PIIR, A TRANSCRIPTIONAL REGULATOR OF PILIATION IN PSEUDOMONAS-AERUGINOSA, BINDS TO A CIS-ACTING SEQUENCE UPSTREAM OF THE PILIN GENE PROMOTER

The PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two-component sensor-regulator family. PilR was overproduced by fusing pilR to the gene for the maltose-binding protein (malE), yielding a MalE-PilR hybrid protein. The plasmid with the malE-pilR fusion, when introduced into a non-piliated pilR mutant strain of P. aeroginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE-PilR protein was purified from Escherichia coli and used in a series of DNA-binding studies. A specific pilin promoter-binding activity of MalE-PilR was observed in a gel retardation assay. Subsequent DNase I footprinting analysis revealed a 40 bp PilR-binding site located at the -120 to -80 region, relative to the transcriptional start site of the pilin gene. This PilR-binding region consists of a nine-base sequence and three consensus sequences of 5'-(N)(4-6)(C)/(G)TGTC-3', in a tandem array in which the first 7-9 bp are bound by the PilR on the noncoding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PHR binds to sequences complementary to the two middle consensus sequences of the non-coding strand. A sequence similar to the NifA recognition site (5'-TGT-(N)(11)-ACA-3') is also found within the PIIR-binding region. Deletion analysis and disruption of the individual consensus PilR-binding sequences by site-directed mutagenesis revealed that all four PilR-binding sites are absolutely required for the PilS/PilR-mediated pilin gene expression. The presence of four PilR-binding repeat sequences suggests that PilR protein may bind co-operatively or as a multimer.