An integration vector, pORI13, was developed to screen in
Lactococcus lactis for expression signals induced by changes in the environment and to assay transcriptional activity of genes in single copy.
The plasmid carries a promoterless
Escherichia coli lacZ gene preceded by a start codon, a lactococcal ribosome binding site, and a multiple cloning site. Chromosomal
Sau3AI fragments of
L. lactis MG1363 DNA were cloned in pORI13 using a RepA
+
E. coli as host. The resulting bank of plasmids was used for Campbell-type integration into the chromosome of
L. lactis MG1363. The relatively large size of the chromosomal fragments used increases the chance of retaining complete genes in the
targeted region. Screening of integrants in the presence of 0.3 M NaCl resulted in the isolation of a clone (NS3) in which
expression of
lacZ was dependent on the concentration of chloride ions.
Key words Single copy transcriptional lacZ fusions - NaCl regulation - Lactococcus lactis
Received: 9 May 1997 / Accepted: 20 September 1997