FIG 3.

Expression of PA5487 and its effect on intracellular c-di-GMP. (A) c-di-GMP concentration measurement by fluorescence determination. The fluorescence from P. aeruginosa PAO1 and PA5487 mutant strains was measured, and the relative fluorescence unit values are arbitrary fluorescence intensity units corrected for cell density. The results are averages of triplicate measurements on test tube cultures in stationary growth phase (12 h). (B) c-di-GMP concentration measurement by HPLC-MS/MS determination. Bacterial cultures of PAO1 and the PA5487 mutant were collected from the stationary growth phase (12 h), and the c-di-GMP concentration was detected using the HPLC-MS/MS method as described in the text. Results are averages of triplicate measurements on test tube cultures. (C) The expression of PA5487 is consistent with its growth stage. PAO1::mini-CTX-dgcH-lacZ was used to detect the expression of PA5487, and control strain PAO1::mini-CTX-lacZ was used to calibrate the background expression. A standard β-galactosidase assay was performed in LB broth for 24 h. We also tested the expression in Jensen’s medium, which showed the same result as that using obtained LB medium (data not shown).