Mutations affecting gluconate catabolism in Escherichia coli. Genetic mapping of loci for the low affinity transport and the thermoresistant gluconokinase

Abstract

The isolation and properties of strains of Escherichia coli carrying mutations affecting either the low affinity transport for gluconate (gene gntU) or the thermoresistant gluconokinase (gene gntK) are described. A lesion of each type was genetically characterized by transduction experiments. Both mutations mapped in the asd region, and the order was malA-glpD-asd-gntU-gntK, with the last two markers at about min 75.78 and 75.86 on the map, respectively. Mutations altering specifically gntU have not been previously reported.

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

“…This study confirms the gene order gntU, gntK, and gntR in the bioH-asd region of the E. coli genome (9). All three genes are transcribed in a counterclockwise direction and are immediately adjacent to asd, which is located clockwise from asd at 77.0 min on the E. coli genomic restriction map (37).…”

“…Early genetic studies of gluconate metabolism revealed some of the genetic loci involved in gluconate transport and gluconate phosphorylation, as well as the key enzymes of the EntnerDoudoroff pathway (1,9,14,24,32,46). There are two systems for gluconate transport and phosphorylation in E. coli (1,24).…”

“…Chloramphenicol (50 g/ml) was added to prevent de novo protein synthesis. The uptake reaction was started by the addition of 15 l of sodium [6][7][8][9][10][11][12][13][14] C]gluconate (3.4 Ci/mol) to 50 l of cell suspension in order to achieve a predetermined final concentration of isotope (1 to 800 M with a constant specific activity of 5.6 Ci/mol). After 5 s (5-s uptake times used for single-time point kinetics reflect initial velocities, which were found to be linear for the first 20 s or longer), uptake was terminated by quenching with 5 ml of ice-cold 250 mM gluconate in 0.05 M potassium phosphate buffer maintained at a temperature of less than Ϫ3ЊC on a salt-ice mixture (31).…”

“…For determination of the substrate specificity of gluconate transport, 50 l of cell suspension was preincubated with competing sugar at a concentration of 2 mmol at 25ЊC for 3 min, and then uptake of 50 M sodium [6][7][8][9][10][11][12][13][14] C]gluconate was assayed in the presence of a 40-fold excess of competing, unlabeled sugar. The process of filtering, washing, and counting of radioactivity was the same as that described above.…”

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

“…GntI, the main system, contains gntT, gntU, and gntK which code for high-and low-affinity gluconate transporters and a thermoresistant gluconokinase, respectively. The expression of GntI is negatively controlled by the gntR gene product (8,42). GntII, the subsidiary system, contains gntW and gntV, which are believed to encode another high-affinity gluconate transporter (41) and a thermosensitive gluconokinase (17,42), respectively.…”