Cloning, nucleotide sequence, and regulatory analysis of the Lactococcus lactis dnaJ gene

Abstract

The dnaJ gene of Lactococcus lactis was isolated from a genomic library of L. lactis NIZO R5 and cloned into pUC19. Nucleotide sequencing revealed an open reading frame of 1,137 bp in length, encoding a protein of 379 amino acids. The deduced amino acid sequence showed homology to the DnaJ proteins of Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis, and Clostridium acetobutylicum. The level of the dnaJ monocistronic mRNA increased approximately threefold after heat shock. The transcription initiation site of the dnaJ gene was determined and appeared to be preceded by a typical gram-positive vegetative promoter sequence (TTGCCA-17 bp-TAAAAT). Upstream of the promoter region, an inverted repeat is located that is identical to those detected upstream of heat shock genes of other gram-positive organisms. A transcriptional fusion between the dnaJ expression signals and a usp45-amyS secretion cassette caused a significant increase in alpha-amylase activity after heat shock induction. Deletion mutagenesis showed that the inverted repeat is involved in heat shock regulation of the dnaJ gene. The conservation of this palindromic sequence in gram-positive heat shock genes suggests a common regulatory pathway distinct from the system used in gram-negative bacteria.