| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The DNA fragment comprising exlB (PSPA7-4641), exlA (PSPA7_4642), intergenic region and the exlB RBS was synthesized by GenScript (www.genscript.com) and sub-cloned into a pBad miniCTX-1 integrative vector pSW196 (Baynham et al., 2006). The exlB gene was amplified by PCR and subcloned into pSW196. To express only exlA, most of the exlB gene (89%) was deleted from pUC57-exlBA plasmid by cutting it with BglII-MfeI. The plasmid was religated after Klenow treatment. Then the EcoRI-SacI fragment was cloned into pSW196 cut with the same enzymes. The construct was verified by sequencing. Plasmids were transferred into P. aeruginosa by triparental mating using pRK2013 (Figurski and Helinski, 1979). | Get A Quote |
Virulence of Pseudomonas aeruginosa is typically attributed to its type III secretion system (T3SS). A taxonomic outlier, the P. aeruginosa PA7 strain, lacks a T3SS locus, and no virulence phenotype is attributed to PA7. We characterized a PA7-related, T3SS-negative P. aeruginosa strain, CLJ1, isolated from a patient with fatal hemorrhagic pneumonia. CLJ1 is highly virulent in mice, leading to lung hemorrhage and septicemia. CLJ1-infected primary endothelial cells display characteristics of membrane damage and permeabilization. Proteomic analysis of CLJ1 culture supernatants identified a hemolysin/hemagglutinin family pore-forming toxin, Exolysin (ExlA), that is exported via ExlB, representing a putative two-pa... More
