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Journal of Bacteriology, December 2006, p. 8070-8078, Vol. 188, No. 23
0021-9193/06/$08.00+0     doi:10.1128/JB.00404-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress

Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan,¹ Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan,² Laboratory of Functional Food Design, Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan³

Received 11 April 2006/ Accepted 14 September 2006

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB_Tha) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB_Tha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB_Tha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE_Tha) and ATP. Interestingly, the mixture of dimer and monomer ClpB_Tha, which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB_Tha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE_Tha and ATP under poststress conditions.

Published ahead of print on 22 September 2006.

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