Construction of compatible wide-host-range shuttle vectors for lactic acidbacteria and Escherichia coli

A new collection of shuttle cloning vectors has been constructed that can b e used in a broad host range, because they carry replication origins which are functional in Escherichia coli (p15A, pWV01, ColE1). Lactococcus lactis , lactobacilli, and Bacillus subtilis (pAM beta1, pWV01). These plasmids, c ontain the lacZ-T1T2 cassette from pJDC9, which allows the X-gal selection and cloning of DNA fragments that could cause plasmid instability in E. col i. In addition, they have been proved to be structurally and segregationall y stable in Lactobacillus casei, in which their copy number has been determ ined by real-time quantitative PCR. Furthermore, the antibiotic resistance markers (fl-lactamase, chloramphenicol acetyl transferase, and erythromycin transacetylase) and the theta and rolling circle replicating origins have been combined to obtain this set of compatible plasmids (pIA family) that c an be cotransformed, both in lactic acid bacteria and in E. coli. (C) 2001 Academic Press.