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Dimerization and DNA binding of the Salmonella enterica PhoP response regulator are phosphorylation independent

Gregory De Crescenzo^2,

¹ Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada H3A 2B4
² Protein–Protein Interaction Facility, Sheldon Biotechnology Centre, McGill University, Montréal, Québec, Canada H3A 2B4
³ Faculty of Dentistry, McGill University, Montréal, Québec, Canada H3A 2B4

Correspondence
Hervé Le Moual
herve.le-moual{at}mcgill.ca

In Salmonella enterica, PhoP is the response regulator of the PhoP/PhoQ two-component regulatory system that controls the expression of various virulence factors in response to external Mg²⁺. Previous studies have shown that phosphorylation of a PhoP variant with a C-terminal His tag (PhoP_His) enhances dimerization and binding to target DNA. Here, the effect of phosphorylation on the oligomerization and DNA binding properties of both wild-type PhoP (PhoP) and PhoP_His are compared. Gel filtration chromatography showed that PhoP exists as a mixture of monomer and dimer regardless of its phosphorylation state. In contrast, unphosphorylated PhoP_His was mostly monomeric, whereas PhoP_HisP existed as a mixture of monomer and dimer. By monitoring the tryptophan fluorescence of the proteins and the fluorescence of the probe 1-anilinonaphthalene-8-sulfonic acid bound to them, it was found that PhoP and PhoP_His exhibited different spectral properties. The interaction between PhoP or PhoP_His and the PhoP box of the mgtA promoter was monitored by surface plasmon resonance. Binding of PhoP to the PhoP box was barely influenced by phosphorylation. In contrast, phosphorylation of PhoP_His clearly increased the interaction of PhoP_His with target DNA. Altogether, these data show that a His tag at the C-terminus of PhoP affects its biochemical properties, most likely by affecting its conformation and/or its oligomerization state. More importantly, these results show that wild-type PhoP dimerization and interaction with target DNA are independent of phosphorylation, which is in contrast to the previously proposed model.

Present address: Département de Génie Chimique, École Polytechnique de Montréal, Boîte Postale 6079, Station Centre-Ville, Montréal, Québec, Canada H3C 3A7.

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