Premium
Characterization of ExsA and of ExsA‐dependent promoters required for expression of the Pseudomonas aeruginosa type III secretion system
Author(s) -
Brutinel Evan D.,
Vakulskas Christopher A.,
Brady Keith M.,
Yahr Timothy L.
Publication year - 2008
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2008.06179.x
Subject(s) - biology , electrophoretic mobility shift assay , promoter , microbiology and biotechnology , transcription (linguistics) , dna , binding site , transcription factor , gene , genetics , gene expression , linguistics , philosophy
Summary 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 ∼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.