Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO 2 . This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate--decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter 3 aspD 3 aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate--decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate--decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.The gram-positive lactic acid bacterium Tetragenococcus halophila is used to ferment soy sauce, which contains large amounts of amino acids, including L-aspartate (aspartate), sugars, such as hexoses and pentoses, and sodium chloride (ca. 17%) (27). Some strains of T. halophila catalyze decarboxylation of aspartate with nearly stoichiometric release of L-alanine (alanine) and CO 2 (11,27). Based on analogy to our previous work on Lactobacillus subspecies M3 (1), aspartate decarboxylation is thought to be advantageous for tetragenococcal cells because aspartate consumption concomitant with release of alanine generates rather than consumes metabolic energy and regulates the intracellular pH. The net charge movement during the exchange of aspartate with alanine results in a membrane potential of physiological polarity. Furthermore, decarboxylation reactions consume scalar protons and thus generate a pH gradient of physiological polarity. The combined activities of the precursor-product exchange and decarboxylation result in a proton motive force (PMF) that is sufficiently high to drive ATP synthesis via FoF1 ATPase. Such metabolic systems are proposed as proton motive metabolic cycles, and the prototype model is found in the oxalate-formate exchange system of Oxalobacter...
We cloned the aspT gene encoding the L-aspartate:L-alanine antiporter AspTCt in Comamonas testosteroni genomic DNA. Analysis of the nucleotide sequence revealed that C. testosteroni has an asp operon containing aspT upstream of the l-aspartate 4-decarboxylase gene, and that the gene order of the asp operon of C. testosteroni is the inverse of that of Tetragenococcus halophilus. We used proteoliposomes to confirm the transport processes of AspTCt. To elucidate the two-dimensional structure of AspTCt, we analysed its membrane topology by means of alkaline phosphatase (PhoA) and beta-lactamase (BlaM) fusion methods. The fusion analyses revealed that AspTCt has seven transmembrane segments (TMs), a large cytoplasmic loop containing approximately 200 amino acid residues between TM4 and TM5, a cytoplasmic N-terminus, and a periplasmic C-terminus. These results suggest that the orientation of the N-terminus of AspTCt differs from that of tetragenococcal AspT, even though these two AspT orthologues catalyse the same transport reactions.
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