A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels

Abstract

Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.

Fig. 1.

Organization of genes encoding the Wsp chemosensory system in P. aeruginosa. Gene names are shown above, and triangles indicating inframe deletion mutations that were constructed are shown below. The genes are predicted to encode: a membrane-bound methyl-accepting chemotaxis protein (MCP) (wspA, PA3708), a cheR-like methyltransferase (wspC, PA3706), two cheW homologs (wspB, PA3707; wspD, PA3705), a hybrid histidine kinase-response regulator (wspE, PA3704), a cheB-like methylesterase (wspF, PA3703), and a response regulator with a GGDEF domain (wspR, PA3702).

Fig. 2.

Phenotypes associated with loss of wspF. (A) Colony morphologies of PAO1 and a mutant lacking wspF. Photographs were taken after 5 days of growth on tryptone agar containing 40 mg of Congo red and 10 mg of Coomassie brilliant blue per liter of medium. (B) Attachment of P. aeruginosa PAO1 and wsp deletion mutants to microtiter dish wells. Results shown are the mean of at least three independent experiments. Error bars represent the standard deviations.

Fig. 3.

Loss of wspF results in enhanced biofilm formation. Biofilm formation by PAO1 and a wspF mutant. Images were obtained by using a ×60 objective after 24- and 48-h growth in continuous flow chambers. Each square on the grid is 20 μm per side.

Fig. 4.

Loss of wspF results in increased c-diGMP levels. Two-dimensional TLC analysis of acid extracts from ³²P-labeled cells. (Left) PAO1. (Right) wspF mutant. The spot corresponding to published R_f values for c-diGMP is indicated by the arrow in Right.

Fig. 5.

WspR generates c-diGMP from GTP. Time course of formation of c-diGMP by WspR. Arrows on left indicate the relative migration of GTP and c-diGMP. The GTP-only control is shown in the left lane. Reactions with WspR are shown at left, and reactions with acetyl-phosphate-treated WspR are shown at right.

Fig. 6.

Degradation of c-diGMP reverses the phenotypes of a wspF mutant. (A) Two-dimensional TLC analysis of a wspF mutant expressing PA2133 from a plasmid. The location of c-diGMP is indicated with an arrow. (B) Attachment of strains expressing PA2133 from a plasmid (pJN2133) to microtiter dish wells. The background absorbance obtained from uninnoculated control wells is shown at right. pJN105 is the control vector. Absorbance obtained is an average of at least three independent trials. The error bars represent the standard deviation.

Fig. 7.

Degradation of c-diGMP inhibits biofilm formation by PAO1. Biofilm formation by strain PAO1 expressing PA2133 from a plasmid (pJN2133) compared to PAO1 with a control vector. Biofilms were visualized by staining with propidium iodide (4 μM) after 72-h growth.