Effect of mutations causing gluconate kinase or gluconate permease deficiency on expression of the Bacillus subtilis gnt operon.

The gluconate (gnt) operon contains genes for a repressor of the operon, gluconate kinase, and gluconate permease. A nonleaky kinase mutation (gntK4) induced the gnt operon constitutively through interaction of the repressor with an inducer of gluconate which had been endogenously formed and accumulated in the cell owing to the complete deficiency of the kinase even in the absence of gluconate in the medium. In contrast, a nonleaky permease mutation (gntP9) never induced the operon by gluconate likely because it cannot give rise to its inducing concentration in the cell even in the presence of gluconate in the medium.     

Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum

A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum, was analyzed further. In the clone, an open reading frame designated glxR, consisting of 681 nucleotides and encoding a 24,957-Da protein, was found. The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers. The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins. The aceB-repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP. In glucose medium, the intracellular cAMP concentration of C. glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase. The expression of glxR was not affected by the carbon source. Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP. These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.     

Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism.

Three genes involved in gluconate metabolism, gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, respectively, were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map. The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction. The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein. The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases. While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E. coli,gntV. The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis. Kinetic analysis of GntU indicates an apparent Km for gluconate of 212 microM, indicating that this is a low-affinity transporter. Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are separated by only 3 bp, form an operon. Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression. These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.     

Genome-wide identification of in vivo binding sites of GlxR, a cyclic AMP receptor protein-type regulator in Corynebacterium glutamicum

Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant.     

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.     

Efficient utilization and operation of the gluconate-inducible system of the promoter of the Bacillus subtilis gnt operon in Escherichia coli.

A DNA fragment containing the promoter of the Bacillus subtilis gluconate (gnt) operon and its first gene (gntR) was cloned into Escherichia coli. E. coli recognized this promoter efficiently and precisely. Moreover, the gluconate-inducible system of this operon operated even in E. coli.