Fig. 4.

Obstruction of pilus retraction stimulates c-di-GMP synthesis by altering the activity of developmental regulators. (A) Quantification of intracellular c-di-GMP concentrations by mass spectrometry of populations of wild-type and Pil-cys strains with PEG-mal treatment. Bar graph shows the mean ± SEM of three independent biological replicates. Statistical comparisons were made using Sidak’s multiple-comparisons test. *P < 0.05. (B) Percent of piliated cells with delocalized PleC at each time point. Error bars indicate the mean ± SEM of three independent biological replicates (n = at least 30 cells per replicate per time point). (C) Percent of piliated cells with localized DivJ at each time point. Error bars indicate the mean ± SEM of four independent biological replicates (n = at least 30 cells per replicate per time point). (D) Representative microscopy images of cells from data shown in B. The green arrowhead represents delocalized PleC at the piliated pole. (E) Representative microscopy images of cells at the 25-min time point of data shown in C. Blocked pili in Pil-cys PEG-mal–treated samples appear as puncta due to shearing of filaments. The blue arrowhead indicates DivJ localization in a piliated cell. White arrowheads indicate piliated cells. (Scale bars, 2 μm.) Maleimide-positive cells were labeled as piliated as the fluorophore entered the cells via pilus retraction.