Abstract
Bacillus subtilis yheL encodes a Na(+)/H(+) antiporter, whereas its paralogue, yqkI, encodes a novel antiporter that achieves a simultaneous Na(+)/H(+) and malolactate antiport. B. subtilis yufR, a control in some experiments, encodes a Na(+)/malate symporter. YqkI complemented a malate transport mutant of Escherichia coli if Na(+) and lactate were present. YheL conferred Na(+) uptake capacity on everted membrane vesicles from an antiporter-deficient E. coli mutant that was consistent with a secondary Na(+)/H(+) antiport, but YqkI-dependent Na(+) uptake depended on intravesicular malate and extravesicular lactate. YqkI-dependent lactate uptake depended on intravesicular malate and extravesicular Na(+). YqkI mediated an electroneutral exchange, which is proposed to be a malic(-2)-2H(+) (or fully protonated malate)/Na(+)-lactate(-1) antiport. Because the composite YqkI-mediated exchanges could be driven by gradients of the malate-lactate pair, this transporter could play a role in growth of B. subtilis on malate at low protonmotive force. A mutant with a disruption of yqkI exhibited an abrupt arrest in the mid-logarithmic phase of growth on malate when low concentrations of protonophore were present. Thus growth of B. subtilis to high density on a putatively nonfermentative dicarboxylic acid substrate depends on a malolactate exchange at suboptimal protonmotive force.
Highlights
The catalogue of diverse structural and mechanistic types of Naϩ efflux systems in prokaryotes is still expanding rapidly, an eloquent testimony to the importance of maintaining low cytoplasmic concentrations of Naϩ [1,2,3]
Because the composite YqkI-mediated exchanges could be driven by gradients of the malate-lactate pair, this transporter could play a role in growth of B. subtilis on malate at low protonmotive force
The current study focuses on two paralogous Bacillus subtilis genes whose deduced products both have strong sequence similarity to the Naϩ/Hϩ antiporter NhaC
Summary
5Ј-AGTGATCCTCCAAGCTTAGCCTAGTT-3Ј 5Ј-GAAGGGGGAGGATCCATGAAGGATGTAAG-3Ј 5Ј-AGCGGATCCATGATAGCGAAACACATGAT-3Ј 5Ј-GAAGGGGGAGGATCCATGAAGGATGTAAG-3Ј 5Ј-AGTGATCCTCCAAGCTTAGCCTAGTT-3Ј 5Ј-GAAGGGGGAGGATCCATGAAGGATGTAAG-3Ј 5Ј-ATCGCGTCGACATGGGAGCAATTCCAAAAACGGGGAC-3Ј 5Ј-ATGCGGATCCTTATGAAGTGAAAAAACGAAGCAGCAC-3Ј 5Ј-ATGCGGATCCATGGATTCTCAAAAAAAGCTGACG-3Ј 5Ј-AGCAAGCTTATTTTTTAACAAATCCAATC-3Ј a The restriction enzyme sites are underlined. The current study was designed to test the possibility that YqkI is a novel transporter that combines Naϩ/Hϩ and malate/lactate antiports in a malate-proton/Na-lactate exchange. Comparisons were carried out on the YheL-, YqkI-, and YufR-mediated transport activities in cells of a malate-minus Escherichia coli strain and in membrane vesicles from a Naϩ/Hϩ antiporter-deficient E. coli strain. The results support the expected catalytic properties for YheL and YufR and support the proposal that YqkI is a new structural type of malate/ lactate antiporter that has novel ion coupling properties, i.e. coupling Naϩ efflux and Hϩ uptake to the substrate-product antiport. Because the concentration gradients of the organic acid substrate and product could drive the coupled antiport, this combined exchange could be less vulnerable to reductions in the ⌬p such as those accompanying increased cell densities or encounters with protonophoric agents. Studies of a mutant in which yqkI was disrupted support this hypothesis
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