Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ

Abstract

The transcription factor FleQ is a bacterial AAA+ ATPase enhancer-binding protein that is the master activator of flagella gene expression in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Homologs of FleQ are present in all Pseudomonas species and in many polarly flagellated gamma proteobacteria. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls the transition between planktonic and biofilm modes of growth in bacteria in response to diverse environmental signals. C-di-GMP binds to FleQ to dampen its activity, causing down-regulation of flagella gene expression. This action is potentiated in the simultaneous presence of another protein, FleN. We explored the effect of c-di-GMP and FleN on the ATPase activity of FleQ and found that a relatively low concentration of c-di-GMP competitively inhibited FleQ ATPase activity, suggesting that c-di-GMP competes with ATP for binding to the Walker A motif of FleQ. Confirming this, a FleQ Walker A motif mutant failed to bind c-di-GMP. FleN, whose gene is regulated by FleQ, also inhibited FleQ ATPase activity, and FleQ ATPase activity was much more inhibited by c-di-GMP in the presence of FleN than in its absence. These results indicate that FleN and c-di-GMP cooperate to inhibit FleQ activity and, by extension, flagella synthesis in P. aeruginosa. The Walker A motif of FleQ is perfectly conserved, opening up the possibility that other AAA+ ATPases may respond to c-di-GMP.

Keywords: AAA+ protein; competitive inhibition; transcription factor; σ54.

Fig. 1.

FleQ has a cooperative ATPase activity. (A) The specific ATPase activity of FleQ (1 µM) as a function of the concentration of ATP. (B) Initial velocity of the ATPase activities of FleQ WT alone or in the presence of 100 μM ADP, 0.25 μM fleS promoter DNA fragment, and ATPase activities of FleQ variant proteins. Expressed as percentage of the activity obtained with FleQ WT. The ATPase activity was assayed with 1 µM protein and 1 mM ATP. The error bars represent SDs.

Fig. 2.

FleQ forms dimers, tetramers, and hexamers. (Left) BN-PAGE of 50 μg of FleQ mixed with 10 mM AMP, ADP, ATP, ATPγS, or GTP or 1 mM c-di-GMP as indicated at the top of the figure. (Right) The FleQ protein was incubated with 1 mM ATP, and 1 mM ATP was also added to the buffer.

Fig. 3.

FleN inhibits FleQ ATPase activity. (A) The ATPase activity of FleN at different concentrations was assayed in the presence of 1 mM ATP and in the presence or absence of 500 μM ATPγS. (B) The ATPase activity of different concentrations (from 0.1 to 10 μM) of FleN was assayed in the absence or in the presence of 1 μM FleQ and in the presence of 1 mM ATP. The initial velocity of FleN or FleN plus FleQ is plotted against FleN concentration.

Fig. 4.

C-di-GMP inhibits FleQ ATPase activity. (A) The ATPase activity of 1 μM FleQ protein, incubated with different c-di-GMP concentrations from 1 to 200 μM, was assayed with 1 mM ATP. (B) The ATPase activity of 1 μM FleQ, preincubated with 25, 37.5, or 50 μM c-di-GMP, and assayed at different ATP concentrations. The activity curves were analyzed according to the Hill equation (Table 1).

Fig. 5.

Effect of c-di-GMP on the trypsin digestion profile of FleQ WT and mutant proteins. FleQ proteins were incubated in the presence or absence of 500 μM c-di-GMP and subjected to proteolysis with trypsin. Incubation times (min) are indicated. Digestion products were separated by SDS/PAGE, transferred to a nitrocellulose membrane, and visualized with anti-FleQ antiserum.

Fig. 6.

Binding of c-di-GMP to FleQ analyzed by ITC. WT FleQ (A), FleQ_K180A (B), and buffer (C) were titrated with c-di-GMP. The amount of power (µcal/s) needed to maintain a constant temperature during the titration reflects the amount of heat released on c-di-GMP binding. This is integrated over time to give a specific enthalpy vs. ligand:protein molar ratio plot (A, Lower). Heat from the dilution of c-di-GMP was subtracted before data analysis. The thin line represents the fit of the integrated data to a single-site binding model and gives the following parameters: n = 2.35 ± 0.04, K = 1.39E5 ± 1.32E4 M⁻¹, and ΔH = −7,652 ± 209 cal/mol.

Fig. 7.

FleQ ATPase activity is inhibited by FleN and c-di-GMP. The ATPase activity of 1 μM FleQ, 1 μM FleN, or 1 μM FleQ plus 1 μM FleN was measured at different concentrations of c-di-GMP (from 1 to 100 μM) and in the presence of 2 mM ATP. The initial velocity of FleN, FleQ, or FleN plus FleQ ATPase activities are plotted against the log of c-di-GMP concentration, allowing determination of the IC₅₀.