Citrate Uptake in Exchange with Intermediates in the Citrate Metabolic Pathway in Lactococcus lactis IL1403

Abstract

Carbohydrate/citrate cometabolism in Lactococcus lactis results in the formation of the flavor compound acetoin. Resting cells of strain IL1403(pFL3) rapidly consumed citrate while producing acetoin when substoichiometric concentrations of glucose or L-lactate were present. A proton motive force was generated by electrogenic exchange of citrate and lactate catalyzed by the citrate transporter CitP and proton consumption in decarboxylation reactions in the pathway. In the absence of glucose or L-lactate, citrate consumption was biphasic. During the first phase, hardly any citrate was consumed. In the second phase, citrate was converted rapidly, but without the formation of acetoin. Instead, significant amounts of the intermediates pyruvate and ␣-acetolactate, and the end product acetate, were excreted from the cells. It is shown that the intermediates and acetate are excreted in exchange with the uptake of citrate catalyzed by CitP. The availability of exchangeable substrates in the cytoplasm determines both the rate of citrate consumption and the end product profile. It follows that citrate metabolism in L. lactis IL1403(pFL3) splits up in two routes after the formation of pyruvate, one the well-characterized route yielding acetoin and the other a new route yielding acetate. The flux distribution between the two branches changes from 85:15 in the presence of L-lactate to 30:70 in the presence of pyruvate. The proton motive force generated was greatest in the presence of L-lactate and zero in the presence of pyruvate, suggesting that the pathway to acetate does not generate proton motive force.

Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403

“…Citrate, oxaloacetate, and pyruvate were measured as described previously (23), using the commercially available enzymes citrate lyase (CL), L-malate dehydrogenase (L-MDH), and L-lactate dehydrogenase (L-LDH). Briefly, an aliquot of 30 l of the sample was added to 50 mM glycyl-glycine (pH 7.8) buffer containing NADH and L-MDH.…”

“…1), results in a pH gradient (inside alkaline). The pathway from pyruvate to acetate, which has not been resolved completely, does not seem to generate PMF (23). The PMF may be used to produce additional ATP via F 1 F o -ATPase or to maintain a high cytoplasmic pH to improve the acid resistance of the strain.…”

“…The exchange of divalent citrate and monovalent lactate catalyzed by CitP generates membrane po-tential of physiological polarity (i.e., inside negative). In L. lactis IL1403(pFL3), the exchange of citrate and the intermediates/products acetate and ␣-acetolactate was shown to be electrogenic as well (23). Proton consumption in the decarboxylation of oxaloacetate and ␣-acetolactate, as well as the formation of the latter (Fig.…”

“…Acetate is not further metabolized and leaves the cells, while oxaloacetate is decarboxylated to pyruvate by a soluble oxaloacetate decarboxylase encoded by the mae gene and termed CitM (25). In a recent study it was demonstrated that in the lactic acid bacterium Lactococcus lactis IL1403(pFL3), part of the pyruvate formed from citrate was converted into acetoin and part was converted into acetate in parallel pathways (23). In resting cells, the distribution over the two end products strongly depended on the conditions.…”

“…Under physiological conditions, when citrate is cometabolized with a carbohydrate, CitP operates in the fast citrate/L-lactate exchange mode; citrate is taken up and at the same time that L-lactate, the product of glycolysis, is excreted (precursor/product exchange) (2,17,19,23). In the absence of L-lactate, CitP operates in the much slower unidirectional H ϩ -symport mode (19), which makes uptake the rate-determining step for the flux through the pathway (23). It was shown that, in addition to L-lactate, CitP has an affinity for intermediates/products of the pathway, which results in the typical biphasic consumption of citrate in the absence of L-lactate.…”