ANALYSIS OF STREPTOCOCCUS-PYOGENES PROMOTERS BY USING NOVEL TN916-BASED SHUTTLE VECTORS FOR THE CONSTRUCTION OF TRANSCRIPTIONAL FUSIONS TO CHLORAMPHENICOL ACETYLTRANSFERASE

We have developed a series of shuttle vectors based on the conjugative transposon Tn916 that have been designed for the analysis of transcri ptional regulation in Streptococcus pyogenes and other gram-positive b acteria. Designated the pVIT vectors (vectors for integration into Tn9 16), the vectors are small, stable plasmids in Escherichia coli to fac ilitate the fusion of promoters from cloned S. pyogenes genes to a pro moterless gene which encodes chloramphenicol acetyltransferase. The ve ctors each contain one or more small regions of Tn916 to direct the in tegration of the transcriptional fusion into the transposon via homolo gous recombination following transformation of S. pyogenes or other su itable gram-positive hosts. Integration can be monitored by the inacti vation or replacement of an antibiotic resistance determinant in modif ied derivatives of Tn916. Promoter activity can then be quantitated by the determination of chloramphenicol acetyltransferase-specific activ ity. In addition, since integration is into loci that do not disrupt t he conjugative transpositional functions of Tn916, the vectors are use ful for analysis of regulation in strains that are difficult or imposs ible to transform and can be introduced into these strains by conjugat ion following transformation of an intermediate host. The promoters fo r the genes which encode both the M protein and protein F of S. pyogen es were active in pVIT vectors, as was the region which controls trans cription of mry, a trans-acting positive regulator of M protein expres sion. However, neither of the two characterized promoters for mry demo nstrated activity when independently analyzed in pVIT-generated partia l diploid strains, suggesting that regulation of mry is more complex t han predicted by current models. The broad host range of Tn916 should make the pVIT vectors useful for analysis of regulation in numerous ot her bacterial species.