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

Article c-di-GMP inhibits rRNA methylation and impairs ribosome assembly in the presence of kanamycin Siqi Yu1,2,3,7,ZheyaoHu 4,7, Xiaoting Xu1,3,5,7, Xiaoran Liang1,2,3, Jiayi Shen1,2,3,MinLiu 1,2,3, Mingxi Lin1,2,3, Hong Chen6,JordiMarti 4✉,Sheng-ceTao 6✉& Zhaowei Xu 1,2,3 ✉ Abstract 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-diGMP in regulating ribosome assembly under stress through the inhibition of rRNA methyltransferases. Keywords c-di-GMP; rRNA Methyltransferase; Ribosome Assembly; Antibiotic Tolerance Subject Category Translation & Protein Quality https://doi.org/10.1038/s44319-025-00377-w Received 18 July 2024; Revised 20 December 2024; Accepted 15 January 2025 Introduction Cyclic diguanosine monophosphate (c-di-GMP) was first identified in Gluconacetobacter xylinus, where it regulates cellulose synthesis (Ross et al, 1987). Subsequent research revealed that c-di-GMP plays a crucial role in a wide range of bacterial biological processes, including motility, virulence, and host-microbe symbiosis (Hengge, 2009; Jenal et al, 2017; Obeng et al, 2023;Romlingetal,2013). In a previous study, we conducted a global screening of c-di-GMP binding proteins using an Escherichia coli proteome microarray, uncovering the interplay loop between c-di-GMP and protein acetylation (Xu et al, 2019). Interestingly, the microarray assay also identified that the 23S rRNA methyltransferases RlmI and RlmE are c-di-GMP binding proteins, suggesting a functional link between c-di-GMP and ribosome assembly. Ribosome assembly involves the processing and folding of rRNA, along with assembly with ribosomal proteins. As part of rRNA processing, rRNA methylation plays a significant role in regulating ribosome assembly. For example, the inactivation of RlmE is associated with defects in large subunit assembly (Arai et al, 2015), and RsmA, also known as KsgA, fulfills quality control requirements in the final stages of small subunit assembly (Connolly et al, 2008). Overall, there are 23 ribosomal RNA methyltransferases in E. coli, most of which have unresolved physiological functions. Therefore, studying the functions and regulatory factors of rRNA methyltransferase is crucial for understanding the mechanism underlying ribosome assembly. Ribosome biogenesis is a fundamental cellular process that equips cells with molecular factories for protein production. Inhibiting ribosome assembly is considered a vital source of new drug targets (Champney, 2022;Champney,2020). Therefore, investigating the relationship between ribosome assembly and bacterial persistence or antibiotic tolerance is essential for designing antibiotics that target ribosome assembly pathways. In gram-positive bacteria, (p)ppGpp negatively impacts ribosome assembly by inhibiting GTPase activity, thereby influencing growth and antibiotic tolerance (Corrigan et al, 2016). In addition, in Gram-negative bacteria, the effects of (p)ppGpp on antibiotic persistence primarily involve nucleotide and amino acid synthesis (Wang et al, 2020; Zhang et al, 2019). The regulatory relationship between ribosome assembly and bacterial persistence or antibiotic tolerance in Gram-negative bacteria remains poorly understood. rRNA methylation is a significant mechanism for bacterial resistance against ribosome-targeting antibiotics. Two clinically relevant examples are 16S and 23S rRNA methyltransferases, which confer resistance by modifying conserved rRNA residues in site A or PTC, respectively. These modifications render bacteria insensitive to aminoglycosides and streptogramin B (Jeremia et al, 2023). For instance, aminoglycoside resistance in E. coli is conferred by the methylation of the G1405 and A1408 residues in the 16S rRNA by RsmF (Gutierrez et al, 2012) and NpmA (Wachino et al, 2007), respectively. However, the upstream regulatory factors of rRNA 1Key Laboratory of Gastrointestinal Cancer (Fujian Medical University), Ministry of Education, Fuzhou, China. 2Laboratory of Scientific Research, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China. 3Fujian Key Laboratory of Tumor Microbiology, Department of Medical Microbiology, Fujian Medical University, Fuzhou, China. 4Department of Physics, Polytechnic University of Catalonia-Barcelona Tech, Barcelona, Catalonia, Spain. 5Department of Endoscopy, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China. 6Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai, China. 