Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli

Abstract

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a BglII-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to Ap R at a frequency of (8.2 ± 1.2) × 10 5 colony forming units (CFU)/μg DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded Cm R, Ap S clones at a frequency of (2.6 ± 0.3) × 10 1 CFU/μg DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to Ap R and S. thermophilus to Cm R with frequencies of (4.8 ± 0.1) × 10 5 and (8.1 ± 0.2) × 10 2 CFU/μg DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) SalI fragment. Transformation frequencies of pDBN183 were (5.0 ± 1.3) × 10 5 and (4.6 ± 0.2) × 10 2 CFU/μg DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17–18 per chromosome. The three plasmids conferred Ap R and Cm R to E. coli, but only Cm R to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene ( choA) into pDBN183 did not affect the plasmid's stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.