| Summary: | The fidelity of protein synthesis is generally considered to be upheld in conditions which would not directly compromise its integrity. Although mistranslation is assumed to occur at some basal level due to inherent errors in biological processes, mistranslation is generally considered deleterious and therefore avoided at all times. However, emerging evidence has demonstrated that levels of mistranslation can increase under certain conditions. Furthermore, artificial mistranslation has shown to confer various phenotypic benefits in the presence of stress. Mistranslation with methionine engendered at the tRNA charging level by the methionyl-tRNA synthetase (MetRS) was implicated as being an especially prevalent after it was discovered in both yeast and mammalian cells as an inducible process. Yet, its prevalence and benefit in the bacterial and archaeal domains was unexplored.
Here we show that the most intensively studied organism, Escherichia coli, readily performs methionine mistranslation under anaerobic conditions that mimic its natural colonic environment and in the presence of antibiotics. We discovered that the E. coli MetRS inherently accepts nonmethionyl-tRNAs and requires a post-translational succinyl modification in one of two fidelity regulating lysine residues along the tRNA binding interface to perform high-fidelity tRNA charging. Glutamic acid mutations---which mimic the terminal carboxyl group of succinyllysine---in these lysine regulatory positions are sufficient to irreversibly render the MetRS accurate in vitro and in vivo. Strains with permanently accurate MetRSs are more sensitive to antibiotics and chemical stressors, indicating that methionine mistranslation affords powerful phenotypic benefits. To establish that methionine mistranslation could be a relevant process for colonization and subsistence in E. coli's natural habitat in the mammalian colon, we showed that several prevalent Firmicutes and Bacteroidetes members of the mouse gut microbiota performed constitutive methionine mistranslation in situ. Intriguingly, these microbiotal organisms lack the lysine MetRS regulatory sites, which mediate tRNA misacylation in E. coli .
Bioinformatic analysis of these homologous regulatory sites in all sequenced bacteria revealed diverse amino acid identities, yet most organisms contain aspartic or glutamic acids in these sites (which should mediate high-fidelity tRNA charging based on the findings in E. coli). Assessment of the Staphylococcus aureus MetRS, which contains aspartic and glutamic acids at the MetRS regulatory sites revealed that this MetRS class does indeed have inherently high-fidelity tRNA charging fidelity. However, one bacterial genus assayed for methionine mistranslation in the mouse gut microbiome had such a high-fidelity MetRS and was capable of methionine mistranslation nonetheless. This indicates that multiple molecular mechanisms exist for performing methionine mistranslation, which demonstrates the importance of evolving and maintaining such a response.
We also confirmed methionine mistranslation in the two classic archaeal phyla (Euryarchaeota and Crenarchaeota) and found that methionine mistranslation was conserved in both cases. In the hyperthermophilic Crenarchaeotal organism Aeropyrum pernix, methionine mistranslation is activated during low temperature growth, where the function of genetically encoded proteins is compromised due to their thermostability. In contrast to the site specific MetRS regulation in E. coli, the A. pernix MetRS is capable of directly responding to temperature variations to conditionally accept nonmethionyl-tRNAs. Mistranslated proteins from A. pernix synthesized during low-temperature mistranslation have increased low-temperature activity than their genetically encoded counterparts, presumably due to their increased flexibility. Altogether, these results reveal that methionine mistranslation is a conserved prokaryotic response that functions through multiple mechanisms to confer distinct phenotypic benefits to diverse organisms.
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