Regulation of the Bacillus subtilis GlcT antiterminator protein by components of the phosphotransferase system

Abstract

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.

FIG. 1

Schematic presentation of mutations in ptsG. (A) Domain structure of EII^Glc. The phosphorylation sites are indicated by small solid circles, and the positions of point mutations used in this work are marked with arrows. (B) Genetic organization of the wild-type pts region cloned in pGP110. (C) Constructs used for the introduction of in-frame deletions in ptsG as described in the text.

FIG. 2

Mutations in GlcT. (Top) Domain structure of GlcT with conserved histidine residues. The N-terminal RNA-binding domain is black, and the duplicated PTS regulation domains (PRD-I and PRD-II) are stippled. The positions of constructed or isolated point mutations in GlcT are indicated below the bar. (Bottom) Alignment of the regions around the conserved histidine residues in PRD-I and PRD-II. The B. subtilis GlcT sequence was compared to those of other known and putative antiterminator proteins with different specificities (ATU, E. coli antiterminator of unknown function; BglG, E. coli BglG; GlcT, B. subtilis GlcT; LacT, Lactobacillus casei LacT; SacT, B. subtilis SacT [see reference for detailed references]). His residues which represent the target of negative regulation in PRD-I are indicated by an arrow. Conserved residues are boxed. Gaps introduced to maximize alignment are indicated by dashes.

FIG. 3

Proposed models of the regulation of GlcT activity (see Discussion). (A) In the presence of glucose, a phosphate residue (P) is transferred from phosphoenolpyruvate (PEP) via EI, HPr, and EIIAB to the sugar which enters the cell upon phosphorylation. GlcT is phosphorylated by EII^Glc in the absence of glucose and thereby inactivated. EII^Glc is able to phosphorylate two different substrates, a sugar and a protein. (B) As proved in this study, HPr–His-15–P is the phosphate donor for GlcT in absence of the substrate. The presence of EII^Glc is essential for a successful phosphate transfer. In the absence of domain B, GlcT is constitutively active, whereas GlcT still is regulated to a certain extent in strains containing point mutations of the phosphorylation sites of PtsG.