Gdx mediates low-affinity Cs⁺/H⁺ antiport and confers cesium resistance in Escherichia coli

• gdx is strongly upregulated in the Cs⁺-resistant strain ZX-1. • A 20-bp deletion in the riboswitch P2 loop causes constitutive gdx expression. • Gdx exports Cs⁺ via H⁺ antiport, at ∼500-fold lower affinity than for Gdm⁺. • Gdm⁺ boosts growth via riboswitch-mediated induction of gdx under Cs⁺ stress. • Gdx expression lowers intracellular Cs⁺ and preserves K⁺ homeostasis. Following the Fukushima Daiichi nuclear power plant accident, radioactive cesium was released into the environment, prompting intensified efforts to identify cesium-resistant microorganisms. During these studies, we isolated a cesium-resistant Escherichia coli strain, designated ZX-1, which exhibits remarkable tolerance to cesium concentrations exceeding 700 mM. As no prior reports of cesium-resistant E. coli exist, this finding suggests the presence of a previously unrecognized resistance mechanism. This study aims to elucidate the molecular basis of cesium resistance in ZX-1. RNA-seq analysis comparing ZX-1 with its parental strain, the commercial E. coli Mach1™, revealed constitutive upregulation of the guanidinium exporter gene gdx in ZX-1. Reanalysis of the whole-genome sequence identified a 20-bp deletion upstream of the gdx open reading frame, likely disrupting formation of the guanidinium riboswitch P2 loop and resulting in constitutive gdx expression. To evaluate gdx function, the gene was cloned into the expression vector pBAD24 and expressed in E. coli . The resulting gdx -expressing strain exhibited even greater cesium resistance than ZX-1. Functional assays demonstrated that this strain mediates not only guanidinium/H⁺ antiport activity but also Cs⁺/H⁺ antiport activity. Cesium resistance was further enhanced in the presence of guanidinium, consistent with riboswitch-mediated induction of gdx . Collectively, these findings provide evidence for a novel cesium efflux mechanism in E. coli and uncover an unexpected role of the guanidinium exporter Gdx in cesium export. These insights may facilitate the discovery of additional cesium-resistant microorganisms and broaden the potential for future applications.