Streptococcus salivarius subsp thermophilus (S thermophilus) is a homo
fermentative, thermophilic lactic acid bacteria, used in dairy starter
cultures. Despite its widespread and long-term use, its molecular bio
logy and genetics have only recently started to be investigated. We re
port here the isolation and characterization of a cryptic, 3-kb plasmi
d which was converted into an E coli - S thermophilus shuttle vector.
Using this and other plasmids, transformation was optimized and used t
o integrate non-replicative plasmids into the bacterial genome. Result
ing cointegrates were able to amplify. Resolution of the cointegrates
was used for gene-replacement by introducing an in vitro generated del
etion 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 f
ollowed lactose regulation. The control region of the lactose operon w
as investigated by analyzing promoter up and down mutants.