Submitted to: Biotechnology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 6, 1997
Publication Date: N/A
Interpretive Summary: Streptococcus thermophilus is an important bacterium used in the manufacture of yogurt and certain cheeses. Genetic engineering of this bacterium provides a potentially quick way for changing these microbes to develop new dairy products. Small, circular DNA molecules called plasmids are useful in transferring new genes between microorganisms for the expression of specific enzyme activities. This report describes the development and characterization of several gene transport vehicles from a small plasmid of a yogurt culture. A section of the DNA molecule critical for the survival of the plasmids in the new host was defined. DNA sequence data revealed signals necessary for the production of a protein, Rep371, needed to maintain the DNA in the cells. These results provide valuable information for making more efficient gene carriers for use in the genetic development of dairy fermentation streptococci and lactobacilli.
Technical Abstract: A native plasmid of Streptococcus thermophilus ST137, pER371 (2.7 kb), was linearized at various unique restriction sites and individually subcloned into Escherichia coli plasmid pUC19 to generate the pUER-series recombinants. A selection cassette consisting of chloramphenicol- and erythromycin-resistance genes was spliced into each construct to generate the pMEU shuttle vectors. Electrotransformation of Streptococcus thermophilus with these vectors showed that a ca. 1.7-kb BstEII/BanII fragment is essential for plasmid replication in the Gram-positive host. A smaller size vector, pMEU14'-1 (5.3 kb), with higher transformation frequency was constructed using the minimally required fragment. Sequence analysis of the replication region revealed the presence of an open reading frame, rep371, with putative promoter and ribosome binding site located upstream. Rep371 is not homologous to RepA in other small S. thermophilus cryptic plasmids. The pER371-based cloning vector provides an alternative gene delivery vehicle for the genetic engineering of lactic acid bacteria.