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

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.

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