Fig 6.

dipA encodes a membrane-associated phosphodiesterase. (A) Immunoblot analysis of subcellular cell fractions (15 μg) demonstrating that V5/6×His-tagged DipA is located in the cytoplasm and associated with the membrane. Subcellular localization was achieved by ultracentrifugation. (B) V5/6×His-tagged DipA purified in the presence of the detergent Tween 20 is partly soluble, as indicated by a portion of the protein precipitating after ultracentrifugation and not remaining detectable in the supernatant. A total of 28 μg of purified DipA and 10 μg of the resulting supernatant fraction (Sup) and precipitate (pellet) were loaded onto the SDS gel. (C) DipA is a phosphodiesterase, as determined using purified V5/6×His-tagged DipA and bis(p-nitrophenyl) phosphate as a substrate. c-di-GMP concentrations ranging from 4 to 100 pmol were added to the enzyme assay. cAMP and cGMP were added at a final concentration of 100 μM. *, significantly different from DipA (P < 0.05). (D) Degradation of c-di-GMP by purified DipA and DipA-NoGAF (100 μg each) over a period of 240 min, as determined by HPLC analysis. cAMP was added at a final concentration of 1 μM. *, significantly different from DipA (P < 0.05). (E) Effect of overexpression of intact and truncated dipA and mutated dipA on aggregative behavior of PAO1/pJN-PA4843, as indicated by turbidity of liquid growth culture. *, significantly different from PAO1 harboring empty vector (P < 0.05). Aggregative behavior was assessed by turbidity. Following 3 h of growth under planktonic conditions at 37°C in LB, arabinose was added to a final concentration of 1%. Following 3.5 h of continued incubation, the bacterial suspensions were allowed to settle at room temperature for 10 min before the absorbance of the suspension was measured at 600 nm. Cultures not induced by arabinose were used as controls. (F) Effect of overexpression of intact and truncated dipA and mutated dipA on swarming motility of PAO1/pJN-PA4843 following 24 h of growth on M8 agar. *, significantly different from PAO1/pJN105 (P < 0.05). All experiments were carried out at least 5 times. Error bars indicate standard deviations. C, cytoplasmic fraction; M, membrane fraction; sup, supernatant after ultracentrifugation; pellet, pellet after ultracentrifugation; TCE, total cell extract; purified, DipA was purified using Ni-NTA affinity chromatography; No DipA, PAO1/pMJT1 cell extract not expressing any His-tagged DipA was purified using Ni-NTA affinity chromatography, and the respective eluates were used as controls; N.D., not detected. Significance was determined by ANOVA and SigmaStat.