Control of effective expression of the phage φCTX-encoded ctx gene in Pseudomonas aeruginosa by a promoter upstream of the cos site

Abstract

In the DNA of bacteriophage phiCTX, the attachment site (attP), the cohesive end site (cos), and the gene (ctx) encoding the pore-forming cytotoxin, are clustered. Deletion variants and PCR-generated fragments of the DNA were cloned into the Pseudomonas aeruginosa and Escherichia coli vector pHA10. Recombinant plasmids carrying the chloramphenicol acetyltransferase gene, cat, were used for promoter studies. Two promoters responsible for ctx expression are located between attP and cos and between cos and ctx, the promoter upstream of cos being stronger than the one downstream. The promoters and the ribosomal binding site are recognized by both the P. aeruginosa and E. coli transcriptional systems. The results indicate that chromosomal integration of phiCTX DNA, which requires cos site ligation, is necessary for high ctx expression in phiCTX-infected P. aeruginosa strains.

Organization and activation of the late promoters of φCTX, a cytotoxin-converting phage from Pseudomonas aeruginosa

“…Both promoters of ctx are similar to the consensus sequence of r 70 -type promoters, and are recognized by the host RNA polymerases of E. coli and P. aeruginosa (Xiong et al 1998) without additional transcriptional factors, as postulated by McLean et al (1997). In the late promoters of /CTX no similarities were found with the Pseudomonas promoter consensus sequences TTGACA-N 17±1 -TATAAT and TGGC-N 8 -TGCT for recognition by the r 70 and r 54 factors, respectively (Deretic et al 1989;Rothmel et al 1991).…”

“…Bacterial strains, /CTX, plasmids, and growth conditions The bacterial strains used for b-galactosidase assays were E. coli XL1-Blue (Bullock et al 1987), P. aeruginosa GuA18 (Xiong et al 1998) and P. aeruginosa PAO1S-Lac (Rist and Kertesz 1998). XL1-Blue and PAO1S-Lac contain the mutant allele lacZDM15, which encodes a fragment of b-galactosidase that complements the product of lacZa encoded by the promoter-probe vector pME4510 [Gen R ] (Rist and Kertesz 1998).…”

“…The methods used for the preparation of phage stocks and the isolation of /CTX DNA were as described by Xiong et al (1998).…”