Single-stranded DNA plasmid, vector construction and cloning of Bacillus stearothermophilus alpha-amylase in Lactobacillus

Abstract

Vector plasmids were constructed by ligating chloramphenicol and erythromycin resistance genes to TaqI-digested DNA of a cryptic plasmid from Lactobacillus plantarum. The minimal region of Lactobacillus plasmid DNA that was required for DNA replication was defined and a single-stranded DNA intermediate replication system was observed. Homologies with other origins of replication of plasmids from Gram-positive bacteria, replicating via rolling circle mechanism, were found. It was shown that the constructed vectors, named pPSC20 and pPSC22, were transformable into L. plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus helveticus, Lactococcus lactis subsp. lactis, Bacillus subtilis, and Escherichia coli. Using plasmid pPSC22, the alpha-amylase gene of Bacillus stearothermophilus was cloned and expressed in several Lactobacillus species.