7These authors contributed equally: Siqi Yu, Zheyao Hu, Xiaoting Xu. ✉E-mail: [email protected];[email protected];[email protected] 1234567890();,: © The Author(s) EMBO reports 1 Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. methylation in the context of antibiotic pressure remain unclear, and the impact of rRNA methylation on bacterial antibiotic tolerance is not well understood. In this study, we demonstrated that c-di-GMP binds to two 23S rRNA methyltransferases, with RlmI identified as the main effector of c-di-GMP in regulating ribosome assembly. Structural analysis revealed that c-di-GMP binds to RlmI at residues R64, R103, G114, and K201, inducing the closure of the catalytic pocket of RlmI. We further showed that c-di-GMP regulates ribosomal assembly to promote antibiotic tolerance by inhibiting RlmI activity. Finally, a sequence comparison of RlmI orthologues among bacteria indicated that some important human pathogens are conserved in the c-di-GMP-based rRNA regulatory mechanism. Results Ribosomal RNA large subunit methyltransferases are cdi-GMP effectors In a previous study, we screened c-di-GMP-binding proteins in E. coli using a proteomic microarray and identified the rRNA methyltransferases RlmI and RlmE as potential c-di-GMP effectors (Xu et al, 2019) (Fig. 1A). Based on the observed binding between c-di-GMP and these methyltransferases, we hypothesized that c-diGMP might influence rRNA methylation activity. To test this hypothesis, we assessed the activity of two methyltransferases in the presence of c-di-GMP, using rRNA methylation as our indicator. Specifically, we synthesized unmethylated 23S rRNA at positions 1932–1991 and 2522–2581 for m5C1962 by RlmI (Purta et al, 2008) and m2U2552 by RlmE (Caldas et al, 2000), respectively. These methyltransferases catalyzed the production of methylated rRNA, which was detected specific peaks in HPLC (Fig. 1B,C; Appendix Fig. S1). When c-di-GMP was introduced, methylation activity was significantly inhibited in a dose-dependent manner. When we compared the effects of additional c-di-GMP on methylation products, using the group without c-di-GMP treatment as a reference, we found that 5 μM c-di-GMP inhibited the activity of RlmI and RlmE by 49% and 31%, respectively (Fig. 1D). c-di-GMP inhibited ribosome assembly, with RlmI being the main effector rRNA methylation is a prerequisite for the accurate assembly of ribosomes. We hypothesized that c-di-GMP might affect ribosomal assembly in E. coli (using E. coli BW25113 as a reference strain) by inhibiting methylation activity. To investigate the regulatory role of c-di-GMP, we constructed strains with dgcZ knockout and overexpression. Compared to wild-type (WT) strains, the c-diGMP level in the dgcZ overexpressing strains increased by 12.2 times (Appendix Fig. S2A). We employed a sucrose density gradient (SDG) assay to detect ribosome particles, which revealed that neither the knockout nor overexpression of dgcZ affected the abundance of the 50S ribosomal subunit compared to the WT strain without antibiotic treatment (Fig. 2A). Since c-di-GMP as a stress response factor, we further hypothesized that the regulation of ribosome assembly by c-di-GMP might occur under antibiotic stress. We treated E. coli cells with kanamycin, a ribosome-targeted antibiotic known to increase the cellular c-di-GMP level in E. coli by elevating dgcZ mRNA level (Ho et al, 2013;Xuetal,2019). This increase is regulated by the RNA-binding protein CsrA (Boehm et al, 2009; Lacanna et al, 2016). Following kanamycin treatment, the c-di-GMP concentration in WT cells was 6.2-fold higher than in untreated cells (Appendix Fig. S2A). Notably, the c-di-GMP levels in dgcZ-defective cells did not respond to kanamycin treatment (Appendix Fig. S2A) because DgcZ functions as a synthase that mediates the kanamycin-induced increase in c-diGMP levels. The SDG assay revealed that the ribosome disintegrated into 30S and 50S particles at low Mg2+concentrations, and ~45S particles (Corrigan et al, 2016) were observed in kanamycintreated WT cells and the dgcZ-defective strain complemented with a functional dgcZ gene (ΔdgcZ::dgcZ) (Fig. 2A). In contrast, the strain with the dgcZ inactivation mutation did not show the presence of 45S particles (ΔdgcZ::dgcZG206A,G207A). These results indicated that the increase in c-di-GMP levels induced by kanamycin inhibited the assembly of large ribosomal subunits in E. coli. In addition, we found that c-di-GMP inhibited the activity of two methyltransferases and downregulated the methylation of 23S RNA in vitro (Fig. 1D). To elucidate the role of methylation enzymes in c-di-GMP-regulated ribosome assembly, we overexpressed the two methyltransferases in kanamycin-treated WT cells. Notably, the overexpression of these methyltransferases did not affect c-di-GMP levels (Appendix Fig. S2B), but the overexpression of RlmI weakened the effect of c-di-GMP on ribosomal assembly (Fig. 2B). Thus, we conclude that c-di-GMP inhibits ribosome assembly by inactivating RlmI. To confirm that RlmI is directly regulated by c-di-GMP and not by other c-di-GMP analogs. We employed a 100-fold excess of unlabeled c-di-GMP and its analogs (GTP, GMP, ATP, AMP, cAMP, and ppGpp) as competitive inhibitors to assess their effects on the interaction between RlmI and biotinylated c-di-GMP. The results demonstrated that unlabeled c-di-GMP effectively blocked the interaction between biotinylated c-di-GMP and RlmI, while none of the other analogs exhibited similar effects (Fig. 2C). c-diGMP binds to its effectors via Arg residues (Chou and Galperin, 2016). To identify the binding sites on RlmI, we mutated all Arg residues to Ala in RlmI. We subsequently developed an in vitro assay in which purified RlmI mutants were incubated with biotin-cdi-GMP, subjected to UV-crosslinked, and probed with fluorescent streptavidin (Kramer et al, 2014;Shuetal,2012). We observed that RlmI mutants with R64A and R103A exhibited a significantly weakened interaction with c-di-GMP (Fig. 2D; Appendix Fig. S3). Furthermore, when we determined the activity of RlmI mutants, both RlmIR64A and RlmIR103A displayed methylation activities slightly lowerthanthatofRlmIunder5μM rRNA substrate. Upon treatment with 20 μM c-di-GMP, the methylation activity decreased by 80%, 6%, and 32% in RlmI, RlmIR64A,andRlmI R103A, respectively (Fig. 2E). The diminished effect of c-di-GMP on RlmI in the R64A and R103A mutant strains, compared to the WT strain, confirmed that R64 and R103 are the key sites involved in cdi-GMP binding to RlmI. To validate whether RlmI is the primary effector of c-di-GMP in ribosome assembly, we eliminated the effect of c-di-GMP on RlmI by mutating the binding sites R64A and R103A. Under kanamycin treatment, we analyzed WT, rlmIR64A,rlmIR103A,andΔrlmI strains using the sucrose density gradient (SDG) assay. We examined the EMBO reports Siqi Yu et al 2EMBO reports © The Author(s) Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. impact of c-di-GMP on ribosome biogenesis by performing SDG to analyze changes in the 70S and 50S ribosomal populations under 10 mM MgCl₂conditions. When c-di-GMP levels were elevated, we observed a significant accumulation of 50S subunits. However, this accumulation was markedly reduced when mutations were introduced at the R64 or R103 sites of RlmI, indicating that c-diGMP acts through RlmI to influence the formation of intact 70S ribosomes (Fig. 3A). In addition, we compared methylation levels of C1962 in the 50S and 70S ribosomes under high c-di-GMP expression and 10 mM MgCl₂conditions. We found that the 50S subunits displayed significantly lower levels of m⁵C1962 methylation compared to the 70S particles, suggesting that proper methylation at C1962 is associated with the maturation of the ribosomal large subunit (Fig. 3B). To more accurately observe ribosome assembly intermediates, we performed density gradient centrifugation under 0.5 mM MgCl₂ conditions. The results demonstrated that under high c-di-GMP expression, there was a significant accumulation of 45S ribosomal intermediates. However, when the RlmI R64A or R103A mutations were present, the accumulation of 45S ribosomes was significantly reduced (Fig. 3A). Furthermore, we observed that in the context of high c-di-GMP expression, the 45S ribosomes exhibited significantly lower levels of m⁵C1962 methylation compared to the 50S subunits. These findings suggest that methylation at C1962 is correlated with the proper assembly and maturation of the ribosomal large subunit (Fig. 3B). Taken together, these results demonstrate that RlmI plays a crucial role in ribosomal assembly under kanamycin stress and that c-di-GMP regulates RlmI’s activity. Figure 1. Ribosomal RNA large subunit methyltransferases are c-di-GMP effectors. (A)E. coli proteome microarrays were probed with biotin-c-di-GMP and biotin. Obvious differences in the binding of RlmI and RlmE on the microarrays incubated with biotin-c-di-GMP and biotin were observed. Two spots per protein were observed, and the positive signal-to-noise ratio [(SNR) (+)] represented the average SNR of the two duplicate spots. (B,C) In vitro methylation reaction. The synthesized rRNA fragments were used for in vitro methylation enzyme activity testing. The HPLC peaks are derived from RlmI (B) and RlmE (C) after treatment with 0, 5, 10, and 20 μM c-di-GMP, respectively. The second peak (black arrow) represents the methylated rRNA, which is used to calculate the activity of the methyltransferase. (D) Quantitative results of the HPLC peak. The methylated rRNAs, indicated by the second peak, were detected by HPLC and quantified by area under the curve. The bar chart shows the relative enzyme activity with the data points, using the reaction without the addition of c-di-GMP as the baseline (n=3 biological replicates, mean ± s.e.m.; **p< 0.01 (p=0.0011 between 0 μM c-di-GMP and 20 μM c-di-GMP in RlmI; p=0.00059 between 0μM c-di-GMP and 20 μM c-di-GMP in RlmE), two‐tailed Student’st‐test). Source data are available online