The utilisation of gluconate by a thermophilic bacillus.
2015-11-19T09:07:51Z (GMT) by
A strain of Bacillus stearothermophilus grows on D-gluconate as sole carbon and energy source with a minimum doubling time of 90 min at 55°C. Gluconate is taken up by an inducible transport system, phosphorylated by an inducible gluconokinase and the 6-phosphogluconate formed is metabolised via a constitutive 6-phosphogluconate dehydrogenase. The inclusion of glucose or acetate in growth media represses the formation of activities induced by gluconate, and each is shown to reduce the activity of a preformed gluconate transport system. The isolation and properties of a mutant are described in which gluconate catabolism has lost sensitivity to glucose despite the occurrence of normal glucose catabolism, but which retains sensitivity to the effects of acetate. A mutant devoid of gluconokinase activity was isolated which, after growth in the presence of gluconate, retains high intracellular concentrations of gluconate even after repeated centrifugation and washing. Use of this mutant and of another strain derived from it which grows on gluconate only at elevated temperatures, has allowed study of the gluconate transport system without further gluconate metabolism. The transport system is highly specific, shows a Km of 8 ?M for gluconate and requires energy derived from respiration to perform concentrative transport. Evidence is presented that energy coupling is mediated by a proton gradient. Temporary accumulation of [14C] gluconate occurs under anaerobic conditions in the presence of high intracellular [12C] gluconate concentrations. Evidence is presented that gluconate transport is the rate limiting step for gluconate catabolism during growth and a close correlation between growth rate and the specific activity of the gluconate transport system at different temperatures is reported. It appears possible that a change in the membrane lipid phase may determine the minimum growth temperature of the organism. The gluconokinase has been purified over 600-fold; its properties include high specificity for phosphate acceptor, great stability at 70°C in the presence of magnesium, inhibition by sulphydryl reagents, and a random bi bi kinetic mechanism.