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Characterization of ExsA and of ExsA-dependent promoters required for expression of the Pseudomonas aeruginosa type III secretion system
Journal article   Open access   Peer reviewed

Characterization of ExsA and of ExsA-dependent promoters required for expression of the Pseudomonas aeruginosa type III secretion system

Evan D Brutinel, Christopher A Vakulskas, Keith M Brady and Timothy L Yahr
Molecular microbiology, Vol.68(3), pp.657-671
05/2008
DOI: 10.1111/j.1365-2958.2008.06179.x
PMID: 18373522
url
https://doi.org/10.1111/j.1365-2958.2008.06179.xView
Published (Version of record) Open Access

Abstract

Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is activated by ExsA, a member of the AraC/XylS family of transcriptional regulators. In the present study we examine the DNA-binding properties of ExsA. ExsA was purified as a histidine-tagged fusion protein (ExsA(His)) and found to be monomeric in solution. ExsA(His) specifically bound T3SS promoters with high affinity as determined by electrophoretic mobility shift assays (EMSA). For each promoter tested two distinct ExsA-DNA complexes were detected. Biochemical analyses indicate that the higher-mobility complex consists of a single ExsA(His) molecule bound to DNA while the lower-mobility complex results from the binding of two ExsA(His) molecules. DNase I protection assays demonstrate that the ExsA(His) binding site overlaps the -35 RNA polymerase binding site and extends upstream an additional approximately 34 bp. An alignment of all 10 ExsA-dependent promoters revealed a number of highly conserved nucleotides within the footprinted region. We find that most of the highly conserved nucleotides are required for transcription in vivo; EMSA-binding assays confirm that several of these nucleotides are essential determinants of ExsA(His) binding. The combined data support a model in which two ExsA(His) molecules bind adjacent sites on the promoter to activate T3SS gene transcription.
 Consensus Sequence Recombinant Fusion Proteins - isolation & purification Transcription Factors - chemistry Open Reading Frames Transcriptional Activation Bacterial Proteins - chemistry Molecular Sequence Data Trans-Activators - isolation & purification Trans-Activators - chemistry Recombinant Fusion Proteins - metabolism DNA-Binding Proteins - metabolism Base Sequence Trans-Activators - genetics Electrophoretic Mobility Shift Assay Binding Sites Genes, Reporter Promoter Regions, Genetic Bacterial Proteins - genetics Recombinant Fusion Proteins - chemistry Transcription Factors - genetics DNA-Binding Proteins - genetics DNA-Binding Proteins - isolation & purification DNA-Binding Proteins - chemistry Protein Transport DNA Footprinting Transcription Factors - metabolism Sequence Alignment Pseudomonas aeruginosa - genetics Recombinant Fusion Proteins - genetics Bacterial Proteins - metabolism Trans-Activators - metabolism Transcription Factors - isolation & purification Gene Expression Regulation, Bacterial Bacterial Proteins - isolation & purification

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