Anti-activator ExsD forms a 1:1 complex with ExsA to inhibit transcription of type III secretion operons.
The ExsA protein is a Pseudomonas aeruginosa transcriptional regulator of the AraC/XylS family that is responsible for activating the type III secretion system operons upon host cell contact. Its activity is known to be controlled in vivo through interaction with its negative regulator ExsD. Using a heterologous expression system, we demonstrated that ExsD is sufficient to inhibit the transcriptional activity of ExsA. Gel shift assays with ExsA- and ExsD-containing cytosolic extracts revealed that ExsD does not block DNA target sites but affects the DNA binding activity of the transcriptional activator. The ExsA-ExsD complex was purified after coproduction of the two partners in Escherichia coli. Size exclusion chromatography and ultracentrifugation analysis revealed a homogeneous complex with a 1:1 ratio. When in interaction with ExsD, ExsA is not able to bind to its specific target any longer, as evidenced by gel shift assays. Size exclusion chromatography further showed a partial dissociation of the complex in the presence of a specific DNA sequence. A model of the molecular inhibitory role of ExsD toward ExsA is proposed, in which, under noninducing conditions, the anti-activator ExsD sequesters ExsA and hinders its binding to DNA sites, preventing the transcription of type III secretion genes.
Commissariat à l'Energie Atomique, Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Biochimie et de Biophysique des Systèmes Intégrés, F-38054 Grenoble.
Julie
J
Thibault
Eric
E
Faudry
Christine
C
Ebel
Ina
I
Attree
Sylvie
S
Elsen
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000382:microbiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D009876:Operon; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; C095093:ExsA protein, Pseudomonas aeruginosa; C470740:ExsD protein, Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D001616:beta-Galactosidase