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

Abstract

The dncJ gene ofLactococcus lactis was isolated from a genomic library ofL. lactis NIZO R5 and An abrupt increase in growth temperature usually causes the induction of synthesis of a small group of proteins called the heat shock proteins. This response is a common feature in eubacterial, archaebacterial, and eukaryotic organisms. Not only is the reaction to heat shock similar, but the structure and function of the induced proteins are highly conserved (for a recent review, see reference 1).The dnaJ gene of Escherichia coli was originally discovered to be essential for bacteriophage lambda replication (39). Recently, it was demonstrated that DnaJ is also involved in the replication of phage P1 (49) and oriC plasmids (22). One of the major activities of DnaJ is to stimulate the ATPase activity of DnaK, the prokaryotic member of the HSP70 family. This enhanced ATPase activity may result in an efficient recycling of DnaK (20). Furthermore, DnaJ is also believed to target other proteins for action by DnaK (48). Because of its cooperation with DnaK, DnaJ also plays a role in protein folding (11) and in the facilitation of export of homologous and hybrid proteins (29,50).Analysis of the heat shock response of Lactococcus lactis has revealed the induction of 13 to 16 proteins after a shift in temperature from 30'C to 37 or 420C (2, 47). Immunological screening of these induced proteins showed the presence of GroEL-and DnaK-like heat shock proteins in L. lactis (2,47). In addition, the lactococcal counterparts of the heat shock proteins GrpE and DnaJ could also be detected (2). Recently, the groELS operon of L. lactis was cloned and its nucleotide sequence was determined (17).In this report, we describe the cloning and characterization of the dnaJ gene of L. lactis. We show that its expression is regulated at the transcriptional level and is critically dependent on the presence of a palindromic structure immediately preceding its promoter. * Corresponding author. MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. E. coli JM83 (45) (27). Antibodies against this partially purified protein fraction were raised and were used to screen the genomic library. Immunoblotting was performed as described previously (42).

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

“…lactis DnaK protein appears to be approximately 3-fold on heat shock at 42 "C based on the Western blot data. This value corresponds well with the 2 to 3-fold induction of the dnaJ gene on heat shock (Van Asseldonk et al, 1993). Another immunoreactive protein of 50 kDa can be seen in L. lactis and although HSPs of 51 and 49 kDa have been observed in L. lactis (Whitaker & Batt, 1991) the 50 kDa cross-reacting protein is not induced on heat shock.…”

“…The L. lactis dnaK operon is unusual in that the dnaJ gene is not part of it and forms a completely separate transcriptional unit having its own promoter (Van Asseldonk et al, 1993) immediately upstream. In L. lactis the ORF downstream of dnaK (or@) appears to form a separate transcriptional unit, being separated from the dnaK gene by a potential transcription terminator and its own vegetative promoter.…”

Efficient insertional mutagenesis in lactococci and other gram-positive bacteria

“…Lactococcus lactis is widely used in dairy fermentations and also serves as a model organism for biological studies of lactic acid bacteria. Most genes thus far identified in L. lactis have been cloned by (i) complementation (2,10,19,35), (ii) immunoscreening of DNA libraries (11,34,48), (iii) PCR amplification of conserved genes (1,14,15,17,30), and (iv) DNA sequencing of regions adjacent to genes of interest (3,26,36). The chromosome and its genetic regulatory networks, however, remain for the most part unknown.…”

Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures

“…Total RNA from Lc. mesenteroides cells was isolated using, with a few modifications, the Macaloid clay method (van Asseldonk et al, 1993). For the isolation, the cells were pelleted by centrifugation (2000 g, 10 min, 4˚C) and immediately frozen at 280˚C.…”