Methionyl-tRNA synthetase synthetic and proofreading activities are determinants of antibiotic persistence.

Bacterial antibiotic persistence is a phenomenon where bacteria are exposed to an antibiotic and the majority of the population dies while a small subset enters a low metabolic, persistent, state and are able to survive. Once the antibiotic is removed the persistent population can resuscitate and continue growing. Several different molecular mechanisms and pathways have been implicated in this phenomenon. A common mechanism that may underly bacterial antibiotic persistence is perturbations in protein synthesis. To investigate this mechanism, we characterized four distinct metG mutants for their ability to increase antibiotic persistence. Two metG mutants encode changes near the catalytic site of MetRS and the other two mutants changes near the anticodon binding domain. Mutations in metG are of particular interest because MetRS is responsible for aminoacylation both initiator tRNAMet and elongator tRNAMet indicating that these mutants could impact translation initiation and/or translation elongation. We observed that all the metG mutants increased the level of antibiotic persistence as did reduced transcription levels of wild type metG. Although, the MetRS variants did not have an impact on MetRS activity itself, they did reduce translation rates. It was also observed that the MetRS variants affected the proofreading mechanism for homocysteine and that these mutants' growth is hypersensitive to homocysteine. Taken together with previous findings, our data indicate that both reductions in cellular Met-tRNAMet synthetic capacity and reduced proofreading of homocysteine by MetRS variants are positive determinants for bacterial antibiotic persistence.

What is microbial dormancy?

Life can be stressful. One way to deal with stress is to simply wait it out. Microbes do this by entering a state of reduced activity and increased resistance commonly called 'dormancy'. But what is dormancy? Different scientific disciplines emphasize distinct traits and phenotypic ranges in defining dormancy for their microbial species and system-specific questions of interest. Here, we propose a unified definition of microbial dormancy, using a broad framework to place earlier discipline-specific definitions in a new context. We then discuss how this new definition and framework may improve our ability to investigate dormancy using multi-omics tools. Finally, we leverage our framework to discuss the diversity of genomic mechanisms for dormancy in an extreme environment that challenges easy definitions - the permafrost.

The Pros of changing tRNA identity.

The notion that errors in protein synthesis are universally harmful to the cell has been questioned by findings that suggest such mistakes may sometimes be beneficial. However, how often these beneficial mistakes arise from programmed changes in gene expression as opposed to reduced accuracy of the translation machinery is still unclear. A new study published in JBC shows that some bacteria have beneficially evolved the ability to mistranslate specific parts of the genetic code, a trait that allows improved antibiotic resistance.

At the Crossroad of Nucleotide Dynamics and Protein Synthesis in Bacteria.

Nucleotides are at the heart of the most essential biological processes in the cell, be it as key protagonists in the dogma of molecular biology or by regulating multiple metabolic pathways. The dynamic nature of nucleotides, the cross talk between them, and their constant feedback to and from the cell's metabolic state position them as a hallmark of adaption toward environmental and growth challenges. It has become increasingly clear how the activity of RNA polymerase, the synthesis and maintenance of tRNAs, mRNA translation at all stages, and the biogenesis and assembly of ribosomes are fine-tuned by the pools of intracellular nucleotides. With all aspects composing protein synthesis involved, the ribosome emerges as the molecular hub in which many of these nucleotides encounter each other and regulate the state of the cell. In this review, we aim to highlight intracellular nucleotides in bacteria as dynamic characters permanently cross talking with each other and ultimately regulating protein synthesis at various stages in which the ribosome is mainly the principal character.

Oxidative stress strongly restricts the effect of codon choice on the efficiency of protein synthesis in Escherichia coli.

Introduction: The response of enterobacteria to oxidative stress is usually considered to be regulated by transcription factors such as OxyR and SoxR. Nevertheless, several reports have shown that under oxidative stress the levels, modification and aminoacylation of tRNAs may be altered suggesting a role of codon bias in regulation of gene expression under this condition. Methods: In order to characterize the effects of oxidative stress on translation elongation we constructed a library of 61 plasmids, each coding for the green fluorescent protein (GFP) translationally fused to a different set of four identical codons. Results: Using these reporters, we observed that GFP production levels vary widely (~15 fold) when Escherichia coli K-12 is cultured in minimal media as a consequence of codon choice variations. When bacteria are cultured under oxidative stress caused by paraquat the levels of GFP produced by most clones is reduced and, in contrast to control conditions, the range of GFP levels is restricted to a ~2 fold range. Restricting elongation of particular sequences does not increase the range of GFP production under oxidative stress, but altering translation initiation rates leads to an increase in this range. Discussion: Altogether, our results suggest that under normal conditions the speed of translation elongation is in the range of the speed of initiation and, consequently, codon choice impacts the speed of protein synthesis. In contrast, under oxidative stress translation initiation becomes much slower than elongation, limiting the speed of translation such that codon choice has at most only subtle effects on the overall output of translation.

