ABSTRACT Several widespread mechanisms enable cellular communication and coordinate the behavior of bacterial populations. The modules responsible for cellular communication are often found in matching pairs: an enzyme that synthesizes a signal molecule and a specific receptor that decodes it. A notable exception is Escherichia coli SdiA, a homolog of known N-acyl-homoserine lactone (AHL) receptors, for which no corresponding AHL synthase exists. SdiA is an orphan receptor that enables cross-talk by sensing AHLs from other bacterial species. In this work, we investigated whether homocysteine thiolactone (HTL), an AHL-like molecule arising from a proofreading reaction of methionyl tRNA synthetase (MetRS) to correct misactivation of homocysteine, participates in E. coli communication through SdiA. A comprehensive analysis of MetRS variants producing different levels of HTL revealed more extended lag periods at lower HTL levels, which were restored by HTL supplementation. SdiA mutants abolished the HTL-induced regulation of the transition from the lag to the exponential phase. Growth phenotypes were HTL specific and were not induced by supplementation of other non-endogenous AHLs previously characterized as SdiA ligands. The gene expression of gadY and rmf , regulated by SdiA, was modulated by HTL supplementation. Through differential scanning fluorimetry, we show the SdiA-HTL interaction in vitro . Together, our observations describe HTL as an SdiA ligand that modulates cell-cell communication in bacteria. IMPORTANCE Bacteria need to adapt to different environments and stress conditions, and quorum sensing (QS) is a widespread and well-established mechanism that enables cell-to-cell communication to coordinate these changes. The modules responsible for QS usually evolve in pairs: an enzyme that synthesizes the signal and a specific receptor that decodes it. A notable exception is Escherichia coli SdiA, a homolog of known N-acyl-homoserine lactone (AHL) receptors for which no corresponding AHL synthase exists. It has been hypothesized that SdiA is an orphan receptor that enables crosstalk by sensing AHLs from other bacterial species. We present data suggesting that homocysteine thiolactone, a ubiquitous molecule arising from homocysteine editing during translation proofreading, can mimic AHLs and participate in bacterial cell-cell communication via SdiA. The near-universal distribution of this proofreading mechanism suggests that homocysteine thiolactone provides a common link between protein synthesis and cell-cell communication in both homologous and heterogeneous microbial populations.

Para-benzoquinone (pBQ) is of growing concern as an emerging redox-active environmental pollutant due to its ubiquitous presence in smoke and combustion byproducts. Recent reports have highlighted its potential role as a redox-driven mitotoxicant, although the involvement of specific mitochondrial protein targets remains unexplored. Here, we investigated the effects of pBQ on human mitochondrial phenylalanyl-tRNA synthetase (hmtPheRS), an essential enzyme required for mitochondrial protein synthesis and linked to severe neurodevelopmental disorders. Our biophysical analyses revealed that pBQ enhanced the formation of covalently modified higher-ordered structures of hmtPheRS by 75% and induced conformational instability, thereby significantly reducing its aminoacylation activity. NMR spectroscopy and molecular docking analyses further supported interactions between pBQ and residues within the catalytic domain of hmtPheRS, indicating the formation of a protein adduct. In parallel, exposure of HEK293 cells to sublethal concentrations of pBQ (20-40 μM) resulted in altered cellular redox homeostasis. It also impaired mitochondrial membrane potential and respiration, disrupted mitochondrial dynamics, and activated mitophagy. Consistent with the broad reactivity of pBQ and its ability to induce oxidative stress, these findings suggest that hmtPheRS is a vulnerable mitochondrial target whose modification may contribute to mitochondrial dysfunction, together with other redox-dependent pathways. Together, this work highlights mitochondrial aminoacyl-tRNA synthetases as an underexplored class of proteins susceptible to redox-active environmental pollutants.

The aminoacyl-tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various translational and non-translational activities beyond aminoacylation, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein. Across the tree of life, the 22 coded amino acids are handled by 16 evolutionary families of Class I aaRS and 21 families of Class II aaRS. We introduce AARS Online, an interactive Wikipedia-like tool curated by an international consortium of field experts. This platform systematizes existing knowledge about the aaRS by showcasing a taxonomically diverse selection of aaRS sequences and structures. Through its graphical user interface, AARS Online facilitates a seamless exploration between protein sequence and structure, providing a friendly introduction to the material for non-experts and a useful resource for experts. Curated multiple sequence alignments can be extracted for downstream analyses. Accessible at www.aars.online, AARS Online is a free resource to delve into the world of the aaRS.

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.

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.

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.

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.

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.

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.

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 proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.

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.

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.

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 β-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.

The bacterial response to oxidative stress requires the adaptation of the proteome to the hostile environment. It has been reported that oxidative stress induces a strong and global inhibition of both, transcription and translation. Nevertheless, whereas it is well known that transcription of a small group of genes is induced thanks to transcription factors such as OxyR and SoxR, an equivalent mechanism has not been described for translation. Here we report that whereas canonical translation that depends on Shine Dalgarno recognition is inhibited by oxidative stress in Escherichia coli, the translation of leaderless mRNA (lmRNA) is enhanced under such conditions. Both, inhibition of canonical translation and enhancement of lmRNA translation, depend on the production of (p)ppGpp. We propose that such a mechanism would allow bacteria to rapidly adapt their proteome to hostile conditions and is, perhaps, a general strategy to confront strong stressful conditions. Significance statement The regulation of translation (the production of proteins based on genetic information) is central for the adaptation to environmental changes. In Escherichia coli translation may begin through two alternative pathways. 1.- A canonical initiation that is well understood and is regulated mostly by changes in the accessibility of ribosomes to specific sequences and 2.- Initiation of leaderless mRNAs (lmRNAs) that lack these sequences and for which we do not understand the regulation process. Our results indicate that under oxidative stress, the production of (p)ppGpp in E. coli inhibits canonical translation and simultaneously enhances translation of lmRNAs, showing for the first time a natural condition where lmRNA translation is regulated and a role for (p)ppGpp in this process.

β-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.

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.

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.