biostudies-literatureYu SR&D Program of Guangzhou National Laboratory福 建 省 科 技 厅 | Natural Science Foundation of Fujian ProvinceMOST | National Natural Science Foundation of China (NSFC)| Natural Science Foundation of Fujian Province (Fujian Natural Science Foundation)R&D Program of Guangzhou National LaboratoryThe Fourteenth Five-Year National Key Reaech and Development Program of ChinaMOST | National Natural Science Foundation of China1367-1384https://www.ebi.ac.uk/biostudies/studies/S-EPMC11894153biostudies-literatureUnknown26(5)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 coEMBO reportsc-di-GMP inhibits rRNA methylation and impairs ribosome assembly in the presence of kanamycin.PMC11894153320000272022J01197GZNL2023A01005923741102023YFC2307200Shen JYu SHu ZLiang XLiu MChen HXu XLin MTao SCXu ZMarti Jfalsec-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 co2025-01-01T00:00:00Z2025 Mar2026-06-02T19:18:17.199Z2025-04-04T02:50:58.473ZS-EPMC118941533987096610.1038/s44319-025-00377-w