CHARACTERIZATION OF PER371-BASED STREPTOCOCCUS-THERMOPHILUS ESCHERICHIA-COLI SHUTTLE VECTORS

Native plasmid of Streptococcus thermophilus ST137, pER371 (2.7 kb) li nearized at various unique restriction sites was individually subclone d into Escherichia coli plasmid pUC19 to generate the pUER-series reco mbinants. A selection cassette consisting of chloramphenicol- and eryt hromycin-resistance genes was spliced into each construct to generate the pMEU shuttle vectors. Electrotransformation of Streptococcus therm ophilus with these vectors showed that a ca. 1.7 kb BstEII/BanII fragm ent is essential for plasmid replication. A shuttle vector, pMEU14'-1 (5.3 kb), was constructed using the minimally required fragment for re plication. A chloramphenicol acetyltransferase (cat) gene was successf ully expressed in the ultimate S. thermophilus host by using pMEU14'-1 Cloning vectors derived from pER371 should provide valuable alternati ve gene delivery vehicles for the genetic engineering of lactic acid b acteria.