram-positive bacteria, such as Bacillus subtilis, an entirely different CR mechanism is operative. This process involves catabolite control protein A (CcpA) (18, 19), which interacts with metabolites and a phosphorylated regulatory protein [HPr(Ser-P) or Crh(Ser-P)] to effect CR by a negative control mechanism (9, 20, 27, 36). Although the general features of this gram-positive bacterial CR mechanism are recognized, the fine details have yet to be elucidated. We recently reported the isolation of a novel B. subtilis mutant exhibiting partial resistance to CR due to the insertion of a mini-Tn10 element into a gene which proved to encode a protein that was 30% identical to CcpA (4). We designated this protein catabolite control protein B (CcpB). While CcpA solely mediates CR during growth in liquid media with a high level of agitation, both CcpA and CcpB mediate CR during growth under the same conditions but with a low level of agitation or during growth on solid media (4). The ccpB mutation was isolated by mini-Tn10 insertional mutagenesis of a B. subtilis strain carrying a translational gluconate (gnt) operon fusion (gntRK′-′lacZ), selecting for blue colonies on solid media containing gluconate plus glucose plus X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (4). The success of this method in identifying the ccpB gene prompted us to isolate and characterize additional mutants by the same procedure. In this paper we describe the isolation and characterization of three classes of mutants, two of which exhibit full resistance to glucose-promoted gnt operon CR and the other of which exhibits partial resistance. The former two classes of mutants proved to carry mini-Tn10 elements in (i) the ptsG gene encoding the well-characterized glucose permease (glucose enzyme II) of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) and (ii) the glcT gene encoding the transcriptional antiterminator that positively controls ptsG gene expression (39). The third class of mutants proved to carry the mini-Tn10 element in a novel, previously unsequenced gene that we have designated glcP. The product of glcP, the GlcP protein, is shown to be a glucose/mannose:H+ symport permease, a member of the fucose/glucose/galactose family of the major facilitator superfamily (28). We present evidence suggesting that expression of glcP is glucose inducible and that induction depends upon the integrity of the PTS.