Isolation and Cs resistance mechanism of strain ZX-1.

This research aims to elucidate the physiological mechanisms behind the accidental acquisition of high-concentration cesium ions (Cs) tolerance of and apply this understanding to develop bioremediation technologies. Bacterial Cs resistance has attracted attention, but its physiological mechanism remains largely unknown and poorly understood. In a prior study, we identified the Cs/H antiporter TS_CshA in sp. TS-1, resistant to high Cs concentrations, exhibits a low Cs affinity with a value of 370 mM at pH 8.5. To enhance bioremediation efficacy, we conducted random mutagenesis of using Error-Prone PCR, aiming for higher-affinity mutants. The mutations were inserted downstream of the P promoter in the pBAD24 vector, creating a mutant library. This was then transformed into -competent cells. As a result, we obtained a Cs-resistant strain, ZX-1, capable of thriving in 400 mM CsCl-a concentration too high for ordinary . Unlike the parent strain Mach1, which struggled in 300 mM CsCl, ZX-1 showed robust growth even in 700 mM CsCl. After 700 mM CsCl treatment, the 70S ribosome of Mach1 collapsed, whereas ZX-1 and its derivative ΔZX-1/pBR322ΔAp remained stable. This means that the ribosomes of ZX-1 are more stable to high Cs. The inverted membrane vesicles from strain ZX-1 showed an apparent value of 28.7 mM (pH 8.5) for Cs/H antiport activity, indicating an approximately 12.9-fold increase in Cs affinity. Remarkably, the entire plasmid isolated from ZX-1, including the region, was mutation-free. Subsequent whole-genome analysis of ZX-1 identified multiple SNPs on the chromosome that differed from those in the parent strain. No mutations in transporter-related genes were identified in ZX-1. However, three mutations emerged as significant: genes encoding the ribosomal bS6 modification enzyme RimK, the phage lysis regulatory protein LysB, and the flagellar base component protein FlgG. These mutations are hypothesized to affect post-translational modifications, influencing the value of TS_CshA and accessory protein expression. This study unveils a novel Cs resistance mechanism in ZX-1, enhancing our understanding of Cs resistance and paving the way for developing technology to recover radioactive Cs from water using TS_CshA-expressing inverted membrane vesicles.