Functional Analysis of the Na⁺/H⁺ Antiporter Encoding Genes of the Cyanobacterium Synechocystis PCC 6803

I. V. Elanskaya^1*, I. V. Karandashova¹, A. V. Bogachev², and M. Hagemann³

¹Department of Genetics, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 138-5207; E-mail: irina@selansky.home.bio.msu.ru

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-0338; E-mail: sodiumc@genebee.msu.su

³Universität Rostock, FB Biowissenschaften, A.-Einstein-Str. 3, D-18057 Rostock, Germany; fax: +49 (0) 381-498-6172; E-mail: martin.hagemann@biologie.uni-rostock.de

^* To whom correspondence should be addressed.

Received October 25, 2001; Revision received December 3, 2001

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The role of putative Na⁺/H⁺ antiporters encoded by nhaS1 (slr1727), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415) in salt stress response and internal pH regulation of the cyanobacterium Synechocystis PCC 6803 was investigated. For this purpose the mutants (single, double, and triple) impaired in genes coding for Na⁺/H⁺ antiporters were constructed using the method of interposon mutagenesis. PCR analyses of DNA demonstrated that mutations in nhaS1, nhaS4, and nhaS5 genes were segregated completely and the mutants contained only inactivated copies of the corresponding genes. Na⁺/H⁺ antiporter encoded by nhaS3 was essential for viability of Synechocystis since no completely segregated mutants were obtained. The steady-state intracellular sodium concentration and Na⁺/H⁺ antiporter activities were found to be the same in the wild type and all mutants. No differences were found in the growth rates of wild type and mutants during their cultivation in liquid media supplemented with 0.68 M or 0.85 M NaCl as well as in media buffered at pH 7.0, 8.0, or 9.0. The expression of genes coding for Na⁺/H⁺ antiporters was studied. No induction of any Na⁺/H⁺ antiporter encoding gene expression was found in wild type or single mutant cells grown under high salt or at different pH values. Nevertheless, in cells of double and triple mutants adapted to high salt or alkaline pH some of the remaining Na⁺/H⁺ antiporter encoding genes showed induction. These results might indicate that some of Na⁺/H⁺ antiporters can functionally replace each other under stress conditions in Synechocystis cells lacking the activity of more than one antiporter.

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KEY WORDS: cyanobacterium Synechocystis PCC 6803, Na⁺/H⁺ antiporters, salt stress, pH adaptation, interposon mutagenesis, gene expression

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