Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system.

Abstract

The transcriptional and translational initiation signals of a portion of the Staphylococcus aureus plasmid pUB110 were analyzed. An Mbo I-Pvu II fragment was sequenced and the site of transcriptional initiation was determined by in vitro mapping. To convert the plasmid into a cloning vector, a multilinker was introduced in different positions relative to a detected reading frame. The Gram-negative beta-galactosidase gene and the Gram-positive chloramphenicol acetyltransferase (cat) gene were fused and the level of expression was determined in Bacillus subtilis. Hybrid proteins consisting of corresponding CAT polypeptides were produced in each translational reading frame. Therefore this vector system can be used to express cloned DNA in the Gram-positive host Bacillus subtilis. Furthermore a derived lacZ fusion plasmid may be used for rapid screening of inserts.