title: Improved vector for promoter screening in lactococci.
abstract: Fragments of Lactococcus lactis subsp. lactis NP45 chromosomal DNA provided promoter activity in Escherichia coli when cloned into the promoter probe vector pGKV210. Only 13% of these recombinant plasmids promoted detectable cat-86 activity when transferred to L. lactis, i.e., expressed chloramphenicol resistance. In these promoter-containing versions of pGKV210, the cat-86 gene specifies chloramphenicol-inducible chloramphenicol acetyltransferase expression. This could be a limiting factor for cloning of promoters with lower activity in L. lactis. Therefore, we have constructed a new promoter probe vector, pBV5030, with the mutated version of the cat-86 gene, which is constitutively expressed when transcriptionally activated by the insertion of a promoter. We found that in L. lactis IL1403 the constitutively expressed cat-86 gene (on a pBV5030 derivative) has four times higher activity than the inducible version of the same gene (on a pGKV210 derivative) when both have the same promoter inserted upstream of the cat-86 gene. These results suggest that plasmid pBV5030 could be a more efficient vector for the cloning of promoters from lactococci.
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, Belgrade, Yugoslavia.
authors.forename: B
authors.initials: B
authors.lastname: Bojovic
authors.forename: G
authors.initials: G
authors.lastname: Djordjevic
authors.forename: L
authors.initials: L
authors.lastname: Topisirovic
mesh_terms: D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic
chemical_list: D004269:DNA, Bacterial; D015500:Chloramphenicol O-Acetyltransferase