Translation efficiency of antiterminator proteins is a determinant for the difference in glucose repression of two β-glucoside phosphotransferase system gene clusters in Corynebacterium glutamicum R

Abstract

Corynebacterium glutamicum R has two β-glucoside phosphoenolpyruvate, carbohydrate phosphotransferase systems (PTS) encoded by bglF and bglF2 located in the respective clusters, bglF-bglA-bglG and bglF2-bglA2-bglG2. Previously, we reported that whereas β-glucoside-dependent induction of bglF is strongly repressed by glucose, glucose repression of bglF2 is very weak. Here, we reveal the mechanism behind the different effects of glucose on the two bgl genes. Deletion of the ribonucleic antiterminator sequence and transcriptional terminator located upstream of the translation initiation codon of bglF markedly relieved the glucose repression of a bglF-lacZ fusion, indicating that glucose affects the antitermination mechanism that is responsible for the β-glucoside-dependent induction of the bglF cluster. The glucose repression of bglF mRNA was also relieved by introducing a multicopy plasmid carrying the bglG gene encoding an antiterminator of the bglF cluster. Moreover, replacement of the GUG translation initiation codon of bglG with AUG was effective in relieving the glucose repression of bglF and bglG. Inversely, expression of bglF2 and bglG2 was subject to strict glucose repression in a mutant strain in which the AUG translation initiation codon of bglG2 encoding antiterminator of the bglF2 cluster was replaced with GUG. These results suggest that the translation initiation efficiency of the antiterminator proteins, at least in part, determines whether the target genes are subject to glucose repression. We also found that bglF expression was induced by glucose in the BglG-overexpressing strains, which may be explained by the ability of BglF to transport glucose.

FIG. 1.

Organization of the two bgl gene clusters. Open arrows represent the coding regions of indicated genes. Functions of each gene are indicated below these genes. RAT-like sequences and putative transcriptional terminators are indicated as closed boxes and stem-loops, respectively.

FIG. 2.

Effect of glucose on the salicin-inducible expression of the bgl PTS genes. The wild-type strain was grown in nutrient-rich A medium supplemented with indicated concentrations of glucose (Glc) and salicin (Sal). The levels of bglF (open bars) and bglG (filled bars) (A) and of bglF2 (open bars) and bglG2 (filled bars) (B) mRNAs were determined by qRT-PCR. The relative mRNA levels were normalized to the level in the presence of 1% (wt/vol) salicin without glucose. The values are the means of three independent experiments, and standard deviations are indicated by error bars.

FIG. 3.

Effects of deletion of the RAT transcriptional terminator region on expression of bgl-lacZ. Strains carrying the wild-type bglF promoter-lacZ YT117; bglF-lacZ), the mutant bglF promoter-lacZ (YT121; bglFΔRAT-lacZ), the wild-type bglF2 promoter-lacZ (YT119; bglF2-lacZ), and the mutant bglF2 promoter-lacZ (YT123; bglF2ΔRAT-lacZ) were grown in nutrient-rich A medium supplemented with 1% (wt/vol) glucose (Glc) and/or 1% (wt/vol) salicin (Sal). The expression levels of these lacZ fusion genes were determined by qRT-PCR using primers specific for lacZ. The relative mRNA levels were normalized to the level in the strain carrying the respective wild-type promoter-lacZ grown in the medium supplemented with salicin alone. The values are the means of three independent experiments and standard deviations are indicated on the bar tops.

FIG. 4.

Effects of overexpression of the bglG on the glucose repression of bglF expression. The wild-type strains transformed with either pCRB1 vector or pCRC806 carrying bglG under the control of a constitutive promoter were grown in nutrient-rich A medium supplemented with 1% (wt/vol) glucose (Glc) and/or 1% (wt/vol) salicin (Sal). The levels of bglF (open bars) and bglG (filled bars) mRNAs were determined by qRT-PCR. The relative mRNA levels were normalized to the level in the pCRB1-carrying strain grown in the presence of salicin. The values are the means of three independent experiments and standard deviations are indicated on the bar tops.

FIG. 5.

Effects of substitution in the translation initiation codon on the expression of antiterminator BglG and BglG2. Translation initiation codons are shown in the sequences of the antiterminator genes, bglG and bglG2 (A; boxed). Strains carrying a gene for the respective FLAG-tagged antiterminator protein with or without substitution of the translation initiation codon, i.e., bglG-FL-GTG (YT111), bglG-FL-ATG (YT113), bglG2-FL-ATG (YT129), and bglG2-FL-GTG (YT136), were grown in nutrient-rich A medium with or without 1% (wt/vol) salicin, and the FLAG-tagged proteins were detected by immunoblot analyses using anti-FLAG M2 antibody (B).

FIG. 6.

Effects of substitution of the translation initiation codon of antiterminator proteins on expression of the bgl genes. Strains carrying the respective antiterminator gene with or without substitution of the translation initiation codon, i.e., bglG-ATG and bglG2-GTG, were grown in nutrient-rich A medium supplemented with 1% (wt/vol) glucose (Glc) and/or 1% (wt/vol) salicin (Sal). The levels of bglF (open bars) and bglG (filled bars) (A) and of bglF2 (open bars) and bglG2 (filled bars) (B) were determined by qRT-PCR. Relative mRNA levels were normalized to the level in the wild-type cells grown in the presence of 1% (wt/vol) salicin. The values are the means of three independent experiments, and standard deviations are indicated by error bars.

FIG. 7.

Effects of deletion of ptsG, bglF, and bglF2 on consumption of glucose. The wild-type strain (open circles), the ptsG mutant (open squares), the bglF mutant (open triangles), the ptsG bglF double mutant (filled triangles), the ptsG bglF2 double mutant (crosses), and the ptsG bglF bglF2 triple mutant (filled diamonds) were grown in nutrient-rich A medium with 0.2% (wt/vol) glucose. Concentrations of glucose in the culture medium were monitored. The results shown are representative of two independent reproducible experiments.

FIG. 8.

Effects of deletion of ptsG on expression of the bgl genes. The wild-type (WT) and the ptsG mutant strains were grown in nutrient-rich A medium supplemented with 1% (wt/vol) glucose (Glc) or salicin (Sal). The levels of bglF (open bars) and bglG (closed bars) (A) and of bglF2 (open bars) and bglG2 (filled bars) (B) were determined by qRT-PCR. The relative mRNA levels were normalized to the level in the wild-type cells grown in the presence of 1% (wt/vol) salicin. The values are the means of three independent experiments, and standard deviations are indicated by error bars.