PMC

Virulence of P. aeruginosa PA14 strains with mutations in genes encoding the DGC, PDE, and DGC-PDE domain proteins in a murine model of acute infection. Survival of mice after thermal injury and infection by 2 × 10⁶ wild-type and various P. aeruginosa mutants was monitored daily.

Phenotypic analysis of P. aeruginosa PA14 transposon mutants in genes encoding the DGC, PDE, and DGC-PDE domain proteins. (A) Cytotoxic effect of P. aeruginosa PA14 transposon mutants on CHO cells after infection, monitored by the release of LDH. (B) Biofilm phenotypes were measured as the attachment of P. aeruginosa to tubes and formation of the biofilm rings and pellicles after 20-h static incubation at 30°C.

Phenotypic analysis of P. aeruginosa PA14 overexpressing genes encoding the DGC, PDE, and DGC-PDE domain proteins. (A) Cytotoxic effect of PA14 transposon mutants on CHO cells after infection, monitored by the release of LDH. (B) Biofilm phenotypes were measured as the attachment of P. aeruginosa to glass tubes and the formation of the biofilm rings and pellicles after 12-h static incubation at 30°C.

Analysis of enzymatic activities of proteins with DGC and PDE domains. Extracts of P. aeruginosa PA14, overexpressing genes with DGC or DGC-PDE domains, were fractionated by reverse-phase HPLC, and levels of c-di-GMP were quantified. For the assessment of PDE activity, synthetic c-di-GMP was added to lysates of cultures overexpressing proteins with PDE and DGC-PDE domains, and loss of the c-di-GMP peak in the HPLC analysis was quantified. (A) Representative traces from the HPLC analysis of the PA14 extract with the pMMB67EHGent vector or overexpressing PA5487. Trace of synthetic c-di-GMP. (B) Traces of extracts of reaction mixtures containing synthetic c-di-GMP and lysates of PA14 with pMMB67EHGent vector, overexpressing pvrR, or PA2133.