Induced succinate uptake and transport (defined as transport of a compound followed by its metabolism and transport in the absence of subsequent metabolism) by Pseudomonas putida are active processes resulting in intracellular succinate concentrations 10-fold that of the initial extracellular concentration. Uptake was studied with the wild-type strain P. putida P2 and transport with a mutant deficient in succinate dehydrogenase activity. Addition of succinate, fumarate, or malate to the growth medium induces both processes above a basal level. Induction is dependent on protein synthesis and subject to catabolite repression. When extracts of induced and noninduced wild-type cells were assayed for succinate dehydrogenase, fumarase, and malate dehydrogenase only malate dehydrogenase increased in specific activity. Transport is inhibited by iodoacetamide, KCN, NaN3, and 2,4-dinitrophenol and shows pH and temperature optima of 6.2 and 30 °C. Kinetic parameters are: basal uptake (cells grown on glutamate) Km 11.6 μm, v 0.32 nmoles per min per mg dry cell mass; induced uptake (cells grown on succinate plus NH4Cl) Km 12.5 μm, v 5.78 nmoles per min per mg dry cell mass; induced transport (cells grown on nutrient broth plus succinate) Km 10 μm, V 0.98 nmoles per min per mg dry cell mass. It was not possible to determine the kinetic parameters of basal transport. Malate and fumarate were the only compounds exhibiting competitive inhibition of uptake and transport suggesting common transport system for all three compounds. The Ki values for competitive inhibition and the Km for succinate indicate the order of affinity for both uptake and transport are succinate > malate > fumarate. Data from kinetic parameters of uptake and transport and studies on succinate metabolism provide evidence consistent with concurrent increases in transport and metabolism to account for induced succinate uptake by P. putida.
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Part of this work was supported by Gulf Research and Development Corporation and Mead Johnson Research Corporation. We thank Diane G. Toscano and Karen L. Walker for technical assistance in preparation of this manuscript, Dr. Govind K. Garg for critically reading the manuscript, and Dr. Francis W. Liegey and the Biology Department of Indiana University of Pennsylvania for their generosity in allowing us use of their equipment.
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