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PilR, a transcriptional regulator of piliation in Pseudomonas aeruginosa, binds to a cis-acting sequence upstream of the pilin gene promoter (1994)

Jin, Shouguang ; Ishimoto, Karyn S. ; Lory, Stephen

Oxford, UK : Blackwell Publishing Ltd

Molecular microbiology 14 (1994), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: 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 gef retardation assay. Subsequent DNase I footprinting analysis revealed a 40bp 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-Goding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds of 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 PilRbinding 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.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.1994.tb01338.x

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Identification and characterization of PilS, an essential regulator of pilin expression in Pseudomonas aeruginosa (1994)

Boyd, Jessica M. ; Koga, Tetsuro ; Lory, Stephen

Springer

Molecular genetics and genomics 243 (1994), S. 565-574

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ISSN: 1617-4623

Keywords: Pseudomonas aeruginosa ; Two-component regulatory systems ; Pili ; pilS ; pilR

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Expression of the pilin gene, pilA, of Pseudomonas aeruginosa requires the alternative sigma factor, σ54, and also two other transcriptional regulators encoded by the pilS and pilR genes. These two linked genes, which have been identified by transposon insertion mutagenesis, share significant amino acid sequence homology with members of the two-component family of regulators. The transcriptional regulator, PilR, has been described previously. PilS, a 37285 Dalton protein, shares significant homology with the protein kinase sensors of the two-component regulatory family. PilS, however, has no hydrophobic domains which might be membrane-spanning alpha-helices, suggesting that PilS is a cytoplasmic protein. Characterization of the pilS gene revealed that when overexpressed in Escherichia coli by the bacteriophage T7 promoter it specifies a protein of approximately 40000 daltons, corresponding to the molecular weight of Pi1S predicted from the deduced amino acid sequence. Deletion analysis of the pilS promoter fused to a promoterless lacZ gene further showed that a significant region upstream of pilS is essential for expression of pilS and pilR, suggesting a need for transcriptional activation. The pilA promoter can be activated in E. coli but only when PilR and σ54 are present. This work suggests that the Pi1S activation signal is received in the bacterial cytoplasm, and that the mechanism of Pi1S/PiIR-mediated signal transduction resulting in activation of the pilin gene promoter is likely to be similar to that of other two-component systems.