Characterization of ExsC and ExsD self-association and heterocomplex formation.
Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced by calcium depletion and is positively regulated by the ExsA transcriptional activator and negatively regulated by the ExsD antiactivator. Under conditions permissive for expression of the T3SS, the negative regulatory activity of ExsD is antagonized by a direct binding interaction with ExsC. In the present study, the ExsC-ExsD binding interaction was characterized. Individually, both ExsC and ExsD form self-associated complexes, as judged by bacterial monohybrid and gel filtration experiments. A mixture of purified ExsC and ExsD readily formed a complex that elutes from gel filtration medium as a single included peak. The calculated molecular weight of the ExsC-ExsD complex is consistent with a complex containing multiple copies of ExsC and ExsD. Isothermic titration calorimetry experiments found formation of the ExsC-ExsD complex to be thermodynamically favorable, with a Kd of approximately 18 nM and a likely binding ratio of 1:1. To identify amino acid residues important for the regulatory activities of ExsC and ExsD, self-association, and complex formation, charged-cluster mutagenesis was performed. Two of the resulting ExsD charged-cluster mutants (DM2 and DM3) demonstrated a hyperrepressive phenotype for expression of the T3SS. By two-hybrid and copurification assays, the DM3 mutant was found to be impaired in its interaction with ExsC. This finding demonstrates that the binding of ExsC to ExsD is required for transcriptional induction of the T3SS under calcium-limiting growth conditions.
Lykken
Guinevere L
GL
Department of Microbiology, University of Iowa, Iowa City, IA 52242-1101, USA.
Chen
GuoZhou
G
Brutinel
Evan D
ED
Chen
Lingling
L
Yahr
Timothy L
TL
D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D017930:Genes, Reporter; D016296:Mutagenesis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D001426:Bacterial Proteins; C470740:ExsD protein, Pseudomonas aeruginosa; D012097:Repressor Proteins; D001616:beta-Galactosidase