Cloning and sequence analysis of the dnaK gene region of Lactococcus lactis subsp. lactis.

Abstract

A 5.4 kb HindIII fragment of Lactococcus lactis subsp. lactis was identified using a homologous dnaK probe generated by PCR and cloned in Escherichia coli. Upstream sequences were generated by inverse PCR. The two cloned fragments partially overlapped, and sequencing of 5915 bp revealed the presence of four open reading frames in the order orf1-grpE-dnaK-orf4. orf1 encodes a 39 kDa protein of unknown function which shows considerable sequence homology with the Orf39 and Orfa proteins of Bacillus subtilis and Clostridium acetobutylicum, respectively. The downstream ORFs showed high homology to the grpE and dnaK genes of other prokaryotes. The DnaK protein has a characteristic 24-amino-acid deletion exhibited by all the known DnaK proteins of Gram-positive species. In many bacteria the dnaK and dnaJ genes are found as part of the same operon. The L. lactis dnaK operon is unusual in that the dnaK gene is followed by a putative transcription terminator and a fourth large ORF which shares no homology with the dnaJ genes of other bacteria but has a small degree of homology with various membrane proteins. Vegetative promoter sequences are found upstream of both orf1 and orf4. A 12 bp inverted repeat is found upstream of the putative promoter of orf1 and an 8 bp inverted repeat is found between this promoter and the orf1 initiation codon. These repeats are thought to be involved in regulation of the heat-shock genes. The DnaK homologue is induced approximately 3-fold on heat shock at 42 degrees C.