Construction of a new sensitive molecular tool for the study of gene expression in Enterococcus faecalis.

ABSTRACT

A promoter-probe vector designated pVEPhoZ-P has been developed to provide a convenient system to analyze transcription activities of promoters. It was constructed on the basis of pVE14218, a plasmid which lacks the replication protein and in which the 5' part of alkaline phosphatase (AP) phoZ gene of Enterococcus faecalis was inserted. The pVEPhoZ vector was used to clone promoters of interest. The resulting promoter-probe vectors were integrated in the phoZ chromosomal locus by homologous recombination. This procedure generates recombinant clones with a single copy of phoZ functional allele placed under the control of the desired promoter. This system was investigated with different promoters of E. faecalis genes, namely those of sigV encoding an ECF sigma factor, croRS encoding a two-component system and dhaK operon. In all cases, expression data obtained previously for the three promoters were properly reported for this new tool. The pVEPhoZ-P promoter-probe vector is easy to use and it showed higher reporter activity in comparison to systems based on ß-galactosidase. Therefore, this system constitutes a useful molecular tool for the study of promoters in E. faecalis or other bacterial species that possess a homologous phoZ gene.