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Journal of Bacteriology, September 2005, p. 6370-6378, Vol. 187, No. 18
0021-9193/05/$08.00+0     doi:10.1128/JB.187.18.6370-6378.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of the N-Terminal Domain of ExeA: a Novel ATPase Involved in the Type II Secretion Pathway of Aeromonas hydrophila

Ian C. Schoenhofen,¹ Gang Li,² Timothy G. Strozen,² and S. Peter Howard^2*

National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada, K1A 0R6,¹ College of Medicine, Department of Microbiology and Immunology, H.Sc. A216, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5E5²

Received 13 April 2005/ Accepted 4 July 2005

Aeromonas hydrophila secretes a number of degradative enzymes and toxins into the external milieu via the type II secretory pathway or secreton. ExeA is an essential component of this system and is necessary for the localization and/or multimerization of the secretin ExeD. ExeA contains two sequence motifs characteristic of the Walker superfamily of ATPases. Previous examination of substitution derivatives altered in these motifs suggested that ATP binding or hydrolysis is required for ExeAB complex formation and subsequent secretion function. To directly examine ExeA function, the N-terminal cytoplasmic domain of ExeA with the addition of a C-terminal hexahistidine tag (cytExeA) was overproduced in Escherichia coli and purified by metal chelate affinity and anion-exchange chromatographic techniques. Purified preparations of cytExeA exhibited ATPase activity in the presence of several divalent cations, Mg²⁺ being the preferred cation, with an optimum reaction temperature of 37 to 42°C and an optimum pH of 7 to 8. cytExeA exhibited an apparent K_m for Mg-ATP of 0.22 mM and a V_max of 0.72 nmol min^–1 mg^–1 of protein. cytExeA displayed low specificity for nucleoside triphosphate substrates and was significantly inhibited by F-type ATPase inhibitors. Gel filtration analyses of cytExeA, ExeA, and ExeAB indicated that ExeA dimerizes and forms a very large complex with ExeB. These findings support a model whereby ExeAB utilizes energy derived from ATP hydrolysis to facilitate the correct localization and multimerization of the ExeD secretin.

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* Corresponding author. Mailing address: College of Medicine, Dept. of Microbiology and Immunology, H.Sc. A216, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E5. Phone: (306) 966-2548. Fax: (306) 966-4311. E-mail: peter.howard{at}usask.ca.

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Journal of Bacteriology, September 2005, p. 6370-6378, Vol. 187, No. 18
0021-9193/05/$08.00+0     doi:10.1128/JB.187.18.6370-6378.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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