Bacillus subtilis can utilize several sugars as single sources of carbon and energy. Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate :sugar phosphotransferase system (PTS). In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B. subtilis. In this study, an analysis of the complete set of PTS proteins encoded within the B. subtilis genome is presented. Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants. Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified. Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B. subtilis ; by contrast only five Enzyme IIA Glc -like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the EIIA Glc -like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families. In addition, 13 PTS-associated proteins are encoded within the B. subtilis genome. These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction. The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.

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Bacillus subtilis transports glucose by the phosphotransferase system (PTS). The genes for this system are encoded in the ptsGHI operon, which is induced by glucose and depends on a termination/antitermination mechanism involving a riboswitch and the RNA-binding antitermination protein GlcT. In the absence of glucose, GlcT is inactive, and a terminator is formed in the leader region of the ptsG mRNA. If glucose is present, GlcT can bind to its RNA target and prevent transcription termination. The GlcT protein is composed of three domains, an N-terminal RNA binding domain and two PTS regulation domains, PTS regulation domain (PRD) I and PRD-II. In this work, we demonstrate that GlcT can be phosphorylated by two PTS proteins, HPr and the glucose-specific enzyme II (EII Glc ). HPr-dependent phosphorylation occurs on PRD-II and has a slight stimulatory effect on GlcT activity. In contrast, EII Glc phosphorylates the PRD-I of GlcT, and this phosphorylation inactivates GlcT. This latter phosphorylation event links the availability of glucose to the expression of the ptsGHI operon via the phosphorylation state of EII Glc and GlcT. This is the first in vitro demonstration of a direct phosphorylation of an antiterminator of the BglG family by the corresponding PTS permease.Glucose is the preferred source of carbon and energy for many bacteria. The sugar is transported into the cell and phosphorylated. The resulting glucose 6-phosphate can immediately feed into glycolysis. In Escherichia coli, Bacillus subtilis, and several other bacteria, glucose is taken up and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS).1 This system is made up of two general energy-coupling proteins, Enzyme I (EI) and HPr, and several multi-domain sugar specific permeases (Enzyme II, EII), which may exist as individual proteins or fused in a single polypeptide. In E. coli, the glucose-specific EII is composed of a membrane-bound protein comprising the actual transporter domain EIIC and the phosphotransfer domain EIIB. In addition, the EIIA domain is present as a cytoplasmic protein. In B. subtilis, all domains of the glucose permease are fused to form a single polypeptide with the domain arrangement EIICBA (1, 2).It was long considered that the genes encoding the components of the glucose PTS are constitutively expressed in bacteria. Although this is the case for the genes encoding the general proteins, ptsI and ptsH, the gene encoding EII Glc , ptsG, is induced by glucose in both E. coli and B. subtilis (1-3). In E. coli, regulation of ptsG expression is accomplished by the transcriptional repressor Mlc. In the presence of glucose, this repressor is sequestered by the non-phosphorylated EIICB Glc and, thus, is unable to repress ptsG transcription (3). In B. subtilis, glucose induction of ptsG expression is mediated by transcriptional antitermination. In the absence of glucose, transcription initiated at the ptsG promoter is terminated in the leader region of the mRNA. If glucose is present, an antiter...

The ptsG operon of Staphylococcus carnosus consists of two adjacent genes, glcA and glcB, encoding glucose-and glucoside-specific enzymes II, respectively, the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The expression of the ptsG operon is glucose-inducible. Putative RAT (ribonucleic antiterminator) and terminator sequences localized in the promoter region of glcA suggest regulation via antitermination. The glcT gene was cloned and the putative antiterminator protein GlcT was purified. Activity of this protein was demonstrated in vivo in Escherichia coli and Bacillus subtilis. In vitro studies led to the assumption that phosphoenolpyruvate-dependent phosphorylation of residue His105 via the general PTS components enzyme I and HPr facilitates dimerization of GlcT and consequently activation. Because of the high similarity of the two ptsG-RAT sequences of B. subtilis and S. carnosus, in vivo studies were performed in B. subtilis. These indicated that GlcT of S. carnosus is able to recognize ptsG-RAT sequences of B. subtilis and to cause antitermination. The specific interaction between B. subtilis ptsG-RAT and S. carnosus GlcT demonstrated by surface plasmon resonance suggests that only the dimer of GlcT binds to the RAT sequence. HPr-dependent phosphorylation of GlcT facilitates dimer formation and may be a control device for the proper function of the general PTS components enzyme I and HPr necessary for glucose uptake and phosphorylation by the corresponding enzyme II.

Bacillus subtilis utilizes glucose as the preferred source of carbon and energy. The sugar is transported into the cell by a specific permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI operon. Expression of this operon is induced by glucose and requires the action of a positive transcription factor, the GlcT antiterminator protein. Glucose availability is sensed by glucose-specific enzyme II (EIIGlc), the product of ptsG. In the absence of inducer, the glucose permease negatively controls the activity of the antiterminator. The GlcT antiterminator has a modular structure. The isolated N-terminal part contains the RNA-binding protein and acts as a constitutively acting antiterminator. GlcT contains two PTS regulation domains (PRDs) at the C terminus. One (PRD-I) is the target of negative control exerted by EIIGlc. A conserved His residue (His-104 in GlcT) is involved in inactivation of GlcT in the absence of glucose. It was previously proposed that PRD-containing transcriptional antiterminators are phosphorylated and concomitantly inactivated in the absence of the substrate by their corresponding PTS permeases. The results obtained with B. subtilis glucose permease with site-specific mutations suggest, however, that the permease might modulate the phosphorylation reaction without being the phosphate donor.