title: TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa.
abstract: Type VI secretion systems (T6SSs) contribute to interactions of bacterial pathogens and symbionts with their hosts. Previously, we showed that Pseudomonas aeruginosa T6S is posttranslationally activated upon phosphorylation of Fha1, an FHA domain protein, by PpkA, a membrane-spanning threonine kinase. Herein, additional structural, enzymatic and genetic requirements for PpkA-catalysed T6SS activation are identified. We found that PpkA plays an essential structural role in the T6SS, and that this role is intimately linked to its ability to promote secretion and phosphorylate Fha1. Protein localization and protein-protein interaction studies show that a complex containing Fha1 and the T6S ATPase, ClpV1 is recruited to the T6S apparatus in a phosphorylation-dependent manner. The mechanism of PpkA activation was also investigated. We identified critical PpkA autophosphorylation sites and showed that small molecule-induced dimerization of the extracellular domains of PpkA is sufficient to activate the T6SS. Finally, we discovered TagR, a component of the T6S posttranslational regulatory pathway that functions upstream of PpkA to promote kinase activity. We present a model whereby an unknown cue causes dimerization of the extracellular domains of PpkA, leading to its autophosphorylation, recruitment of the Fha1-ClpV1 complex, phosphorylation of Fha1, and T6SS activation. Our findings should facilitate approaches for identifying physiological activators of T6S.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA.
authors.forename: FoSheng
authors.initials: F
authors.lastname: Hsu
authors.forename: Sandra
authors.initials: S
authors.lastname: Schwarz
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Mougous
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C116685:ppkA protein, Pseudomonas aeruginosa; D017346:Protein Serine-Threonine Kinases