MOLECULAR-GENETIC CHARACTERIZATION OF THE ESCHERICHIA-COLI GNTT GENE OF GNTI, THE MAIN SYSTEM FOR GLUCONATE METABOLISM

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression sas characterized, The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transpo rt and directed the formation of a high-affinity gluconate transporter with a measured apparent K-m of 6 mu M for gluconate. Primer extensio n analysis indicated two transcriptional start sites for gntT, which a re separated by 66 bp and which give rise to what appears on a Norther n blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. T hus, it was concluded that gntT is monocistronic and is regulated by t wo promoters, Both of the promoters have -10 and -35 sequence elements typical of sigma(70) promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite rep ression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearl y identical palindromic sequences in the regulatory regions of all kno wn gluconate-inducible genes, and these seven putative gnt operators w ere used to derive a consensus gnt operator sequence. A gntT::lacZ ope ron fusion was constructed and used to examine gntT expression. The re sults indicated that gntT is maximally induced by 500 mu M gluconate, modestly induced by very low levels of gluconate (4 mu M), and partial ly catabolite repressed by glucose. The results also showed a pronounc ed peak of gntT expression very early in the logarithmic phase, a patt ern of expression similar to that of the Fis protein, Thus, it is conc luded that GntT is important for growth on low concentrations of gluco nate, for entry into the logarithmic phase, and for cometabolism of gl uconate and glucose.