Functional Expression in Escherichia coli of Low-Affinity and High-Affinity Na + (Li + )/H + Antiporters of Synechocystis

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Abstract
Synechocystis sp. strain PCC 6803 has five genes for putative Na+/H+ antiporters (designatednhaS1, nhaS2, nhaS3,nhaS4, and nhaS5). The deduced amino acid sequences of NhaS1 and NhaS2 are similar to that of NhaP, the Na+/H+ antiporter of Pseudomonas aeruginosa, whereas those of NhaS3, NhaS4, and NhaS5 resemble that of NapA, the Na+/H+ antiporter ofEnterococcus hirae. We successfully induced the expression of nhaS1, nhaS3, andnhaS4 under control of an Na+-dependent promoter in Escherichia coli TO114, a strain that is deficient in Na+/H+ antiport activity. Inverted membrane vesicles prepared from TO114nhaS1 and TO114 nhaS3 cells exhibited Na+(Li+)/H+antiport activity. Kinetic analysis of this activity revealed thatnhaS1 encodes a low-affinity Na+/H+ antiporter with a Km of 7.7 mM for Na+ ions and a Kmof 2.5 mM for Li+ ions, while nhaS3encodes a high-affinity Na+/H+ antiporter with a Km of 0.7 mM for Na+ ions and a Kmof 0.01 mM for Li+ ions. Transformation ofE. coli TO114 with the nhaS1 andnhaS3 genes increased cellular tolerance to high concentrations of Na+ and Li+ ions, as well as to depletion of K+ ions during cell growth. To our knowledge, this is the first functional characterization of Na+/H+ antiporters from a cyanobacterium. Inverted membrane vesicles prepared from TO114nhaS4 cells did not have Na+/H+ antiport activity, and the cells themselves were as sensitive to Na+ and Li+ ions as the original TO114 cells. However, the TO114 nhaS4 cells were tolerant to depletion of K+ ions. Taking into account these results and the growth characteristics ofSynechocystis mutants in which nhaS genes had been inactivated by targeted disruption, we discuss possible roles of NhaS1, NhaS3, and NhaS4 in Synechocystis.

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