A new integration vector, pBGK, was constructed for delivery of heterologous DNA into the chromosome of Streptococcus mutans. A kanamycin resistance element (OmegaKm), which is flanked by transcriptional and translational terminators and which is selectable in both Escherichia coli and streptococci, was inserted into a 2.4-kb EcoRI fragment carrying the S. mutans gtfA gene. A unique SmaI site flanking OmegaKm is available for cloning of promoter:reporter gene fusions or foreign genes, which can then be integrated into the S. mutans chromosome by allelic exchange with the gtfA gene. The vector was used to analyze the activity of an S. mutans promoter by fusing it to a chloramphenicol acetyltransferase gene. The reporter fusions could readily be cloned into the vector at a unique SmaI site and the vector and passenger DNA were stable in E. coli. DNAs flanked by gtfA sequees integrated efficiently into the chromosome of S. mutans and were stably maintained in the absence of selective pressure. Expression levels of the reporter gene were consistent regardless of orientation or repeated subculturing.
Copyright 2001 Academic Press.