Self-association is required for occupation of adjacent binding sites in Pseudomonas aeruginosa type III secretion system promoters

Anne E Marsden; Florian D Schubot; Timothy L Yahr

doi:10.1128/JB.01969-14

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Self-association is required for occupation of adjacent binding sites in Pseudomonas aeruginosa type III secretion system promoters

Journal article

Open access

Peer reviewed

Self-association is required for occupation of adjacent binding sites in Pseudomonas aeruginosa type III secretion system promoters

Anne E Marsden, Florian D Schubot and Timothy L Yahr

Journal of bacteriology, Vol.196(20), pp.3546-3555

10/2014

DOI: 10.1128/JB.01969-14

PMCID: PMC4187695

PMID: 25070741

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Abstract

ExsA is a member of the AraC/XylS family of transcriptional regulators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). All P. aeruginosa T3SS promoters contain two adjacent binding sites for monomeric ExsA. The amino-terminal domain of ExsA (NTD) is thought to mediate interactions between the ExsA monomers bound to each site. Threading the NTD onto the AraC backbone revealed an α-helix that likely serves as the primary determinant for dimerization. In this study, we performed alanine scanning mutagenesis of the ExsA α-helix (residues 136 to 152) to identify determinants required for self-association. Residues L137, C139, L140, K141, and L148 exhibited self-association defects and were required for maximal activation by ExsA. Disruption of self-association resulted in decreased binding to T3SS promoters, particularly loss of binding by the second ExsA monomer. Removing the NTD or increasing the space between the ExsA-binding sites restored the ability of the second ExsA monomer to bind the PexsC promoter. This finding indicated that, in the absence of self-association, the NTD prevents binding by a second monomer. Similar findings were seen with the PexoT promoter; however, binding of the second ExsA monomer in the absence of self-association also required the presence of a high-affinity site 2. Based on these data, ExsA self-association is necessary to overcome inhibition by the NTD and to compensate for low-affinity binding sites, thereby allowing for full occupation and activation of ExsA-dependent promoters. Therefore, ExsA self-association is indispensable and provides an attractive target for antivirulence therapies.

Bacterial Proteins - chemistry

Bacterial Proteins - genetics

Models, Molecular

Recombinant Fusion Proteins - chemistry

Trans-Activators - chemistry

Recombinant Fusion Proteins - metabolism

Gene Expression Regulation, Bacterial - physiology

Protein Transport

Pseudomonas aeruginosa - genetics

Pseudomonas aeruginosa - metabolism

Trans-Activators - genetics

Protein Binding

Recombinant Fusion Proteins - genetics

Bacterial Proteins - metabolism

Protein Conformation

Trans-Activators - metabolism

Details

Title: Subtitle: Self-association is required for occupation of adjacent binding sites in Pseudomonas aeruginosa type III secretion system promoters
Creators: Anne E Marsden - Department of Microbiology, University of Iowa, Iowa City, Iowa, USA
Florian D Schubot - Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA
Timothy L Yahr - Department of Microbiology, University of Iowa, Iowa City, Iowa, USA tim-yahr@uiowa.edu
Resource Type: Journal article
Publication Details: Journal of bacteriology, Vol.196(20), pp.3546-3555
Publisher: United States
DOI: 10.1128/JB.01969-14
PMID: 25070741
PMCID: PMC4187695
ISSN: 0021-9193
eISSN: 1098-5530
Grant note: T32 AI007511 / NIAID NIH HHS R01-AI055042 / NIAID NIH HHS R01 AI055042 / NIAID NIH HHS T32 AI07511 / NIAID NIH HHS
Language: English
Date published: 10/2014
Academic Unit: Microbiology and Immunology
Record Identifier: 9984001211502771

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