OsaR (PA0056) Functions as a Repressor of the Gene fleQ Encoding an Important Motility Regulator in Pseudomonas aeruginosa.

FleQ plays a crucial role in motility and biofilm formation by regulating flagellar and exopolysaccharide biosynthesis in Pseudomonas aeruginosa. It has been reported that the expression of FleQ is transcriptionally downregulated by the virulence factor regulator Vfr. Here, we demonstrated that a LysR-type transcriptional regulator, OsaR, is also capable of binding to the promoter region of fleQ and repressing its transcription. Through gel shift and DNase I footprinting assays, the OsaR binding site was identified and characterized as a dual LysR-type transcriptional regulator box (AT-N11-AT-N7-A-N11-T). Mutation of the A-T palindromic base pairs in the fleQ promoter not only reduced the binding affinity of OsaR in vitro but also derepressed fleQ transcription in vivo. The OsaR binding site was found to cover the Vfr binding site; knockout of osaR or vfr separately exhibited no effect on the transcriptional level of fleQ; however, fleQ expression was repressed by overexpression of osaR or vfr. Furthermore, simultaneously deleting both osaR and vfr resulted in an upregulation of fleQ, but it could be complemented by the expression of either of the two repressors. In summary, our work revealed that OsaR and Vfr function as two transcriptional repressors of fleQ that bind to the same region of fleQ but work separately. IMPORTANCE Pseudomonas aeruginosa is a widespread human pathogen, which accounts for serious infections in the hospital, especially for lung infection in cystic fibrosis and chronic obstructive pulmonary disease patients. P. aeruginosa infection is closely associated with its motility and biofilm formation, which are both under the regulation of the important transcription factor FleQ. However, the upstream regulatory mechanisms of fleQ have not been fully elucidated. Therefore, our research identifying a novel regulator of fleQ as well as new regulatory mechanisms controlling its expression will be significant for better understanding the intricate gene regulatory mechanisms related to P. aeruginosa virulence and infection.