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 chloramphenicolinducible 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 transcriptionaHly 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.

Efficient transformation of Lactococcus lactis IL1403 and generation of knock‐out mutants by homologous recombination

“…However, there exist large differences in transformability, not only between species, but also between different strains of the same species. In particular, transformation of Lactococcus lactis IL1403 in excess of 10 4 cfu/µg could not be obtained in our and several other laboratories working with this organism (personal communication, Bojovic et al 1991). This is in contrast to a report by Kim et al, describing the electrotransformation of L. lactis IL1403 at an efficiency of 10 8 cfu/µg (Kim et al 1996).…”

Comparative analysis of gene expression in Streptococcus pneumoniae and Lactococcus lactis

“…The high AT content (61%) of the pneumococcal chromosome predicts a more stringent consensus sequence requirement for the S. pneumoniae RNA polymerase than that for the E. coli enzyme. This higher stringency has been observed in the AT-rich Lactococcus lactis system, since most of the lactococcal sequences, isolated in E. coli by their promoter activity, were not func-SSDI 0378-1097(94)00338-6 290 tional in the original host [4]. At present, three transcriptional units corresponding to the pneumococcal polA, lytA and malX genes have been characterized in S. pneumoniae [5][6][7], but the strength of these promoter sequences was not investigated.…”

Casitone-mediated expression of the prtP and prtM genes in Lactococcus lactis subsp. lactis BGIS29

“…Plasmid DNA from lactococci was isolated by the method of Bojovic et al (1991). Large plasmids from lactococci were isolated by the method of O'Sullivan and Klaenhammer (1993).…”