Functional domains of ExsA, the transcriptional activator of the Pseudomonas aeruginosa type III secretion system.
The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to evade phagocytosis and damage eukaryotic cells. Transcription of the T3SS regulon is controlled by ExsA, a member of the AraC/XylS family of transcriptional regulators. These family members generally consist of an approximately 100-amino acid carboxy-terminal domain (CTD) with two helix-turn-helix DNA binding motifs and an approximately 200-amino acid amino-terminal domain (NTD) with known functions including oligomerization and ligand binding. In the present study, we show that the CTD of ExsA binds to ExsA-dependent promoters in vitro and activates transcription from ExsA-dependent promoters both in vitro and in vivo. Despite possessing these activities, the CTD lacks the cooperative binding properties observed for full-length ExsA at the P(exsC) promoter. In addition, the CTD is unaffected by the negative regulatory activity of ExsD, an inhibitor of ExsA activity. Binding studies confirm that ExsD interacts directly with the NTD of ExsA. Our data are consistent with a model in which a single ExsA molecule first binds to a high-affinity site on the P(exsC) promoter. Protein-protein interactions mediated by the NTD then recruit an additional ExsA molecule to a second site on the promoter to form a complex capable of stimulating wild-type levels of transcription. These findings provide important insight into the mechanisms of transcriptional activation by ExsA and inhibition of ExsA activity by ExsD.
University of Iowa, Department of Microbiology, Iowa City, IA 52242-1101, USA.
Evan D
ED
Brutinel
Christopher A
CA
Vakulskas
Timothy L
TL
Yahr
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C095093:ExsA protein, Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators