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Journal of Bacteriology, December 1998, p. 6642-6648, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Putative Multisubunit Na+/H+ Antiporter from Staphylococcus aureus

Toshiaki Hiramatsu,1 Kazuyo Kodama,1 Teruo Kuroda,1,2 Tohru Mizushima,1 and Tomofusa Tsuchiya1,2,*

Department of Microbiology, Faculty of Pharmaceutical Sciences,1 and Gene Research Center,2 Okayama University, Tsushima, Okayama 700-8530, Japan

Received 10 August 1998/Accepted 9 October 1998

We cloned several genes encoding an Na+/H+ antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na+/H+ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na+/H+ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na+/H+ antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na+/H+ antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na+/H+ antiporter. We observed a large Na+ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na+ extrusion was sensitive to an H+ conductor, supporting the idea that the system is not a respiratory Na+ pump but an Na+/H+ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan. Phone and Fax: 81-86-251-7957 or 81-86-251-7926. E-mail: tsuchiya{at}pheasant.pharm.okayama-u.ac.jp.

Journal of Bacteriology, December 1998, p. 6642-6648, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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