A Hanks‐type bacterial kinase, <scp>PknS</scp> , directly phosphorylates the alternative sigma factor <scp>EcfK</scp> to promote resistance to protist predation

Serine/threonine (Ser/Thr) kinases of the Hanks-type family are widespread in bacteria, playing key roles in signal transduction. The transmembrane Ser/Thr kinase PknS (XAC4127) from the phytopathogenic bacterium Xanthomonas citri is required for the expression of a type VI secretion system, which confers resistance to predation by the soil amoeba Dictyostelium discoideum. PknS exerts its function via activation of the cognate ECF-type alternative sigma factor EcfK, ultimately triggering the expression of type VI secretion system (T6SS) genes. In this study, we characterize PknS, demonstrating its ability to undergo autophosphorylation both in vitro and within X. citri cells. Structural analysis of the PknS kinase domain revealed the conservation of the canonical fold characteristic of Hanks-type kinases. PknS directly phosphorylates EcfK at five Ser/Thr residues located in two distinct regions of the sigma factor: the conserved σ₂ domain (residue T51) and a nonconserved linker connecting domains σ₂ and σ₄ (residues T104, T106, S108, and S110). The conserved residue T51, previously shown to be essential for sigma factor activity in an EcfK homolog, corresponds to a site that directly interacts with the RNA polymerase β' subunit. Site-directed mutagenesis analyses further revealed that the conserved residue T106 is also critical for EcfK function. Structural studies indicated that, in addition to T51, phosphorylation at T106 activates EcfK by promoting its interaction with a positively charged pocket within the RNA polymerase β' subunit. Collectively, our findings describe a previously unknown signal transduction pathway involving a Hanks-type kinase and a sigma factor, providing new insights into the mechanisms of sigma factor activation via phosphorylation in bacteria.