PMC

WspA–YFP localization in cells of wild-type and various mutant strains grown on LB agar.
A. Phase-contrast images are shown on left and fluorescent images on the right. The marker bar is 1 μm.
B. Colony morphologies of strains.

WspR–YFP and WspA–CFP sometimes colocalize in ΔwspF mutant cells. WspR–YFP is coloured green. WspA-CFP is coloured red and the merged image of the two is shown in the middle. A phase-contrast image of the cells is shown on the left. Cells were grown on LB agar. The marker bar is 1 μm.

WspR–YFP and WspA–YFP fusion proteins are functional. The introduction of the fusion constructs into the chromosome of a ΔwspF strain by homologous recombination to replace wild-type wspR or wild-type wspA does not alter the (A) colony morphologies or (B) motility in soft agar swim plates, of the strains. This indicates that the Wsp signal transduction pathway has remained intact.

Phosphorylation is required for WspR–YFP cluster formation.
A. The localization of WspR–YFP in ΔwspF wspR–yfp or ΔwspF ΔwspA wspR–yfp mutant cells and the localization of various WspR–YFP mutant proteins in ΔwspF cells. Phase-contrast images of cells are shown on the left. The marker bar is 1 μm.
B. Colony morphologies of the strains.
C. WspR–YFP cluster formation is restored in a ΔwspF ΔwspA wspR–yfp mutant strain by providing WspA in trans.
D. The colony morphologies of the ΔwspF ΔwspA wspR–yfp strain complemented with empty vector or wspA.

Time-lapse fluorescence and phase-contrast images of surface-grown wild-type cells expressing the WspR–YFP fusion protein. Images were acquired every 30 s. Individual cells were followed over a 150 s time period in (A), (B) and (C). Cartoons depicting the movement and change in fluorescence intensity of the spots in each cell are shown below each panel. The marker bar is 1 μm.

WspR–YFP localization in (A) wild-type cells grown in liquid cultures. The phase-contrast (left) and fluorescence (right) images of cells expressing wspR–yfp are shown.
B. Single cell measurements of fluorescence signal intensities expressed as the ratio of maximum to average signal intensities plotted against the area of the respective single cells in wild-type cells expressing WspR–YFP.
C. ΔwspF mutant cells grown in liquid cultures and expressing WspR–YFP.
D. Single cell measurements of fluorescence signal intensities in ΔwspF mutant cells expressing WspR–YFP. Single cell fluorescence data were collected from three independent experiments.

Surface growth stimulates WspR–YFP cluster formation in wild-type cells.
WspR–YFP localization in (A) wild-type cells grown on LB agar. The phase-contrast (left) and fluorescence (right) images of cells expressing wspR–yfp are shown.
B. Single cell measurements of fluorescence signal intensities expressed as the ratio of maximum to average signal intensities plotted against the area of the respective single cells in wild-type cells expressing WspR–YFP.
C. ΔwspF mutant cells grown on agar and expressing WspR–YFP.
D. Single cell measurements of fluorescence signal intensities in ΔwspF mutant cells expressing WspR–YFP. Single cell fluorescence data were collected from three independent experiments.

Characteristics of the Wsp signal transduction system.
A. Organization of the genes encoding the Wsp system.
B. The Wsp proteins are predicted to be a membrane-bound methyl-accepting chemotaxis protein (WspA), a CheR-like methyltransferase (WspC), a CheB-like methylesterase (WspF), two CheW homologues (WspB and WspD) that are predicted to serve as linkers between WspA and a hybrid histidine kinase response regulator (WspE). The response regulator protein, WspR, has a GGDEF domain and catalyses the synthesis of c-di-GMP when phosphorylated. As described in the text, a wspF mutation is predicted to lock the Wsp system into a configuration where WspR is constantly phosphorylated and thus constantly producing c-di-GMP.
C. The physical organization of homologous Che proteins (encoded by PA1456–1459, PA1464 and PA3349) and (D) colony morphologies of P. aeruginosa PAO1 wild-type and wsp deletion strains. wspF mutants have high levels of intracellular c-di-GMP relative to wild-type cells ().