Ca ²⁺ -Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334

Abstract

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. Lactobacillus represents a large genus of Gram-positive bacteria that contains over 100 species (1). Among these, Lactobacillus casei is a facultative heterofermentative lactic acid bacterium (LAB) that can be isolated from diverse environments. It is an important nonstarter lactic acid bacterium commonly present in cheese and dairy products. The most important energy and carbon source for LAB in general is lactose, which is abundantly present in milk, but few LAB have in addition the capacity to metabolize citrate, which is present in cheese curd at a concentration of approximately 10 mmol · kg Ϫ1 . Citrate fermentation by bacteria proceeds via two main routes that are catalyzed by a variety of enzymes and transporters. The genes encoding the citrate lyase complex and accessory proteins are diagnostic for citrate fermentation by bacteria. Citrate lyase catalyzes the common, first metabolic step in the pathways, i.e., the splitting of internalized citrate in oxaloacetate and acetate. The two main pathways diverge at oxaloacetate yielding succinate or pyruvate. The route to succinate via malate and fumarate is found in many lactobacilli but also in the gammaproteobacterium Escherichia coli (2). The pathway is characterized by a transporter that takes up citrate in exchange for the end product succinate (precursor-product exchange). The transporter is a member of the divalent anion:Na ϩ symporter (DASS) family of secondary transporters (3, 4). The second pathway involves decarboxylation of oxaloacetate, yielding pyruvate, which is added to the central pyruvate pool in the glycolytic pathway during carbohydrate/citrate cometabolism. In LAB, redundant pyruvate in the pool is converted to the flavor compound acetoin. In different bacteria, the pathway up to pyruvate is catalyzed by different combinations of a transporter responsible for the uptake of citrate and an enzyme (complex) that decarboxylates oxaloacetate, which has consequences predominantly for the energetics of the pathways. In Gram-negative bacteria like Klebsiella pneumoniae, citrate is taken up by an Na ϩ -dependent symporter, termed CitS, and oxaloacetate is decarboxylated by a membrane-bound complex, termed OAD, that conserves the free energy released in the reaction by pumping out Na ϩ ions (5, 6). Subsequently, pyruvate is converted to acetate via the acetate kinase route, which yields 1 ATP per molecule of pyruvate. The free-energy yield is high enough to allow the organism to grow on citrate as the sole energy source (7,8). In LAB like Lactococcus lactis and Leuconostoc mesenteroides, a cytoplasmic oxaloacetate decarboxylase, termed CitM, which does not conserve the free energy released in the reaction (9), is combined with a transporter, termed CitP, that functions as a citrate...

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Potential of three probiotic lactobacilli in transforming star fruit juice into functional beverages

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