Escherichia coli K-12 structural kdgT mutants exhibiting thermosensitive 2-keto-3-deoxy-D-gluconate uptake

Abstract

A specific method is described for selecting thermosensitive mutants of Escherichia coli K-12 able to grow on 2-keto-3-deoxy-D-gluconate (KDG) and D-glucuronate at 2, but not at 42 degrees C. The extensive analysis of one such mutant is consistent with the conclusion that the carrier molecule responsible for KDG and glucuronate uptake becomes thermolabile. (i) Growth on a variety of carbon sources is perfectly normal at 28 and 42 degrees C, whereas in the same temperature range it gradually diminishes on KDG and glucuronate. (ii) The apparent Km value for KDG is about twofold in the range 25 to 40 degrees C. In the same temperature range, the Vmax values for KDG influx are higher for the mutant compared with those of the wild-type strain, but the optimum temperature is 34 degrees C instead of 38 degrees C. On the contrary, the Vmax values for glucuronate influx are lower for the mutant than for the parental strain, and the optimum temperature for both strains is shifted beyond 40 degrees C. (iii) The activation energies for KDG and glucuronate uptake are about twofold higher in the mutant than in the wild-type strain. (iv) Kinetics of counterflow under deenergized conditions (overshoot) at different temperatures indicate that the defect is located in the translocation step rather than in the processes involved in energy coupling. (v) The first-order rate constants for thermal denaturation are, respectively, 2.5- and 5-fold higher at 40 and 30 degrees C in the mutant than in the wild-type strain, and the activation energy for thermal denaturation is lower. (vi) The carrier molecule in the mutant is also much more sensitive to denaturation by N-ethylmaleimide. (vii) Four independent thermosensitive mutations and one revertatn were located by transduction in or near the kdgT locus, defined previously as the site of nonconditional KDG transport-negative mutations. These results support the conclusion that kdgT represents the structural gene coding for the KDG transport system.