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  • 1
    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.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Transcription of the type IV pilus subunit gene of Pseudomonas aeruginosa is controlled by a two-component signal transduction system. PilS, the histidine kinase, is membrane bound and PilR, its cognate response regulator, is cytoplasmic. The signal that activates PilS is unknown. PilS has three domains: (i) The N-terminus, predicted to form six transmembrane (TM) helices; (ii) a central linker domain; and (iii) the C-terminal transmitter domain containing all the conserved residues of sensor kinases. A translational fusion of the gfp gene (green fluorescent protein) to the 3′ end of pilS was used to determine the position of PilS in the bacterial cell. Epifluorescence microscopy revealed that PilS is retained to the poles of P. aeruginosa but is distributed evenly about the membrane of Escherichia coli. Deletions of the PilS–GFP fusion revealed that the TM domain was sufficient and necessary to bring GFP to the membrane of P. aeruginosa and E. coli but was not sufficient to confine GFP to the poles. Retention to the poles of P. aeruginosa required both the TM and linker domains. Replacement of the PilS TM domain with an E. coli membrane protein, MalG, still allowed polar localization. Therefore, the PilS TM domain positions the linker domain close to the membrane allowing it to interact with the putative polar anchor which is specific to P. aeruginosa.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In Pseudomonas aeruginosa, synthesis of pilin, the major protein subunit of the pili, is regulated by a two-component signal transduction system in which PilS is the sensor kinase. PilS is an inner membrane protein found at the poles of the bacterial cell. It is composed of three domains: an N-terminal hydrophobic domain; a central cytoplasmic linker region; and the C-terminal transmitter region conserved among other sensor kinases. The signal that activates PilS and, consequently, pilin transcription remains unknown. The membrane topology of the hydrophobic domain was determined using the lacZ and phoA gene fusion approach. In this report, we describe a topological model for PilS in which the hydrophobic domain forms six transmembrane helices, whereas the N- and C-termini are cytoplasmic. This topology is very stable, and the cytoplasmic C-terminus cannot cross the inner membrane. We also show that two of the six transmembrane segments are sufficient for membrane anchoring and polar localization of PilS.
    Type of Medium: Electronic Resource
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