PilR, 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. aeruginosa, 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)4-6C/GTGTC-3', in a tandem array in which the first 7-9 bp are bound by the PilR on the non-coding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR 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 PilR-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Jin
S
S
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Ishimoto
K S
KS
Lory
S
S
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors; C074774:pilR protein, Pseudomonas aeruginosa; D036781:Fimbriae Proteins