Regulation of flagellar motor switching by c-di-GMP phosphodiesterases in Pseudomonas aeruginosa

Abstract

The second messenger cyclic diguanylate (c-di-GMP) plays a prominent role in regulating flagellum-dependent motility in the single-flagellated pathogenic bacterium Pseudomonas aeruginosa The c-di-GMP-mediated signaling pathways and mechanisms that control flagellar output remain to be fully unveiled. Studying surface-tethered and free-swimming P. aeruginosa PAO1 cells, we found that the overexpression of an exogenous diguanylate cyclase (DGC) raises the global cellular c-di-GMP concentration and thereby inhibits flagellar motor switching and decreases motor speed, reducing swimming speed and reversal frequency, respectively. We noted that the inhibiting effect of c-di-GMP on flagellar motor switching, but not motor speed, is exerted through the c-di-GMP-binding adaptor protein MapZ and associated chemotactic pathways. Among the 22 putative c-di-GMP phosphodiesterases, we found that three of them (DipA, NbdA, and RbdA) can significantly inhibit flagellar motor switching and swimming directional reversal in a MapZ-dependent manner. These results disclose a network of c-di-GMP-signaling proteins that regulate chemotactic responses and flagellar motor switching in P. aeruginosa and establish MapZ as a key signaling hub that integrates inputs from different c-di-GMP-signaling pathways to control flagellar output and bacterial motility. We rationalized these experimental findings by invoking a model that postulates the regulation of flagellar motor switching by subcellular c-di-GMP pools.

Keywords: Pseudomonas aeruginosa (P. aeruginosa); adaptor protein; cell motility; chemotaxis; cyclic di-GMP (c-di-GMP); flagellum; methyltransferase; molecular motor; phosphodiesterases.

Figure 1.

Expression of the exogenous diguanylate cyclase YedQ in P. aeruginosa PAO1 suppresses flagellar motor switching and motor speed. A, illustration of MapZ-mediated and c-di-GMP–dependent chemotaxis pathways and the bacterial cell-tethering assay. The MCPs from the chemoreceptor array detect chemoattractants and repellents and transduce the chemical signals to the autokinase CheA via the scaffolding protein CheW. A decrease in attractant binding activates the autokinase activity of CheA, and an increase in repellent binding suppresses the autokinase activity. When activated, CheA undergoes autophosphorylation and transfers the phosphoryl group to CheY, and the phosphorylated CheY binds to the flagellar switching complex to control flagellar rotation. The ligand-binding activity of MCPs is modulated by CheR and CheB, with CheR methylating specific glutamyl residues in MCPs and CheB removing the methyl groups. CheR and CheB constitutes an adaptation mechanism that constantly resets the MCPs to a prestimulus state as the bacterium travels through a ligand gradient (55). The adapter protein MapZ putatively senses the c-di-GMP pools, which are governed by the DGC and PDE proteins, to control the methylation of MCPs and the autokinase activity of CheA. B, bar graphs showing the percentage of time the bacterial cells spent in clockwise rotation obtained from the cell-tethering assays. The data suggest that motor switching in PAO1/pYedQ cells are greatly suppressed, whereas motor switching in PAO1 and mapZ^R13A/pYedQ cells is not suppressed. C, average motor speed for the PAO1, PAO1/pYedQ, and mapZ^R13A/pYedQ cells. The mean ± S.D. (n = 12–37 cells) data were obtained from two independent experiments. There was a significant effect of YedQ expression on average motor speed (two-way ANOVA, YedQ expression F_1,103 = 109.29, p < 0.0001; *, p < 0.01 with LSD post hoc analysis). There was no significant effect of mapZ^R13A mutation or interaction on average motor speed (two-way ANOVA, mapZ^R13A mutation F_1,103 = 0.89, p = 0.3477; interaction F_1,103 = 0.12, p = 0.7297; p > 0.01 with LSD post hoc analysis). D, relative global cellular c-di-GMP levels of PAO1, PAO1/pYedQ, and mapZ^R13A/pYedQ strains measured using the HPLC method. The data were obtained from two independent experiments. There were significant effect of YedQ expression, mapZ^R13A mutation, and interaction on the relative global cellular c-di-GMP level (two-way ANOVA, YedQ expression F_1,16 = 195.02, p < 0.0001; mapZ^R13A mutation F_1,16 = 20.46, p < 0.001; interaction F_1,16 = 17.96, p < 0.001; *, p < 0.01 with LSD post hoc analysis).

Figure 2.

