title: Molecular characterization and regulatory analysis of dnaK operon of halophilic lactic acid bacterium Tetragenococcus halophila.
abstract: We have cloned and characterized the dnaK operon of Tetragenococcus halophila JCM5888. Nucleotide sequence analysis of cloned fragments showed that the dnaK operon consists of four open reading frames with the organization hrcA-grpE-dnaK-dnaJ. Two regulatory CIRCE (Controlling Inverted Repeat of Chaperone Expression) elements were identified in the region up-stream of hrcA. The T. halophila dnaK encoded a protein of 618 amino acids with a calculated molecular mass of 67 kDa. The deduced amino acid sequence of T. halophila DnaK showed high similarities with those of the corresponding DnaK homologues of Lactococcus lactis, Lactobacillus sakei and Bacillus subtilis. Using a pET expression system, the T. halophila DnaK was overexpressed in Escherichia coli and the purified DnaK was found to exhibit ATPase and refolding activities. Northern hybridization analysis revealed that the transcription of the dnaK gene was induced by heat shock, and several transcripts were detected including a tetra-cistronic mRNA with a maximum size of 4.9-kb which corresponds to the transcript of the complete dnaK operon. The amount of dnaK transcripts increased about 3.5-fold at high NaCl concentration of 3-4 M, but not at the same KCl concentrations. These results suggest that the cloned DnaK acts as a functional molecular chaperone and plays an important role in salinity adaptation.
authors.affiliation: 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.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Fukuda
authors.forename: Maki
authors.initials: M
authors.lastname: Watanabe
authors.forename: Shino
authors.initials: S
authors.lastname: Sonezaki
authors.forename: Shinya
authors.initials: S
authors.lastname: Sugimoto
authors.forename: Kenji
authors.initials: K
authors.lastname: Sonomoto
authors.forename: Ayaaki
authors.initials: A
authors.lastname: Ishizaki