for this figure. Siqi Yu et al EMBO reports © The Author(s) EMBO reports 3 Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. c-di-GMP induces the closure of the catalytic pocket of RlmI To elucidate the structural mechanism by which c-di-GMP regulates RlmI enzyme activity, we investigated the conformational changes of RlmI during its interaction with c-di-GMP in an aqueous ionic solution using molecular dynamics (MD) simulations. The root mean square deviation (RMSD) indicated the fluctuations and stability of the conformations of RlmI, while the root mean square fluctuation (RMSF) revealed flexibility throughout the simulation period. Analysis showed that residues 160–170, 302–320, and 370–390 were mainly involved in the conformational fluctuations of RlmI (Fig. 4A). We labeled residues 160–170 as “Domain-I”(DM-I), residues 302–320 as “Domain-II” EMBO reports Siqi Yu et al 4EMBO reports © The Author(s) Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. (DM-II), and residues 370–390 as “Domain-III”(DM-III). DM-I and DM-III are the regions of the protein that correspond to the RNA-binding area, whereas DM-II is located near the S-adenosylL-methionine (AdoMet) binding-related area. An overall view of the evolution of RlmI fluctuations revealed a distinct conformational fluctuation of approximately 0.8 μs during the simulation (average of Trajectory #1 and Trajectory #2) (Fig. 4B). Combining RMSD, RMSF, and trajectory analysis results, we identified two states for RlmI during its interaction with c-di-GMP: State-I and State-II. c-di-GMP interacts with the RNA-binding area (State-I), and the domain DM-III shut down after 0.8 μs. The results suggested that (1) c-di-GMP can interact with the RNA-binding domains and then induce the closure of DM-III, and that (2) the “on-off”of the RNA-binding area was mainly embodied by DM-III to a large extent (Fig. 4CandMovieEV1–2). The dynamic process of c-di-GMP-induced conformational rearrangements in the active domain of RlmI was similar to that of other c-di-GMP effectors such as YcgR (Hou et al, 2020), FleQ (Matsuyama et al, 2015), and CheR1 (Yan et al, 2018). Interactions of R64, R103, G114, and K201 residues of RlmI with bound c-di-GMP We employed Gibbs free energy analysis to identify the dominant conformation of RlmI and c-di-GMP complex via molecular dynamics simulations. The Gibbs free energy surfaces for the two runs and their average values are shown (Fig. 5A), using RMSD and radius of gyration used as the variables. We identified the free energy basin, the one with the lowest free energy (set to 0 kJ/mol) (Fig. 5A, yellow point), and found that the corresponding regions were almost overlapping for the three sets (Fig. 5B). Thus, the results indicated the two independent simulated trajectories as convergent and physically equivalent. To explore the binding sites of c-di-GMP and RlmI, we superimposed the stable-state configurations of RlmI and c-diGMP for the three sets (Fig. 5B). Two independent trajectories, #1 and #2, were taken into consideration for the computational analysis and the average was selected for convergence and physical equivalence analysis. The average conformation showed that R64 and G114 together stabilize the guanosine moiety of c-di-GMP. Correspondingly, R103 and K201 act to stabilize the negatively charged region of c-di-GMP. It is evident that R103 and K201 formed a stable hydrogen bond with the oxygen atom of the phosphate group of c-di-GMP. Noncovalent interactions, including hydrogen bonds, coordination bonds, and salt bridges, are crucial for maintaining the tertiary structure of proteins. The all-atom-level precision of the molecular dynamics simulations, we analyzed the hydrogen bond interaction map of c-di-GMP with RlmI using time-dependent atomic site distances between selected atomic sites to uncover the interaction mode of c-di-GMP with RlmI, providing guidance for further experimental verification. Atomic detail sketches of c-di-GMP and the main residues described in this section are provided. While labeling the amino acid residues in the hydrogen bond interaction map of c-di-GMP with RlmI, we also labeled the lifetime of hydrogen-bonding interactions between c-di-GMP and the corresponding amino acid residues. Considering that our molecular dynamics simulation spanned a timeframe of 2 μs, we subsequently performed site mutation verification on residues with hydrogen bond interaction lifetimes exceeding 400 ns. Six amino acid residues from RlmI were selected as the potential binding sites for c-di-GMP: R64, R103, E108, G114, T116, and K201 (Fig. 5C). Atom-atom distances as a function of time and bond lifetimes are presented in Appendix Figs. S5–15. We employed the streptavidin blotting assays to determine the interaction between c-di-GMP and RlmI mutants, aiming to validate the