D-Glucose and D-gluconate transport in vesicles from Pseudomonas putida

Abstract

Vesicles prepared from glucose-grown cells of Pseudomonas putida (ATCC, 12633) retain glucose oxidase (GOX) and gluconate dehydrogenase (GADH) activity and actively transport D-glucose, 2-deoxy-D-glucose (2DOG), 3-deoxy-3-fiuoro-D-glucose (3FG), and D-gluconate by saturable processes. The transport of these substrates is stimulated by the addition of L-malate or reduced phenazine methosulphate (PMS). Vesicles prepared from succinate-grown cells of P. putida lose their capacity to transport D-glucose, 2DOG, and 3FG by a saturable process. The transport and accumulation of D-gluconate, however, is retained with a Kx value of 65 μM and a Vmax of 1.0 nmol∙mg protein−1∙min−1. The rate of D-gluconate transport is stimulated by the addition of reduced PMS or L-malate with a reduction in the Kx value to 42.0 μM. Respirometric studies with these vesicles indicate the presence of an active GOX and L-malate dehydrogenase but a defective GADH. Thus a reductase activity is detected in the presence of D-gluconate and either 2,6-dichloroindophenol (DCIP) or ferricyanide, as measured by a decrease in absorbance at 600 and 420 nm, respectively. Measurements on these vesicles with the oxygen electrode, however, indicate that no electron transfer from GADH to oxygen occurs. This is in contrast to the results with glucose-grown vesicles or with L-malate or D-glucose as substrates in the succinate-grown vesicles. A comparison between glucose and gluconate oxidase activity in native and detergent-treated vesicles is made. The significance of these results in relation to the presence of a glucose carrier in P. putida and other pseudomonads is presented.