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 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.
Sanders
J W
JW
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands. sandersj@biol.rug.nl
Venema
G
G
Kok
J
J
Leenhouts
K
K
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D007763:Lac Operon / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012965:Sodium Chloride / Q000378:metabolism*
D012965:Sodium Chloride