The antiactivator FleN uses an allosteric mechanism to regulate σ(54)-dependent expression of flagellar genes in Pseudomonas aeruginosa

Diverse sigma factors associate with the RNA polymerase (RNAP) core enzyme to initiate transcription of specific target genes in bacteria. σ(54)-Mediated transcription uses AAA+ activators that utilize their ATPase activity for transcription initiation. FleQ is a σ(54)-dependent master transcriptional regulator of flagellar genes in Pseudomonas aeruginosa. The ATPase activity of FleQ is regulated via a P-loop ATPase, FleN, through protein-protein interaction. We report a high-resolution crystal structure of the AAA+ domain of FleQ in complex with antiactivator FleN. The data reveal that FleN allosterically prevents ATP binding to FleQ. Furthermore, FleN remodels the region of FleQ essential for engagement with σ(54) for transcription initiation. Disruption of the conserved protein-protein interface, by mutation, shows motility and transcription defects in vivo and multiflagellate phenotype. Our study provides a detailed mechanism used by monoflagellate bacteria to fine-tune the expression of flagellar genes to form and maintain a single flagellum.