Regulation of Galactose Metabolism through the HisK:GalR Two-Component System in Thermoanaerobacter tengcongensis

Abstract

ABSTRACT Thermoanaerobacter tengcongensis could utilize galactose as a carbon source via the enzymes encoded by a novel gal operon, whose regulation mechanism has yet to be elucidated. We propose here that the gal operon in T. tengcongensis is regulated through a HisK:GalR two-component system. By using radioactive isotope assay and genetic analysis, we found that the kinase of this system, HisK, is phosphorylated by ATP, and the regulator, GalR, accepts a phosphoryl group during phosphorelay, in which the phosphoryl group at HisK-His-259 is transferred to GalR-Asp-56. Two-dimensional electrophoresis, followed by Western blotting, revealed that phosphorylation status of GalR is uniquely dependent on the galactose stimulus in vivo . Furthermore, DNA pulldown assays demonstrated that the phosphorylated GalR prefers binding to the operator DNA O 2 , whereas the unphosphorylated GalR to O 1 . A model of HisK:GalR is proposed to explain how galactose mediates the expression of the gal operon in T. tengcongensis.