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Journal of Bacteriology, July 2006, p. 5045-5054, Vol. 188, No. 14
0021-9193/06/$08.00+0     doi:10.1128/JB.00128-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biochemical and Molecular Characterization of a Na⁺-Translocating F₁F_o-ATPase from the Thermoalkaliphilic Bacterium Clostridium paradoxum

Scott A. Ferguson, Stefanie Keis, and Gregory M. Cook^*

Department of Microbiology and Immunology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand

Received 23 January 2006/ Accepted 19 April 2006

Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56°C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum. The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an F₁F_o-ATP synthase but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q²⁸, E⁶¹, and S⁶²). On the basis of these properties, the F₁F_o-ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of Na⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the subunit that has been shown to be essential for coupled ATP synthesis.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin, New Zealand. Phone: 64 3 4797722. Fax: 64 3 4798540. E-mail: greg.cook{at}stonebow.otago.ac.nz.

This paper is dedicated to Professor Peter Dimroth on the occasion of his 65th birthday.

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Journal of Bacteriology, July 2006, p. 5045-5054, Vol. 188, No. 14
0021-9193/06/$08.00+0     doi:10.1128/JB.00128-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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