results obtained from the molecular dynamics simulations. The results revealed that the amino acid residues R64, R103, G114, and K201 were crucial for the binding of c-diGMP to RlmI. In addition, E108A and T116A of RlmI slightly affected c-di-GMP binding (Fig. 5D). We next performed isothermal titration calorimetry (ITC) titrations with these mutants and determined K d values of 1.3, 102.3, 76.5, 148.6, and 401.2 μM for RlmI, RlmIR64A,RlmI R103A,RlmI G114A,andRlmI K201A, respectively (Fig. 5E; Appendix Fig. S16). We found that the stoichiometries of RlmI and its variants with c-di-GMP are not significantly different, each showing a 1:1 binding ratio (Fig. 5E). Furthermore, we observed that the activity of RlmIK201A did not significantly differ between the 20 μM c-di-GMP treatment group and the c-di-GMP free group (Fig. 5F). The results suggested that the R64, R103, G114, and K201 residues of RlmI were the critical sites for c-diGMP binding. c-di-GMP regulates RlmI to promote antibiotic tolerance Given that a close correlation exists between c-di-GMP, ribosomal assembly, and antibiotic resistance (Gomez et al, 2017; Gupta et al, 2014), we hypothesized that c-di-GMP regulates ribosomal Figure 2. c-di-GMP inhibits ribosome assembly, with RlmI as the main effector. (A) SDG assay for the strains with elevated c-di-GMP. c-di-GMP was elevated by treatment with kanamycin or overexpression of DgcZ, and the ribosome particles were assayed by SDG. The corresponding three peaks represent the ribosome particles of 30S, pre-50S, and 50S. (B) SDG assay for the strains overexpressing two methyltransferases. RlmI and RlmE were overexpressed under kanamycin treatment, and the ribosome particles were assayed by SDG. (C) c-di-GMP analog competitive assay. Streptavidin represents the interaction signals, and α-His represents the protein levels. The bar chart shows the relative intensity of streptavidin with the data points (n=3 biological replicates, mean ± s.e.m.; ns: no significant difference, two‐tailed Student’st‐test). (D) The arginine on RlmI was mutated to alanine, and the interaction of c-di-GMP and RlmI mutants was determined. The results indicated that R64A and R103A weakened the binding of c-di-GMP and RlmI. Streptavidin represents the interaction signals, and α-His represents the protein levels. The bar chart shows the relative intensity of streptavidin with the data points (n=3 biological replicates, mean ± s.e.m.; **p< 0.01 (p=0.0016 between WT and R64A; p=0.0011 between WT and R103A), two‐tailed Student’st‐test). (E) In vitro methylation assay of the two RlmI mutants. The synthesized rRNA fragments were used as substrates, and the reaction products were analyzed by HPLC. The bar chart shows the relative activity of RlmI with the data points (n=3 biological replicates, mean ± s.e.m.; ns: no significant difference, **p< 0.01 (p=0.00046 between 0 μM c-di-GMP and 20 μM c-di-GMP in WT; p=0.057 between 0 μM c-di-GMP and 20 μM c-di-GMP in R64A; p=0.0082 between 0 μM c-di-GMP and 20 μM c-di-GMP in R103A), two‐tailed Student’st‐ test). Source data are available online for this figure. Siqi Yu et al EMBO reports © The Author(s) EMBO reports 5 Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. EMBO reports Siqi Yu et al 6EMBO reports © The Author(s) Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. assembly to promote bacterial persistence or antibiotic tolerance by inhibiting RlmI activity. To test this, we interfered with the interaction of c-di-GMP and RlmI by introducing the K201 mutation in endogenous RlmI (rlmIK201A), dgcZ depletion (△dgcZ), dgcZ overexpression (△dgcZ dgcZ+) and both mutants (△dgcZ rlmIK201A). We subsequently determined the c-di-GMP level and methylation level of 23S rRNA C1962. The results indicated no significant changes in c-di-GMP levels and C1962 methylation levels across WT, △dgcZ,rlmIK201A,and△dgcZ rlmIK201A without kanamycin treatment (Fig. 6A,B). However, with kanamycin treatment, the c-di-GMP levels of WT and rlmIK201A increased approximately six times compared with △dgcZ and △dgcZ rlmIK201A. The c-di-GMP levels of △dgcZ dgcZ+increased approximately eleven times compared with those of WT (Fig. 6A). Methylation analysis showed that elevated c-di-GMP levels (WT, △dgcZ dgcZ+)significantly decreased C1962 methylation, while the K210A mutation (rlmIK201A) abolished the effect of c-di-GMP (Fig. 6B). These findings provide in vivo evidence that c-di-GMP regulates 23S rRNA methylation through RlmI. To investigate the role of c-di-GMP regulation of RlmI in bacterial antibiotic tolerance, we examined wild-type (WT), rlmIK201A,and△dgcZ strains. The △dgcZ strain, in which the major c-di-GMP synthase DgcZ is deleted in E. coli,exhibitsa reduced intracellular c-di-GMP level, while the rlmIK201A strain carries a mutation at the K201 site of RlmI, abolished