Organization and activation of the late promoters of phiCTX, a cytotoxin-converting phage from Pseudomonas aeruginosa.
The late genes of the temperate phage of Pseudomonas aeruginosa are organized in an analogous fashion to the corresponding transcription units of the Escherichia coli P2 and P2-like phages. Sequence analysis of four putative late promoter regions, PP(phiCTX), PO(phiCTX), PV(phiCTX) and PF(phiCTX), reveals no similarity to sigma(70)-type promoters or promoter consensus sequences found in Pseudomonas, indicating the apparent need for a phage-encoded protein to control the expression of phiCTX late genes. To elucidate the mode of expression of the late genes, we fused the putative late promoter regions to the promoterless lacZalpha gene, which encodes the N-terminal part of beta-galactosidase as a reporter enzyme, in the promoter-probe vector pME4510. The candidate transactivator gene orf34 was cloned into expression vector pHA10, to generate the plasmid pHA34. The two recombinant plasmids were introduced together into E. coli XL1-Blue and P. aeruginosa PAO1S-Lac. Our results demonstrate that in phiCTX three late promoters (PP(phiCTX), PO(phiCTX), and PF(phiCTX)) are activated upon induction by IPTG in PAO1S-Lac carrying the cloned promoters and pHA34. Deletions and base-pair substitutions obtained by PCR-mediated mutagenesis demonstrated that two conserved sequences, TTGTAG-N(9)-cTACAa and GcCGCGCGCGCGgC, are essential for effective late gene expression. Whereas the late promoters were active in P. aeruginosa, only weak beta-galactosidase activity was obtained in E. coli.
Alber
Jörg
J
Institut für Pharmakologie und Toxikologie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 107, 35392 Giessen, Germany, Joerg.Alber@vetmed.uni-giessen.de
Langewische
F W
FW
Adebayo
A A
AA
Lutz
F
F
D001483:Base Sequence; D003603:Cytotoxins / Q000378:metabolism*; D017931:DNA Primers; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017105:Pseudomonas Phages / Q000235:genetics* / Q000378:metabolism
D003603:Cytotoxins; D017931:DNA Primers