Cloning and sequencing of the Lactococcus lactis subsp. lactis dnaK gene using a PCR-based approach

The coding region for the dnaK gene from Lactococcus lactis subsp. lactis LM0230 was isolated and sequenced. An internal 789-bp fragment was amplified by the polymerase chain reaction (PCR) using a pair of degenerate oligodeoxyribonucleotide primers designed on the basis of amino acid (aa) sequences conserved in a number of DnaK. This PCR product was cloned, sequenced and used as a Southern hybridization probe to locate the flanking regions of the gene. The sequence of this central region from dnaK was also used to design two sets of inverse PCR primers to amplify, separately, the upstream and downstream regions. The inverse PCR products were then cloned and partially sequenced. The complete nucleotide sequence was obtained from overlapping cloned fragments of the gene and found to consist of a single 1824-bp open reading frame coding for a 602-aa protein. Alignment of the deduced aa sequence with those of other bacterial DnaK showed a high degree of homology and is most similar to the Bacillus megaterium DnaK.