Dual function of PilS during transcriptional activation of the Pseudomonas aeruginosa pilin subunit gene

The polar pili of Pseudomonas aeruginosa are composed of subunits encoded by the pilA gene. Expression of pilA requires the alternative sigma factor RpoN and a pair of regulatory elements, PilS and PilR. These two proteins are members of the two-component regulatory family, in which PilS is the sensory component and PilR is the response regulator. By using expression and localization analyses, in this work we show that PilS is synthesized as a 59-kDa polypeptide located in the P. aeruginosa cytoplasmic membrane. When the pilS gene is expressed in Escherichia coli, aberrant translational initiation results in a smaller, 40-kDa polypeptide. Unexpectedly, overexpression of pilS in P. aeruginosa results in decreased transcription of the pilA gene. Moreover, fully functional PilS was not required for this inhibitory effect. A mutation in the histidine residue essential for kinase activity resulted in a protein unable to activate transcription, yet when overexpressed in the presence of the wild-type PilS protein, this protein still repressed pilin synthesis. A shorter form of PilS, lacking its transmembrane segments, was active and fully capable of stimulating pilA transcription but when overexpressed did not show the inhibitory effect on pilin expression seen with full-length PilS. We also show that overexpression of pilR can activate transcription of pilA even in the absence of PilS. On the basis of our studies, we propose a complex mechanism of regulation of PilS function, involving other cellular factors that control PilS and its activities during the phosphorelay mechanism of signal transduction.