Construction of a new integration vector for use in Streptococcus mutans

A new integration vector, pBGK, was constructed for delivery of heterologou s DNA into the chromosome of Streptococcus mutans. A kanamycin resistance e lement (Omega Km), which is Banked by transcriptional and translational ter minators 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 Banking Omega Km is available for cloning of promoter: reporter gene fusions or foreign genes, which can then be integrated into t he 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 passeng er DNA were stable in E. coil DNAs Banked by gtfA sequences integrated effi ciently 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, (C) 2001 Aca demic Press.