MOLECULAR-GENETICS IN STREPTOCOCCUS-THERMOPHILUS - FROM TRANSFORMATION TO GENE-EXPRESSION

Streptococcus salivarius subsp thermophilus (S thermophilus) is a homofermentative, thermophilic lactic acid bacteria, used in dairy starter cultures. Despite its widespread and long-term use, its molecular biology and genetics have only recently started to be investigated. We report here the isolation and characterization of a cryptic, 3-kb plasmid which was converted into an E coli - S thermophilus shuttle vector. Using this and other plasmids, transformation was optimized and used to integrate non-replicative plasmids into the bacterial genome. Resulting cointegrates were able to amplify. Resolution of the cointegrates was used for gene-replacement by introducing an in vitro generated deletion into a genomic located structural gene. By the same mechanism, a heterologous, promoter-less marker gene was inserted onto the genome between the permease and beta-galactosidase gene of the lactose operon. It was thereafter expressed as a functional part of the operon and followed lactose regulation. The control region of the lactose operon was investigated by analyzing promoter up and down mutants.