Cloning and characterization of dnaK operon of Tetragenococcus halophila

Daisuke Fukuda, Maki Watanabe, Shino Sonezaki, Kenji Sonomoto, Ayaaki Ishizaki

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We have cloned and characterized the dnaK operon of Tetragenococcus halophila JCM5888. The nucleotide sequence analysis of the 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 upstream 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 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 T. halophila DnaK was found to have ATPase activity. Northern hybridization analysis revealed that the transcription of dnaK gene was induced by heat shock, and several transcripts were detected including a maximum size of tetra-cistronic mRNA 4.9-kb which represents the transcript of complete dnaK operon. The amount of dnaK transcripts was also increased about 3.5-fold by high NaCl condition (3-4M), but not by the same concentration of KCl. These results suggest that the cloned DnaK surely acts as the functional molecular chaperone and play an important role in the salinity adaptation.

Original languageEnglish
Pages (from-to)229-241
Number of pages13
JournalJournal of the Faculty of Agriculture, Kyushu University
Issue number1
Publication statusPublished - Jan 1 2001

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Agronomy and Crop Science


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