Characterisation of pSMA23, a 3.5 kbp plasmid of Lactobacillus casei, and application for heterologous expression in Lactobacillus

The complete nucleotide sequence of plasmid pSMA23 isolated from Lactobacillus casei A23 was determined. Plasmid pSMA23 is a 3497 bp circular molecule with a G+C content of 38.18%. Four putative open reading frames were identified. Based on homology, two orfs were identified as genes encoding replication initiation (Rep) and mobilisation (Mob) protein, respectively. Various regulatory regions like promoters, ribosome binding site (RBS), transcriptional terminators were deduced from the sequences of rep and mob. The origin of replication (dso) was predicted. Shuttle vectors pL142 and pL157 were constructed for Escherichia coli and Lactobacillus using rep gene and ori of pSMA23 for replication in Lactobacillus, the ori of the commercial vector pBluescript SkII+ for replication in E. coli, and the erythromycin and chloramphenicol resistance genes of pE194 and pC194, respectively, as selection markers. Transformants of E. coli and Lactobacillus were obtained on media containing erythromycin and chloramphenicol, respectively, suggesting expression of the ermC and cat194 genes in both organisms. The shsp gene of plasmid pSt04 of Streptococcus thermophilus encoding a small heat shock protein and the Lactobacillus plantarum cbh gene encoding conjugated bile salts hydrolase were cloned and successfully expressed in the heterologous host Lb. casei LK1 with the aid of pSMA23-derived vectors.