New paradigm for teaching scientific writing in STEM.

Written and oral communication are skills graduate students often request training in and supervisors often bemoan the lack of. We describe an approach to address this training gap using an instructional model that integrates experienced research-active PIs with an expert in the study and teaching of technical writing.

Escherichia coli alanyl-tRNA synthetase maintains proofreading activity and translational accuracy under oxidative stress.

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species "sinks" to protect catalytically critical sites from oxidative damage. Combined, our results indicate that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.

Characterizing the amino acid activation center of the naturally editing-deficient aminoacyl-tRNA synthetase PheRS in Mycoplasma mobile.

To ensure that correct amino acids are incorporated during protein synthesis, aminoacyl-tRNA synthetases (aaRSs) use proofreading mechanisms collectively referred to as editing. Although editing is important for viability, editing-deficient aaRSs have been identified in host-dependent organisms. In Mycoplasma mobile, editing-deficient PheRS and LeuRS have been identified. We characterized the amino acid activation site of MmPheRS and identified a previously unknown hyperaccurate mutation, L287F. Additionally, we report that m-Tyr, an oxidation byproduct of Phe which is toxic to editing-deficient cells, is poorly discriminated by MmPheRS activation and is not subjected to editing. Furthermore, expressing MmPheRS and the hyperaccurate variants renders Escherichia coli susceptible to m-Tyr stress, indicating that active site discrimination is insufficient in tolerating excess m-Tyr.

Oxidation alters the architecture of the phenylalanyl-tRNA synthetase editing domain to confer hyperaccuracy.

High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the ß-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.

Role of a cryptic tRNA gene operon in survival under translational stress.

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.

Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response.

Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNAPhe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence. IMPORTANCE Bacterial antibiotic persistence is a transient physiological state wherein cells become dormant and thereby evade being killed by antibiotics. Once the antibiotic is removed, bacterial persisters are able to resuscitate and repopulate. It is thought that antibiotic bacterial persisters may cause reoccurring infections in the clinical setting. The molecular triggers and pathways that cause bacteria to enter into the persister state are not fully understood. Our results suggest that accumulation of deacylated tRNA is a trigger for antibiotic persistence independent of the RelA-dependent stringent response, a pathway thought to be required for persistence in many organisms. Overall, this provides a mechanism where changes in translation quality control in response to physiological cues can directly modulate bacterial persistence.

Modulation of Escherichia coli Translation by the Specific Inactivation of tRNAGly Under Oxidative Stress.

Bacterial oxidative stress responses are generally controlled by transcription factors that modulate the synthesis of RNAs with the aid of some sRNAs that control the stability, and in some cases the translation, of specific mRNAs. Here, we report that oxidative stress additionally leads to inactivation of tRNAGly in Escherichia coli, inducing a series of physiological changes. The observed inactivation of tRNAGly correlated with altered efficiency of translation of Gly codons, suggesting a possible mechanism of translational control of gene expression under oxidative stress. Changes in translation also depended on the availability of glycine, revealing a mechanism whereby bacteria modulate the response to oxidative stress according to the prevailing metabolic state of the cells.

Fine-Tuning of Alanyl-tRNA Synthetase Quality Control Alleviates Global Dysregulation of the Proteome.

One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which corresponds to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. We have previously shown that defects in AlaRS proofreading of Ser-tRNAAla lead to global dysregulation of the E. coli proteome, subsequently causing defects in growth, motility, and antibiotic sensitivity. Here we report second-site AlaRS suppressor mutations that alleviate the aforementioned phenotypes, revealing previously uncharacterized residues within the AlaRS proofreading domain that function in quality control.

Translational quality control and reprogramming during stress adaptation.

Organisms encounter stress throughout their lives, and therefore require the ability to respond rapidly to environmental changes. Although transcriptional responses are crucial for controlling changes in gene expression, regulation at the translational level often allows for a faster response at the protein levels which permits immediate adaptation. The fidelity and robustness of protein synthesis are actively regulated under stress. For example, mistranslation can be beneficial to cells upon environmental changes and also alters cellular stress responses. Additionally, stress modulates both global and selective translational regulation through mechanisms including the change of aminoacyl-tRNA activity, tRNA pool reprogramming and ribosome heterogeneity. In this review, we draw on studies from both the prokaryotic and eukaryotic systems to discuss current findings of cellular adaptation at the level of translation, specifically translational fidelity and activity changes in response to a wide array of environmental stressors including oxidative stress, nutrient depletion, temperature variation, antibiotics and host colonization.

