Cloning and characterization of promoter-active DNA sequences from Streptococcus equisimilis

Genomic fragments of Streptococcus equisimilis exhibiting potent promoter activity in Escherichia coli were isolated by transcriptional fusion to chloramphenicol acetyl transferase (CAT) structural gene. Random S. equanimities DNA, cloned in E. coli, exhibited a higher frequency of strong promoter activity than did similarly cloned E. coli fragments. The determination of the relative promoter strength of randomly selected clones with CAT assay demonstrated the dominance of sequences acting as a strong promoter in E. coli. Removal of downstream terminator from the strong promoter containing plasmid resulted in a twofold to threefold increase in CAT expression in some cases. Structural characteristics of promoter sequences of some representative streptococcal genes clearly indicate that the streptococcal promoter regions are rich in secondary structures and might be one of the factors of their instability in E. coli.