Expression of the exogenous diguanylate cyclase YedQ in P. aeruginosa PAO1 suppresses swimming reversal and decreases swimming speed. A, swimming trajectories show significantly less frequent directional reversal for the PAO1/pYedQ cells than the other two strains. Scale bar, 5 μm. B, average swimming speed of PAO1 and mutant cells to show that expression of YedQ decreases motor speed in a MapZ-independent manner. The data are means ± S.D. (n = 33–37 cells) from two independent experiments. There was a significant effect of YedQ expression on average swimming speed (two-way ANOVA, F_1,132 = 328.05, p < 0.0001; *, p < 0.01 with LSD post hoc analysis). There was no significant effect of mapZ^R13A mutation or interaction on average swimming speed (two-way ANOVA, mapZ^R13A mutation F_1,132 = 2.61, p = 0.1086; interaction F_1,132 = 3.13, p = 0.0792; p > 0.01 with LSD post hoc analysis). C, swimming persistence of PAO1 and the mutant cells to show YedQ increases the persistence of runs in a MapZ-dependent manner. The calculation of the swimming persistence is described under “Materials and methods.” The data are means ± S.D. (n = 32–33 cells) from two independent experiments. There were significant effects of YedQ expression, mapZ^R13A mutation, and interaction on swimming persistence (two-way ANOVA, YedQ expression F_1,127 = 9.34, p < 0.01; mapZ^R13A mutation F_1,127 = 27.22, p < 0.0001; interaction F_1,127 = 132.9, p < 0.0001; *, p < 0.01 with LSD post hoc analysis).

Figure 3.

Identification of c-di-GMP PDEs that control flagellar motor switching by screening 22 transposon mutants. A and B, bar graphs generated from the data of cell-tethering assays showing the percentage of time spent in CW rotation for PAO1 and the three pde::Tn strains that showed severely repressed motor switching. C, bar graphs showing the percentage of time spent in CW rotation for the three complimentary mutants that exhibited a normal motor switching pattern. The data are means ± S.D. (n = 47–50 cells) from two independent experiments. D, domain organization of RbdA, NbdA, and DipA. TM, transmembrane motif. PAS/PAC, GAF, and MHYT are the putative sensory domains. The C-terminal EAL domains are the catalytically active PDE domains responsible for degrading c-di-GMP.

Figure 4.

Inactivation of the PDE domain of RbdA, NbdA, and DipA inhibits flagellar motor switching in a MapZ-dependent manner. A, crystal structure of the EAL domain of RbdA showing the position and crucial role of the ELL motif in binding the metal ions and c-di-GMP (56). B, bar graphs showing the percentage of time spent in CW rotation for the three PDE inactive mutants and the three double mutants that harbor inactive PDE and defective MapZ. The data suggest that MapZ is indispensable for the inhibition of motor switching. The data are means ± S.D. (n = 29 to 34 cells) from two independent experiments.

Figure 5.

Inactivation of the PDE domain of RbdA, NbdA, and DipA suppresses directional reversal in swimming in a MapZ-dependent manner. A, swimming trajectories for the three PDE inactive mutants. Scale bar, 5 μm. B, swimming trajectories for the three double mutants that harbor a catalytically inactive PDE and a defective MapZ. Scale bar, 5 μm. The data suggest that MapZ is indispensable for the suppression of swimming reversal. C, comparison of directional swimming persistence. The calculation of the swimming persistence is described under “Materials and methods.” The data are presented as means ± S.D. (n = 26–34 cells) from two independent experiments. There were significant effects of YedQ expression, mapZ^R13A mutation, and interaction on swimming persistence (two-way ANOVA, YedQ expression F_3,238 = 20.45, p < 0.0001; mapZ^R13A mutation F_1,238 = 62.41, p < 0.0001; interaction F_3,238 = 27.34, p < 0.0001; *, p < 0.01 with LSD post hoc analysis). D, comparison of average swimming speed. The data are presented as means ± S.D. (n = 26–34 cells) from two independent experiments.

Figure 6.

A model for the regulation of chemotaxis and flagellar motor switching by c-di-GMP and the three PDEs in P. aeruginosa. The model depicts the bacterial chemoreceptor arrays as highly organized sensory patches composed of thousands of transmembrane MCP proteins. DipA, RbdA, and NbdA are postulated to be situated in proximity to the MCPs-constituted chemoreceptor array and interacting with different MCPs. The gray sphere represents the sphere of influence of the three PDEs, which allow them to control separate c-di-GMP pools, and thus the methylation level of different sets of MCPs to control distinct chemotactic pathways.