Search

This research aims to elucidate the physiological mechanisms behind the accidental acquisition of high-concentration cesium ions (Cs+) tolerance of Escherichia coli 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 Microbacterium sp. TS-1, resistant to high Cs+ concentrations, exhibits a low Cs+ affinity with a Km value of 370 mM at pH 8.5. To enhance bioremediation efficacy, we conducted random mutagenesis of TS_cshA using Error-Prone PCR, aiming for higher-affinity mutants. The mutations were inserted downstream of the PBAD promoter in the pBAD24 vector, creating a mutant library. This was then transformed into E. coli-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 E. coli. 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 Km 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 TS_cshA 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 Km 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.

This research aims to elucidate the physiological mechanisms behind the accidental acquisition of high-concentration cesium ions (Cs+) tolerance of Escherichia coli 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 Microbacterium sp. TS-1, resistant to high Cs+ concentrations, exhibits a low Cs+ affinity with a Km value of 370  mM at pH 8.5. To enhance bioremediation efficacy, we conducted random mutagenesis of TS_cshA using Error-Prone PCR, aiming for higheraffinity mutants. The mutations were inserted downstream of the PBAD promoter in the pBAD24 vector, creating a mutant library. This was then transformed into E. coli-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 E. coli. 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 ΔZX1/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 Km 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 TS_cshA region, was mutationfree. 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 posttranslational modifications, influencing the Km 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_CshAexpressing inverted membrane vesicles. [ABSTRACT FROM AUTHOR]

Objective: This study aims to evaluate the safety of a short hydration (SH) protocol during concurrent chemoradiotherapy (CCRT) with cisplatin for cervical cancer. While massive hydration is a standard for cisplatin chemotherapy to prevent renal toxicity, the safety of SH in the context of CCRT remains underexplored. Methods: We retrospectively reviewed cervical cancer patients undergoing cisplatin-based CCRT at two hospitals from January 2019 to August 2023, excluding those with poor performance status and pre-existing renal impairment. SH involved infusing up to 2 L of fluid within 5 hours on cisplatin days, while prolonged hydration (LH) exceeded this volume or this time. We compared serum creatinine levels (sCr, mg/dL) at baseline, after each cisplatin dose, and 3-6 months post-CCRT (long term) between the 2 groups. Results: Among 86 patients, 25 received SH, and 61 received LH. The SH group had a higher mean age than the LH group (49 vs. 56, p=0.047). There were no significant differences in body weight, stage, surgery ratio, and total cisplatin dose per body surface area between the 2 groups. None of the SH patients required cisplatin dose reduction or discontinuation due to nephrotoxicity, while one and 2 patients in the LH group, respectively. Mean sCr levels at baseline, the last cisplatin dose, and the long term were 0.62, 0.56, and 0.61 in SH, and 0.61, 0.66, and 0.67 in LH (p=0.72, p=0.04, and p=0.15, respectively). Conclusion: SH during CCRT for cervical cancer demonstrates no inferiority in safety compared to LH, with no significant increase in nephrotoxicity. [ABSTRACT FROM AUTHOR]