the regulation of c-di-GMP on RlmI. To assess antibiotic sensitivity, bacterial killing curves were generated for the three strains in the presence of kanamycin and ampicillin at 20× and 100× MIC concentrations. The results revealed that both the rlmIK201A and △dgcZ strains exhibited increased sensitivity to antibiotics compared to the WT strain, suggesting that both c-di-GMP and RlmI contribute to modulating antibiotic response (Fig. 6C). Notably, the MDK 99 for the rlmIK201A strain was significantly lowerthanthatoftheWTstrainat both 20× and 100× kanamycin concentrations, measuring 104.79 and 87.25 min, respectively. Similarly, the MDK 99.99 for rlmIK201A was markedly reduced to 196.62 and 189.40 min under the same kanamycin concentrations compared to the WT. These reductions were also observed when treated with ampicillin. These results indicate that, compared to the WT, the rlmIK201A strain exhibits significantly decreased antibiotic tolerance. In addition, the △dgcZ strain also displayed significantly reduced MDK 99 and MDK 99.99 values compared to the WT, indicating that c-di-GMP modulates antibiotic sensitivity (Fig. 6D). Based on this evidence, we propose that c-di-GMP promotes bacterial tolerance to antibiotics through its regulation of RlmI. The effect of c-di-GMP on RlmI may be conserved in multiple pathogenic bacteria c-di-GMP is a ubiquitous bacterial secondary messenger, and RlmI is highly conserved in bacteria. Thus, we hypothesized that the binding and inhibition of c-di-GMP with RlmI from E. coli wasthesamefortheRlmI homologs in other bacteria. To test this hypothesis, we aligned RlmI protein sequences from a series of highly diverse bacteria and found that the c-di-GMP binding region well conserved in these bacteria (Fig. 7A). Then, we selected Salmonella typhimurium,Klebsiella pneumoniae,and Vibrio cholerae as the exemplary members of this conserved set. Our analysis revealed that RlmIS. typhimurium,RlmI K. pneumoniae,andRlmI V. cholerae could bind to c-di-GMP, with binding abolished upon mutation of the lysine in RlmI (Fig. 7B). Moreover, the in vitro activity analysis showed that similar to RlmIE.coli, the aforementioned three RlmI homologs exhibited methylase activity for 23S rRNA and this activity could be inhibited by c-di-GMP (Fig. 7C). Thus, the effect of c-di-GMP on RlmI may be conserved in multiple pathogenic bacteria. Discussion c-di-GMP is a crucial secondary messenger in prokaryotes, and rRNA methylation occurs in both prokaryotes and eukaryotes. This study revealed that c-di-GMP binds to two rRNA methyltransferases, inhibiting their activities, with RlmI identified as the primary effector of c-di-GMP in ribosome assembly. Molecular dynamics simulations revealed the binding sites and models of c-diGMP interacting with RlmI. In addition, killing assays demonstrated that c-di-GMP inhibits ribosome assembly, thereby promoting antibiotic tolerance in E. coli. This research establishes a regulatory pathway linking c-di-GMP to ribosomal functions, underscoring the role of c-di-GMP in antibiotic tolerance. Previous studies have reported that c-di-GMP regulates mature ribosome function through RimK in Pseudomonas (Grenga et al, 2020; Little et al, 2016), EF-P in Acinetobacter baumannii (Guo et al, 2022), and Vc2 riboswitches in V. cholerae (Inuzuka et al, 2018). c-di-GMP regulates the glutamate ligase RimK, which catalyzes glutamate residues to the C-terminus of the ribosomal protein RpsF to affect ribosomal function (Grenga et al, 2020; Little et al, 2016). The binding of c-di-GMP enhances the function of EF-P, promoting translation efficiency and modulating bacterial physiology and virulence (Guo et al, 2022). In addition, c-di-GMP binds to the Vc2 riboswitch, inducing structural changes that result in switch-OFF and switch-ON states of translational initiation (Inuzuka et al, 2018). This study revealed that the role of c-di-GMP affects ribosome assembly, offering Figure 3. Ribosome assembly and C1962 methylation in response to varying Mg²⁺concentrations and c-di-GMP levels in the presence of kanamycin. (A) Sucrose density gradient (SDG) analysis of ribosome profiles in RlmI-depleted and RlmI-mutant strains treated with kanamycin. Ribosomal particles were separated by SDG under two different MgCl 2 concentrations (0.5 mM and 10 mM) to evaluate changes in ribosome assembly and stability. Comparison of the resulting profiles highlights how varying MgCl 2 conditions influence ribosome distribution and reveals the effects of RlmI depletion or mutation on ribosome integrity and function (n=3 biological replicates). (B) Methylation levels of C1962 in 23S rRNA were examined under varying MgCl 2 conditions to assess how assembly states affect ribosomal modification. At 10 mM MgCl 2 , both the 50S subunits and fully assembled 70S ribosomes were