Nucleotide sequence of attP and cos sites of phage CTX and expression of cytotoxin in Pseudomonas aeruginosa PA158

Abstract

The gene for Pseudomonas aeruginosa cytotoxin (CTX) has been found to be part of a temperate phage with a total size of 35.5 kb. We have investigated several DNA fragments of this phage for CTX production. For phage integration, the phage genome cohesive (cos) ends covalently associate with host DNA of strain PA158. The cos ends and the CTX gene are found on a 3.4 kb EcoRI fragment B and are included in the 11 kb HindIII fragment A and the 8.5 kb BamHI fragment B of the phage DNA. The cos ends are 20 nucleotides long and are located at 338-357 nucleotides upstream of the CTX transcriptional initiation site. The phage attachment (attP) site is also present on the 3.4 kb EcoRI fragment B. The attP site consists of 34 bp and is located at 974-1007 nucleotides upstream of the CTX gene start site. Replication of the vegetative form of the phage is increased at 37 degrees C compared to that at 30 degrees C, while cytotoxin production in infected cells is similar at 30 degrees C and 37 degrees C. It can be concluded, therefore, that the integrated form of the CTX gene is responsible for CTX production. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed ten proteins in purified phage preparations; however, CTX could not be detected on Western blots using an enzyme-linked immunofluorescence assay.