Translational regulation of environmental adaptation in bacteria.

Bacteria must rapidly respond to both intracellular and environmental changes to survive. One critical mechanism to rapidly detect and adapt to changes in environmental conditions is control of gene expression at the level of protein synthesis. At each of the three major steps of translation-initiation, elongation, and termination-cells use stimuli to tune translation rate and cellular protein concentrations. For example, changes in nutrient concentrations in the cell can lead to translational responses involving mechanisms such as dynamic folding of riboswitches during translation initiation or the synthesis of alarmones, which drastically alter cell physiology. Moreover, the cell can fine-tune the levels of specific protein products using programmed ribosome pausing or inducing frameshifting. Recent studies have improved understanding and revealed greater complexity regarding long-standing paradigms describing key regulatory steps of translation such as start-site selection and the coupling of transcription and translation. In this review, we describe how bacteria regulate their gene expression at the three translational steps and discuss how translation is used to detect and respond to changes in the cellular environment. Finally, we appraise the costs and benefits of regulation at the translational level in bacteria.

Aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases are an essential and universally distributed family of enzymes that plays a critical role in protein synthesis, pairing tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code. Synthetases help to ensure accurate translation of the genetic code by using both highly accurate cognate substrate recognition and stringent proofreading of noncognate products. While alterations in the quality control mechanisms of synthetases are generally detrimental to cellular viability, recent studies suggest that in some instances such changes facilitate adaption to stress conditions. Beyond their central role in translation, synthetases are also emerging as key players in an increasing number of other cellular processes, with far-reaching consequences in health and disease. The biochemical versatility of the synthetases has also proven pivotal in efforts to expand the genetic code, further emphasizing the wide-ranging roles of the aminoacyl-tRNA synthetase family in synthetic and natural biology.

Targeting tRNA-synthetase interactions towards novel therapeutic discovery against eukaryotic pathogens.

The development of chemotherapies against eukaryotic pathogens is especially challenging because of both the evolutionary conservation of drug targets between host and parasite, and the evolution of strain-dependent drug resistance. There is a strong need for new nontoxic drugs with broad-spectrum activity against trypanosome parasites such as Leishmania and Trypanosoma. A relatively untested approach is to target macromolecular interactions in parasites rather than small molecular interactions, under the hypothesis that the features specifying macromolecular interactions diverge more rapidly through coevolution. We computed tRNA Class-Informative Features in humans and independently in eight distinct clades of trypanosomes, identifying parasite-specific informative features, including base pairs and base mis-pairs, that are broadly conserved over approximately 250 million years of trypanosome evolution. Validating these observations, we demonstrated biochemically that tRNA:aminoacyl-tRNA synthetase (aaRS) interactions are a promising target for anti-trypanosomal drug discovery. From a marine natural products extract library, we identified several fractions with inhibitory activity toward Leishmania major alanyl-tRNA synthetase (AlaRS) but no activity against the human homolog. These marine natural products extracts showed cross-reactivity towards Trypanosoma cruzi AlaRS indicating the broad-spectrum potential of our network predictions. We also identified Leishmania major threonyl-tRNA synthetase (ThrRS) inhibitors from the same library. We discuss why chemotherapies targeting multiple aaRSs should be less prone to the evolution of resistance than monotherapeutic or synergistic combination chemotherapies targeting only one aaRS.

The mechanism of ß-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation.

ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome.

Mechanisms have evolved to prevent errors in replication, transcription, and translation of genetic material, with translational errors occurring most frequently. Errors in protein synthesis can occur at two steps, during tRNA aminoacylation and ribosome decoding. Recent advances in protein mass spectrometry have indicated that previous reports of translational errors have potentially underestimated the frequency of these events, but also that the majority of translational errors occur during ribosomal decoding, suggesting that aminoacylation errors are evolutionarily less tolerated. Despite that interpretation, there is evidence that some aminoacylation errors may be regulated, and thus provide a benefit to the cell, while others are clearly detrimental. Here, we show that while it has been suggested that regulated Thr-to-Ser substitutions may be beneficial, there is a threshold beyond which these errors are detrimental. In contrast, we show that errors mediated by alanyl-tRNA synthetase (AlaRS) are not well tolerated and induce a global stress response that leads to gross perturbation of the Escherichia coli proteome, with potentially catastrophic effects on fitness and viability. Tolerance for Ala mistranslation appears to be much lower than with other translational errors, consistent with previous reports of multiple proofreading mechanisms targeting mischarged tRNAAla These results demonstrate the essential role of aminoacyl-tRNA proofreading in optimizing cellular fitness and suggest that any potentially beneficial effects of mistranslation may be confined to specific amino acid substitutions.IMPORTANCE Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in Escherichia coli This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming.