Regulation of bacterial stringent response by an evolutionary conserved ribosomal protein L11 methylation

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics dataset from 48 bacterial strains representing 6 phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. A comparative proteomics analysis combined with a guanosine polyphosphate assay of the Δ prmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. Moreover, we show that the Δ prmA mutant has an abnormal polysome profile, suggesting a role in ribosomal homeostasis during stationary growth phase. Overall, our investigation demonstrates that the evolutionary conserved bL11 methylation is important for stringent response signaling and ribosomal homeostasis. Importance Protein methylation in bacteria was first identified over sixty years ago. Since then, its functional role has been identified for only a few proteins. To better understand the functional role of methylation in bacteria, we analyzed a large phyloproteomics dataset encompassing 48 diverse bacteria. Our analysis revealed that ribosomal proteins are often methylated at conserved residues, suggesting that methylation of these sites may have a functional role in translation. Further analysis revealed that methylation of ribosomal protein L11 is important for stringent response signaling and ribosomal homeostasis. ### Competing Interest Statement Alan J. Wolfe declares membership on the Scientific Advisory Boards of Pathnostics and Urobiome Therapeutics and funding from Pathnostics, the Craig H. Neilsen Foundation, and an anonymous donor. The other authors declare that they have no conflicts of interest.