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Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

ABSTRACT: The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 3.3 ± 0.2 mm for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.

SUBMITTER: Sasahara A

PROVIDER: S-EPMC3190712 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

Sasahara Ayako A Nanatani Kei K Enomoto Masaru M Kuwahara Shigefumi S Abe Keietsu K

The Journal of biological chemistry 20110630 33

The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by mean ...[more]

PMID: 21719707

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Project description:Bacteriophages infecting Tetragenococcus halophilus, a halophilic lactic acid bacterium, have been a major industrial concern due to their detrimental effects on the quality of food products. Previously characterized tetragenococcal phages displayed narrow host ranges, but there is little information on these mechanisms. Here, we revealed the host's determinant factors for phage susceptibility using two virulent phages, phiYA5_2 and phiYG2_4, that infect T. halophilus YA5 and YG2, respectively. Phage-resistant derivatives were obtained from these host strains, and mutations were found at the capsular polysaccharide (CPS) synthesis (cps) loci. Quantification analysis verified that capsular polysaccharide production by the cps derivatives from YG2 was impaired. Transmission electron microscopy observation confirmed the presence of filamentous structures outside the cell walls of YG2 and their absence in the cps derivatives of YG2. Phage adsorption assays revealed that phiYG2_4 adsorbed to YG2 but not its cps derivatives, which suggests that the capsular polysaccharide of YG2 is the specific receptor for phiYG2_4. Interestingly, phiYA5_2 adsorbed and infected cps derivatives of YG2, although neither adsorption to nor infection of the parental strain YG2 by phiYA5_2 was observed. The plaque-surrounding halos formed by phiYA5_2 implied the presence of the virion-associated depolymerase that degrades the capsular polysaccharide of YA5. These results indicated that the capsular polysaccharide is a physical barrier rather than a binding receptor for phiYA5_2 and that phiYA5_2 specifically overcomes the capsular polysaccharide of YA5. Thus, it is suggested that tetragenococcal phages utilize CPSs as binding receptors and/or degrade CPSs to approach host cells. IMPORTANCE T. halophilus is a halophilic lactic acid bacterium that contributes to the fermentation processes for various salted foods. Bacteriophage infections of T. halophilus have been a major industrial problem causing fermentation failures. Here, we identified the cps loci in T. halophilus as genetic determinants of phage susceptibility. The structural diversity of the capsular polysaccharide is responsible for the narrow host ranges of tetragenococcal phages. The information provided here could facilitate future studies on tetragenococcal phages and the development of efficient methods to prevent bacteriophage infections.

Dataset Information

Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

Publications

Substrate specificity of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus in reconstituted liposomes.

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