analyzed, while at 0.5 mM MgCl 2 , the 50S and partially assembled 45S components were examined. This comparison highlights the influence of MgCl 2 concentration and ribosomal assembly state on the C1962 methylation status within the ribosome (n=3 biological replicates, mean ± s.e.m.; **p< 0.01, *p< 0.05; (In the condition of 0.5 mM MgCl 2 ,p=0.0039 between 50S and 45S in WT, p=0.048 between 50S and 45S in △rlmI; In the condition of 10 mM MgCl 2 ,p=0.026 between 50S and 70S in WT, p=0.034 between 50S and 70S in △rlmI), two‐tailed Student’st‐ test). Source data are available online for this figure. Siqi Yu et al EMBO reports © The Author(s) EMBO reports 7 Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. a new perspective on c-di-GMP in ribosome regulation. Numerous accessory factors play a role in guiding the ribosome assembly process, including GTPases, rRNA modification enzymes, helicases, and maturation factors (Davis and Williamson, 2017). Our findings establish c-di-GMP as an upstream regulatory signal for rRNA modification, creating a connection between environmental stimuli and ribosome function. RlmI is a large ribosomal RNA subunit methyltransferase that specifically methylates cytosine at position 1962 (m5C1962) of 23S rRNA. Previous studies indicated that RlmI depletion did not lead to abnormal ribosome assembly or growth arrest of E. coli at 20 °C or 37 °C (Pletnev et al, 2020). Indeed, we found that RlmI depletion did not affect the abundance of 50S ribosome subunits compared with WT strains in the absence of antibiotics. However, upon kanamycin treatment, ~45S particles were observed in the △rlmI cells. Thus, RlmI plays a key role in ribosomal assembly under kanamycin stress. As deletion of most ribosomal methyltransferases does not cause significant phenotypic changes, these studies have demonstrated that the function of methylases under different growth conditions may help understand the physiological significance of ribosome assembly. In addition, we have not yet addressed the specific role of kanamycin in ribosome assembly, a question that remains of significant interest. Understanding how kanamycin interacts with the ribosomal assembly process is a key area of curiosity for us. To investigate this, identifying the components of 45S ribosomal intermediates and analyzing their structures will be crucial. This approach will provide valuable insights into the molecular mechanisms underlying the influence of kanamycin on ribosome assembly and its potential effects on bacterial protein synthesis. RlmE plays a critical role in ribosome assembly by modifying the methylation status of m²U2552, thereby influencing the structural and functional integrity of the ribosome. Our study reveals that the secondary messenger c-di-GMP can bind to RlmE, and in vitro assays demonstrate that c-di-GMP effectively inhibits the methyltransferase activity of RlmE. However, in vivo experiments indicate that the restoration of RlmE expression does not alter the regulatory effect of c-di-GMP on ribosome assembly. This discrepancy suggests Figure 4. The simulated interaction model of c-di-GMP and RlmI. The interaction model of c-di-GMP with RlmI was simulated using the CHARMM-GUI platform. (A) RMSD value of RlmI during interaction with c-di-GMPs. The arrow marks the main changes at the residue level. The solid and dashed lines represent two simulated trajectories, and the degree of overlap represents the convergence of two trajectories. (B) RMSF value of RlmI during interaction with c-di-GMP. The arrow indicates the main changes on the timeline. (C) Representative snapshots of the transition between RlmI State-I and State-II. The arrow marks the main changes in DM-III. Source data are available online for this figure. EMBO reports Siqi Yu et al 8EMBO reports © The Author(s) Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. Siqi Yu et al EMBO reports © The Author(s) EMBO reports 9 Downloaded from https://www.embopress.org on March 4, 2025 from IP 147.83.201.74. Streptavidin blotting assay In this assay, RlmI (0.1 mg/mL) and its mutants were incubated with 10 μM biotin-c-di-GMP in a reaction buffer (20 mM Tris, 50 mM NaCl, 200 mM KCl, pH 7.0) at 37 °C for 1 h. The samples were subjected to UVcross-linking on ice for 0.5 h to further link c-di-GMP to RlmI. These linked samples were divided into two parts for western blot analysis. After incubation with IRDye 800CW Conjugated Streptavidin (#926-32230; LI-COR Biosciences, USA) at room temperature for 2 h, another membrane was incubated with an anti-His antibody (05-949, Millipore, USA) at 4 °C for 12 h and then incubated with an IRDye 800 secondary antibody for 1 h. The resulting membranes were visualized with an Odyssey Infrared Imaging System (LI-COR Biosciences). Isolation and quantification of c-di-GMP in E. coli Isolation