The gluconate operon gnt of Bacillus subtilis encodes its own transcriptional negative regulator.

Abstract

The gluconate (gnt) operon ofBacillus subtilis consists of four gnt genes; the second and third genes code for gluconate kinase (gluconokinase, EC 2.7.1.12) and gluconate permease, respectively. A fragment carrying the promoter of this operon (gnt promoter) and the first gene (gntR) was subcloned into a promoter probe vector (pPL603B). Repression of the expression of cat-86 gene, encoded in the vector portion of a constructed plasmid (pgnt2l), that is under the control of the gnt promoter was removed by gluconate. The results of deletion analysis and of insertional inactivation ofthe gntR gene cloned in pgnt2l suggested that the product of the gntR gene, actually synthesized as a 29-kDa protein in vivo, is involved in repression of the gnt promoter. A 4-base-pair insertional mutation within the gntR gene constructed in vitro was introduced into the B. subtilis chromosomal gnt operon by use of linkage of the 4 base pairs to gntK1O in transformation. The introduced mutation gntRI caused the constitutive expression of the gluconate kinase and gluconate permease genes. S1 nuclease analysis indicated that the mRNA of this operon is synthesized in the gntRI strain and amounts of mRNA are not changed very much by gluconate, which acts as an inducer in the wild-type gene. These results strongly indicate that the gntR gene codes for a transcriptional negative regulator for the gnt operon.Various microorganisms are able to grow on gluconate as the sole carbon source. After entering a cell, gluconate is phosphorylated to gluconate-6-P, which is then catabolized through the pentose cycle or Entner-Doudoroff pathway. Bacillus subtilis does not possess the latter pathway, so only two enzymes, gluconate permease and gluconate kinase (gluconokinase, EC 2.7.1.12), seem to be specifically involved in gluconate catabolism in this organism. Both enzymes are induced by gluconate, and their induction is repressed by carbon sources that are rapidly metabolized such as glucose (1, 2).We have characterized and cloned the gluconate (gnt) operon of B. subtilis, which includes the structural genes for gluconate permease and gluconate kinase (3). Recently, we reported the entire nucleotide sequence of the gnt operon (4). Analysis of the operon sequence revealed the presence of four gnt genes, which were designated from the 5' end as gntR, gntK, gntP, and gntZ (see Fig. 1A). The gntK-and gntP-encoded proteins were identified as gluconate kinase and gluconate permease, respectively. But we had no idea of the functions of the gntR-and gntZ-encoded proteins. Transcription of the gnt operon starts from the gnt promoter 40 base pairs (bp) upstream of the gntR gene and terminates 45 bp downstream of the gntZ gene, resulting in a polycistronic mRNA (gnt mRNA), the product of the four gnt genes (4, 7).This operon appears to be regulated mainly at the transcriptional level because gnt mRNA synthesis is induced by gluconate and is repressed by glucose (4, 7).Induction of the gnt operon by gluconate seems to be negatively controlled through a r...

The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

“…When CT-43 cells were grown in GYS medium containing yeast extract, they selected a third route that relies on the gnt operon that participates in gluconate metabolism. In CT-43, the gnt operon (CH2189 -2191) is composed of gntP (gluconate permease), gntK, and gntZ/gndA (6-phosphogluconate dehydrogenase), and lacks its own transcriptional regulator, which is comparable to the negative regulator gntR in B. subtilis (53). Our RNA-seq results showed that the gnt operon expression level reached a maximum at 7 h, and then gradually decreased.…”

Versatile Transcription of Biphenyl Catabolic bphOperon in Pseudomonas pseudoalcaligenes KF707

“…The orf0 encoded a putative protein of 245 amino acids, and the data base search revealed that the ORF0 protein shares 28% identity to GntR, a regulatory protein known as the transcriptional repressor of the gluconate operon in Bacillus subtilis (29). These two proteins exhibited a high similarity of amino acid sequence in the N-terminal region ( Fig.…”

“…It was first reported that the GntR protein negatively regulates the gnt operon in B. subtilis (29). The GntR-type regulatory proteins involved in the degradation of aromatic compounds are also reported to be repressors (18,31).…”

A developmentally regulated gene encoding a repressor‐like protein is essential for sporulation in Streptomyces coelicolor A3(2)

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