c-di-GMP inhibits rRNA methylation and impairs ribosome assembly in the presence of kanamycin.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial secondary messenger with diverse functions. A previous Escherichia coli proteome microarray identified that c-di-GMP binds to the 23S rRNA methyltransferases RlmI and RlmE. Here we show that c-di-GMP inhibits RlmI activity in rRNA methylation assays, and that it modulates ribosome assembly in the presence of kanamycin. Molecular dynamics simulation and mutagenesis studies reveal that c-di-GMP binds to RlmI at residues R64, R103, G114, and K201. Structural simulations indicate that c-di-GMP quenches RlmI activity by inducing the closure of the catalytic pocket. We also show that c-di-GMP promotes antibiotic tolerance through RlmI. Binding and methylation assays indicate that the inhibitory effect of c-di-GMP on RlmI is conserved across various pathogenic bacteria. Our data suggest an unexpected role for c-di-GMP in regulating ribosome assembly under stress through the inhibition of rRNA methyltransferases. © 2025. The Author(s).

Citation

Siqi Yu, Zheyao Hu, Xiaoting Xu, Xiaoran Liang, Jiayi Shen, Min Liu, Mingxi Lin, Hong Chen, Jordi Marti, Sheng-Ce Tao, Zhaowei Xu. c-di-GMP inhibits rRNA methylation and impairs ribosome assembly in the presence of kanamycin. EMBO reports. 2025 Mar;26(5):1367-1384

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PMID: 39870966

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