ANALYSIS OF STREPTOCOCCUS-PYOGENES PROMOTERS BY USING NOVEL TN916-BASED SHUTTLE VECTORS FOR THE CONSTRUCTION OF TRANSCRIPTIONAL FUSIONS TO CHLORAMPHENICOL ACETYLTRANSFERASE
Rt. Geist et al., ANALYSIS OF STREPTOCOCCUS-PYOGENES PROMOTERS BY USING NOVEL TN916-BASED SHUTTLE VECTORS FOR THE CONSTRUCTION OF TRANSCRIPTIONAL FUSIONS TO CHLORAMPHENICOL ACETYLTRANSFERASE, Journal of bacteriology, 175(23), 1993, pp. 7561-7570
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.