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Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70
Published on: March 8, 2024
A gram-negative characteristic segment in Escherichia coli DnaK is essential for the ATP-dependent cooperative
Shinya Sugimoto1, Chihana Higashi, Kozue Saruwatari
1Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka, Japan.
Abstract:
We describe importance of the characteristic segment in ATPase domain of DnaK chaperone which is present in all gram-negative bacteria but is absent in all gram-positive bacteria. In vitro studies, ATPase activity, luciferase-refolding activity, and surface plasmon resonance analyses, demonstrated that a segment-deletion mutant DnaKDelta74-96 became defective in the cooperation with the co-chaperones DnaJ and GrpE. In addition, in vivo complementation assay showed that expression of DnaKDelta74-96 could not rescue the viability of Escherichia coli DeltadnaK mutant at 43 degrees C. Consequently, we suggest evolutionary significance for this DnaK ATPase domain segment in gram-negative bacteria towards the DnaK chaperone system.
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