Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa.

Publication Type:

Journal Article


Proc Natl Acad Sci U S A, Volume 113, Issue 2, p.E209-18 (2016)


Amino Acid Motifs, Amino Acid Sequence, Bacterial Proteins, Base Sequence, Binding Sites, Biofilms, Calorimetry, Conserved Sequence, Cross-Linking Reagents, Crystallography, X-Ray, Cyclic GMP, DNA, Bacterial, Gene Expression Regulation, Bacterial, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutant Proteins, Promoter Regions, Genetic, Protein Multimerization, Protein Stability, Protein Structure, Quaternary, Protein Structure, Tertiary, Pseudomonas aeruginosa, Sequence Alignment, Solutions, Temperature, Trans-Activators, Transcription, Genetic


<p>Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.</p>