DUAL FUNCTION OF PILS DURING TRANSCRIPTIONAL ACTIVATION OF THE PSEUDOMONAS-AERUGINOSA PILIN SUBUNIT GENE

The polar pill of Pseudomonas aeruginosa are composed of subunits enco ded by the pilA gene. Expression of pilA requires the alternative sigm a factor RpoN and a pair of regulatory elements, PilS and PilR. These two proteins are members of the two-component regulatory family, in wh ich PilS is the sensory component and PilR is the response regulator. By using expression and localization analyses, in this work we show th at PilS is synthesized as a 59-kDa polypeptide located in the P. aerug inosa cytoplasmic membrane. When the pilS gene is expressed in Escheri chia 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 mutatio n in the histidine residue essential for kinase activity resulted in a protein unable to activate transcription, yet when overexpressed in t he presence of the wild-type PilS protein, this protein still represse d pilin synthesis. A shorter form of PilS, lacking its transmembrane s egments, was active and fully capable of stimulating pilA transcriptio n but when overexpressed did not show the inhibitory effect on pilin e xpression seen with full-length PilS. We also show that overexpression of pilR can activate transcription of pilA even in the absence of Pil S. On the basis of our studies, we propose a complex mechanism of regu lation of PilS function, involving other cellular factors that control PilS and its activities during the phosphorelay mechanism of signal t ransduction.