IDENTIFICATION OF A SODIUM-CHLORIDE-REGULATED PROMOTER IN LACTOCOCCUS-LACTIS BY SINGLE-COPY CHROMOSOMAL FUSION WITH A REPORTER GENE

An integration vector, pORI13, was developed to screen in Lactococcus lactis for expression signals induced by changes in the environment an d to assay transcriptional activity of genes in single copy. The plasm id carries a promoterless Escherichia coli lacZ gene preceded by a sta rt 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 plas mids 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 dep endent on the concentration of chloride ions.