Journal of General Microbiology (1973), 76, 375-388 Printed in Great Britain 375 The Metabolism of Lactate and Pyruvate by Pseudomonas AM^ By A. R. SALEM, C. WAGNER," A. J. HACKING AND J. R. QUAYLE Department of Microbiology, University of Shefield, Shefield, S 10 2 TN (Received 29 January 1973) SUMMARY Pseudomonas AMI, which can grow on C, compounds, also grows well on pyruvate and lactate. Extracts of the pyruvate-grown organism catalysed the synthesis of only small amounts of phosphoenolpyruvate directly from pyruvate. Experiments tracing the incorporation of radioactivity into bacterial constituents from pyruvate or lactate, each labelled in the C-I or C-3 position, indicated that these substrates are mainly assimilated after removal of the C-I atom. A mutant, c5, of Pseudornonas AMI was isolated by a procedure intended to select organisms lacking phosphoenolpyruvate carboxylase. Although this enzyme was present in mutant c5, growth on lactate or pyruvate was impaired and there was no growth on C1 compounds or ethanol. Good growth on each of these substrates was restored by a supplement of glyoxylate or glycollate. A scheme is proposed for the assimilation via malate synthase and phosphoenol- pyruvate carboxykinase of the C, fragment produced from pyruvate and lactate. INTRODUCTION It has been shown that the formation of phosphoenolpyruvate from pyruvate is achieved in Enterobacteriaceae in vivo through the sole agency of phosphoenolpyruvate synthetase (Cooper & Kornberg, I 967 ; Kornberg, I 970) : CH,. CO . COzH + ATP + CH,:C(OP) . COBH + AMP + Pi. (1) The phosphoenolpyruvate can then be used for gluconeogenesis or, after carboxylation by phosphoen@firuvate carboxylase, for the anaplerotic formation of C4 intermediates of the tricarboxylic acid cycle (Ashworth & Kornberg, I 966; Kornberg, 1970) : CH,:C(OP). C02H + CO, -+ H0,C. CH,. CO. C02H +Pi. (2) Despite the presence of other enzymes capable of carboxylating pyruvate or phosphoenol- pyruvate in vitro, behaviour of mutants lacking phosphoenolpyruvate carboxylase shows that it is this enzyme which effects the formation of C4dicarboxylic acids under physiological conditions. Such mutants can be isolated on the basis of their inability to grow on glucose, glycerol, lactate or pyruvate except in the presence of a supplement of an intermediate of the tricarboxylic acid cycle or an amino acid related thereto. In Pseudomonas AMI the only enzyme capable of carboxylating a C, compound to a C4 compound, which has so far been detected, is phosphoenolpyruvate carboxylase (Large, Peel & Quayle, 1962). If growth of Pseudomonas AMI on lactate or pyruvate entails a similar process to that of the Enterobacteriaceae then this enzyme would play a similar anaplerotic * Present address : Veterans AdministrationHospital, Department of Microbiology, 13 10 Twenty Fourth Avenue South, Nashville, Tennessee, 37203, U.S.A.