RESUMEN
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 data set from 48 bacterial strains representing six 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. Comparative proteomics analysis combined with inorganic polyphosphate and guanosine tetra/pentaphosphate assays of the ΔprmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. In the stationary phase, we found that the ΔprmA mutant has impaired guanosine tetra/pentaphosphate production. This leads to a reduction in inorganic polyphosphate levels, accumulation of RNA and ribosomal proteins, and an abnormal polysome profile. Overall, our investigation demonstrates that the evolutionarily conserved bL11 methylation is important for stringent response signaling and ribosomal activity regulation and turnover. IMPORTANCE: Protein methylation in bacteria was first identified over 60 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 data set 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.
RESUMEN
Nε-lysine acetylation is a common posttranslational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this posttranslational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of either of the acetyl donors acetyl phosphate and acetyl-coenzyme A inhibits translation but not transcription using an E. coli cell-free system. Further investigations using in vivo assays revealed that acetylation does not appear to alter the rate of translation elongation but, rather, increases the proportions of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunits than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits being most pronounced during late-exponential and early-stationary-phase growth-the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell. IMPORTANCE Numerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from central metabolite acetyl-coenzyme A or acetyl phosphate to a lysine residue in a protein. This posttranslational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering protein synthesis as a new target of regulation and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.