Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism

Abstract

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent K m of 6 M for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have ؊10 and ؊35 sequence elements typical of 70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 M gluconate, modestly induced by very low levels of gluconate (4 M), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

A GntR family transcriptional regulator (PigT) controls gluconate-mediated repression and defines a new, independent pathway for regulation of the tripyrrole antibiotic, prodigiosin, in Serratia

“…We demonstrate that PigT activates transcription of the Pig biosynthetic operon (pigA-O), whereas addition of gluconate causes a reduction in transcription of pigA-O. Furthermore, a putative PigT binding site was identified in the promoter of pigA, based on sequence similarity to the gnt operator site (Porco et al, 1997). Therefore, PigT is predicted to activate pigA-O transcription directly.…”

“…Interestingly, a putative PigT binding site, based on that demonstrated for GntR (Porco et al, 1997), was identified upstream of the 235 element in the pigA promoter (Fig. 3a).…”

“…The bold type represents the two conserved half sites of the predicted PigT/GntR binding sites. This predicted binding site is based on the E. coli GntR palindromic consensus binding sequence , GC-rich)-TAACAT) (Porco et al, 1997) present in the promoter regions of GntR-regulated genes. The position of this binding site suggests that PigT may be binding directly to the pigA-O promoter region and functioning as a Class I activator to regulate transcription (Busby & Ebright, 1999).…”

“…(a) Partial sequence of the pigA promoter that was cloned into contructs pTA15, pTA30, pTA31 and pTA32 (indicated). Features of the promoter include the ROP1 (repressor of pigment) region (bold italics), the transcriptional start site (+1), the predicted "10 and "35 elements (boxed) (Slater et al, 2003), and the proposed PigT binding site (bold boxed, centred at "78?5) based on the GntR binding site (Porco et al, 1997). A putative cAMP receptor protein (CRP) binding site centred at "64?5 relative to the +1 overlaps the PigT box and is shown underlined with the two conserved half sites in italics.…”

Conversion of Glucose and Gluconate to Ethanol in Mineral Salts Medium using Recombinant Escherichia coli Strains

“…The fumarate reductase (frd) gene was deleted to prevent succinate production and further improve the carbon flow toward ethanol production [5]. E. coli strains have distinct pathways to transport glucose and gluconate [8][9][10][11]. Gluconate does not seem to have any obvious effect on carbon catabolite repression of glucose and vice versa.…”