Ca2+-citrate uptake and metabolism in Lactobacillus casei ATCC 334.

The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca(2+) and not as free citrate or the Mg(2+)-citrate complex, thereby identifying Ca(2+)-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca(2+) and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca(2+)-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca(2+)-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca(2+)-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca(2+)-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages.

Uptake of α-ketoglutarate by citrate transporter CitP drives transamination in Lactococcus lactis.

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of L-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.

Rerouting citrate metabolism in Lactococcus lactis to citrate-driven transamination.

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.

Substrate specificity of the citrate transporter CitP of Lactococcus lactis.

The citrate transporter CitP of lactic acid bacteria catalyzes electrogenic precursor-product exchange of citrate versus L-lactate during citrate-glucose cometabolism. In the absence of sugar, L-lactate is replaced by the metabolic intermediates/end products pyruvate, α-acetolactate, and acetate. In this study, the binding and translocation properties of CitP were analyzed systematically for a wide variety of mono- and dicarboxylates of the form X-CR(2)-COO(-), where X represents OH (2-hydroxy acid), O (2-keto acid), or H (acid) and R groups differ in size, hydrophobicity, and composition. It follows that CitP is a very promiscuous carboxylate transporter. A carboxylate group is both essential and sufficient for recognition by the transporter. A C-2 atom is not essential, formate is a substrate, and C-2 may be part of a ring structure, as in benzoate. The R group may be as bulky as an indole ring structure. For all monocarboxylates of the form X-CHR-COO(-), the hydroxy (X = OH) analogs were the preferred substrates. The preference for keto (X = O) or acid (X = H) analogs was dependent on the bulkiness of the R group, such that the acid was preferred for small R groups and the 2-ketoacid was preferred for more bulky R groups. The C(4) to C(6) dicarboxylates succinate, glutarate, and adipate were also substrates of CitP. The broad substrate specificity is discussed in the context of a model of the binding site of CitP. Many of the substrates of CitP are intermediates or products of amino acid metabolism, suggesting that CitP may have a broader physiological function than its role in citrate fermentation alone.

Mechanism of citrate metabolism by an oxaloacetate decarboxylase-deficient mutant of Lactococcus lactis IL1403.

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

Citrate uptake in exchange with intermediates in the citrate metabolic pathway in Lactococcus lactis IL1403.

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 α-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.