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

• Published in: EMBO reports 2025-01
• Main Authors: Yu, Siqi, Hu, Zheyao, Xu, Xiaoting, Liang, Xiaoran, Shen, Jiayi, Liu, Min, Lin, Mingxi, Chen, Hong, Marti, Jordi, Tao, Sheng-Ce, Xu, Zhaowei
• Format: Article
• Language: English
• ISSN: 1469-3178; 1469-3178
• DOI: 10.1038/s44319-025-00377-w

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.