of c-di-GMP was conducted as previously described (Spangler et al, 2010;Xuetal,2019). Briefly, E. coli cells at 50 OD were harvested and resuspended in 2 mL of ddH 2 O. Subsequently, 8 mL of a 50% methanol and 50% acetonitrile mixture was added to extract intracellular c-di-GMP. Moreover, 1 μM cGMP was added as an internal reference. For absolute quantification of c-di-GMP, the density of E. coli sediment was defined as 1 mg/mL, and bacterial concentration was calculated using absorbance measurements, which were used for c-di-GMP quantification. The extracts were analyzed via ultrahigh-performance liquid chromatography coupled with ion mobility mass spectrometry (UPLC-IM-MS), utilizing a Waters UPLC I-class system equipped with a binary solvent delivery manager and a sample manager coupled with a Waters VION IMS Q-TOF mass spectrometer equipped with an electrospray interface (Waters Corporation, CT, USA). Determination of the strain growth curve in Vogel-Bonner medium As previously mentioned, the strains WT, ΔdgcZ,rlmIK201A,and ΔdgcZ rlmIK201A were grown in Vogel-Bonner medium supplemented with 10 mM acetate at 25 °C. For kanamycin treatment, concentrations of 0, 1.5, 3, 6, or 9 μg/mL kanamycin were added to the Vogel‐Bonner medium Cell concentrations were measured at OD 600 using a NanoDrop 2000 spectrophotometer at 8, 12, 16, 24, and 32 h. The growth curve was subsequently plotted using GraphPad Prism 6. Kanamycin and ampicillin killing assay To evaluate the killing kinetics of kanamycin and ampicillin, bacterial cultures were grown to mid-log phase (OD600: 0.4) in LB medium. Equal volumes of bacterial suspensions were distributed into 96-well plates in LB medium. Each well received varying concentrations containing kanamycin (MIC: 8 μg/mL) or ampicillin (MIC: 5 μg/mL), specifically at 20× and 100× MIC. The plates were incubated at 37 °C at 200 rpm for a total duration of 6 h, with measurements taken every 30 min. After each time point, aliquots were collected from each well, serially diluted in PBS, and plated on LB agar for overnight incubation to determine colony-forming units (CFUs). Each measurement was conducted in triplicate, and the average CFU count was calculated. The reduction in CFUs compared with the control was used to evaluate the killing efficiency of each antibiotic. All experiments were independently repeated three times, and the killing curve was plotted using GraphPad Prism 6. Statistical analysis Pairwise comparisons were performed using two‐tailed Student’s t‐test, and statistical significance was set at *p<0.05and**p< 0.01. Error bars represent the means ± s.e.m.s. Data availability The crystal structure files, MD simulation files (input files, parameter files, topology files, etc.), and structures of c-di-GMP are available on the website https://github.com/Zheyao-Hu/RlmIcdiGMP. Moreover, all the software (free to use) packages used in this study were the official release versions without any modifications. The raw protein microarray data have been published in the Protein Microarray Database (www.proteinmicroarray.cn/) with the accession number PMDE226. The source data of this paper are collected in the following database record: biostudies:S-SCDT-10_1038-S44319-025-00377-w. Expanded view data, supplementary information, appendices are available for this paper at https://doi.org/10.1038/s44319-025-00377-w. Peer review information A peer review file is available at https://doi.org/10.1038/s44319-025-00377-w References Arai T, Ishiguro K, Kimura S, Sakaguchi Y, Suzuki T (2015) Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly. Proc Natl Acad Sci USA 112:E4707–E4716 Berendsen HJC, van der Spoel D, van Drunen R (1995) GROMACS: a messagepassing parallel molecular dynamics implementation. 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Author contributions Siqi Yu: Conceptualization; Data curation; Formal analysis; Validation; Investigation; Methodology; Writing—original draft; Writing—review and editing. Zheyao Hu: Conceptualization; Data curation; Formal analysis; Investigation; Visualization; Methodology; Writing—original draft; Writing—review and editing. Xiaoting Xu: Formal analysis; Validation; Investigation; Writing—review and editing. Xiaoran Liang: Investigation; Visualization. Jiayi Shen: Investigation; Visualization. Min Liu: Investigation; Visualization. Mingxi Lin: Investigation; Visualization. Hong Chen: Investigation; Visualization; Methodology. Jordi Marti: Conceptualization; Supervision; Methodology; Writing—review and editing. Sheng-ce Tao: Conceptualization; Funding acquisition; Writing—review and editing. Zhaowei Xu: Conceptualization; Supervision; Funding acquisition; Investigation; Writing—original draft; Project administration; Writing—review and editing. 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