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Applied and Environmental Microbiology, August 2005, p. 4176-4184, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4176-4184.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Halotolerant Cyanobacterium Aphanothece halophytica Contains NapA-Type Na⁺/H⁺ Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH

Nuchanat Wutipraditkul,¹ Rungaroon Waditee,² Aran Incharoensakdi,³ Takashi Hibino,⁴ Yoshito Tanaka,⁴ Tatsunosuke Nakamura,⁵ Masamitsu Shikata,⁶ Tetsuko Takabe,¹ and Teruhiro Takabe^2,4*

Graduate School of Agricultural Science, Nagoya University, Nagoya 464-8601, Japan,¹ Research Institute of Meijo University, Nagoya 468-8502, Japan,² Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand,³ Graduate School of Environmental and Human Sciences, Meijo University, Nagoya 468-8502, Japan,⁴ Faculty of Pharmacy, Niigata University of Pharmacy and Applied Life Science, Niigata 950-2081, Japan,⁵ Genomic Research Center, Shimadzu Corporation, Kyoto 604-8511, Japan⁶

Received 6 December 2004/ Accepted 20 February 2005

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na⁺/H⁺ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na⁺/H⁺ and Li⁺/H⁺ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K⁺ uptake-deficient E. coli mutant and exhibited K⁺ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.

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* Corresponding author. Mailing address: Research Institute of Meijo University, Tenpaku-ku, Nagoya 468-8502, Japan. Phone: 81-52-838-2277. Fax: 81-52-832-1545. E-mail: takabe{at}ccmfs.meijo-u.ac.jp.

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Applied and Environmental Microbiology, August 2005, p. 4176-4184, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4176-4184.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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