Abstract

The 5.8 kb pMMH1, rolling-circle replication (RCR) plasmid of the wild type soil Bacillus mesentericus was developed into a novel secretion vector system in Bacillus subtilis. The pMMH1 turned out to have a replication origin and two open reading frames (ORFs) of the putative [gamma]-GTP and type I signal peptidase (sipP). To characterize the regions necessary for plasmid stability and high copy number, five vectors (pPS, pPP, pEN, pMN, pME) were constructed by disruption or deletion of each region in pMMH1. Like pMMH1, all constructed vectors were stable over 100 generations in a non-selective medium. Since pPS was the smallest (2.3 kb)of all, it was selected for the construction of a novel secretion vector. Using the [alpha]-amylase promoter/signal sequence of B. subtilils, the novel plasmid pJSN was constructed. When [beta]-glucosidase was expressed using pJSN, we found [beta]-glucosidase activity in the medium. This result strongly suggested that plasmid pJSN can be used for the production of bioactive peptides in B. subtilis.

• Keywords: rolling circle replication (RCR) plasmid; Bacillus mesentericus; [beta]-glucosidase