Advertisement

  • Loading metrics

Open Access

Review

Review articles synthesize the best available evidence on a topic relevant to the pathogens community.

See all article types »

tRNA modifications as regulators of bacterial virulence and stress responses

Abstract

Bacteria are recognized for their ability to adapt their lifestyle to the environment. Specifically, when considering pathogenic bacteria, their capacity to respond to stress and switch to a virulent state through gene regulation is crucial. One of the mechanisms that enables regulation of gene expression at the translational level is RNA modification. These chemical changes produced by specific enzymes are present on all types of RNAs and can modulate translational efficiency by influencing the structure of RNA molecules, the codon usage bias, the interaction with other molecules, or the efficiency of ribosome action. Transfer RNA (tRNA) is the most modified RNA in the cell, with modifications in the core body of the tRNA primarily affecting the stability and flexibility of the structure while modifications in the anticodon stem-loop (ASL) are more involved in decoding, as well as the efficiency and fidelity of translation. Given the impacts of these modifications on the translation process and the critical role of modulating translation fidelity during bacterial stress responses and host interactions, tRNA modifications play an important role in regulating the expression of virulence factors in bacterial pathogens, resulting in changes in various phenotypes. This review aims to establish a comprehensive landscape of tRNA modifications and their direct impact on the translation process, emphasizing their significant role in bacterial virulence and stress responses.

Citation: Teixeira C, Vandenesch F, Moreau K (2025) tRNA modifications as regulators of bacterial virulence and stress responses. PLoS Pathog 21(10): e1013600. https://doi.org/10.1371/journal.ppat.1013600

Editor: Hayley Newton, Monash University, UNITED STATES OF AMERICA

Published: October 23, 2025

Copyright: © 2025 Teixeira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the French National Research Agency ANR (SaRNAmod:ANR-21-CE12-0030-01 to FV; RHU IdBioriv: ANR-18-RHUS to FV), and by Fondation pour la Recherche Médicale FRM (FDT202404018387 to CT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interest exists.

Introduction

Pathogenic bacteria are responsible for various infections and quickly adapt to different environments thanks to the numerous virulence factors they possess [1,2]. These virulence factors are finely regulated to be produced at the right time and place during infection thanks to various regulatory systems that impact gene expression at different levels. At the transcriptional level, transcription factors or epigenetic modifications that target DNA are the main actors; at the post-transcriptional level, an important mechanism is the alteration of messenger RNA (mRNA) translation by regulatory RNAs that impact both the structure and stability of mRNA [3]. Finally, at the translational level regulatory systems can modulate the translation process thanks to regulatory RNAs, riboswitches, ribosomal proteins, and RNA modifications [4].

RNA modifications, first discovered in eukaryotes and subsequently in prokaryotes, are present on all types of RNA and can be added or removed by specific modifying enzymes [5]. Since the modifications are present on transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which play a central role in ensuring the fidelity and efficiency of protein synthesis [6], they are involved in modulating translation efficiency and can alter or enhance protein production [7]. tRNA modifications represent a system for pathogenic bacteria to regulate gene expression, particularly in shaping the virulence of bacterial pathogens by impacting the production of virulence factors and stress response proteins. Herein, we illustrate the diversity of tRNA modifications in bacteria, their direct impact on the translation process, and how they regulate the virulence program and stress responses during bacterial infection.

Variety and complexity of tRNA modifications in bacteria

The identification of the first DNA modification in eucaryotes was made in 1950 with the finding of a non-canonical base (5-methylcytosine) [8]. This was followed closely by the discovery in 1956 of the first RNA modification in yeast with the transformation of uridine in pseudouridine [9], and there has since been a steady increase in the number of new types of RNA modification [5]. To date, a total of 433 different modified RNA residues have been identified in the organisms of the three domains of life, including 71 modifications specific to eukaryotes, 17 to archaea, and 68 to bacteria; the remaining 277 are common to all three domains, as listed in the MODOMICS database. tRNA modifications represent slightly more than half (53.6%) of these RNA modifications [7].

There is an increased interest in studying tRNA modifications owing to their role in bacterial physiology. Thus, recent reviews have primarily focused either on specific tRNA modifications [10,11] or on the functional consequences of these modifications on the translation process [12]. Other recent contributions have highlighted the technical challenges associated with studying tRNA modifications [13], or have provided broader overviews of RNA modifications across different RNA species [14,15]. Given the rapidly evolving knowledge in this field, we present herein an update on the tRNA modifications identified in bacteria, with a focus on pathogenic bacteria, and illustrate their notable impacts with various examples.

Which modifications are present on bacterial tRNA?

A diverse range of modifications can be made to tRNAs and the first glossary cataloguing these modifications was published in 1994 by Limbach and colleagues [16]. In the domain of bacteria, these modifications, which can be permanent or transitory, vary from elementary chemical alterations to more intricate modifications involving the incorporation of complex groups (Table 1).

thumbnail
Download:
Table 1. Summary of bacterial tRNA modifications and their associated enzymes.

https://doi.org/10.1371/journal.ppat.1013600.t001

Nucleotide methylation is the most common type of modification, as it occurs on all four canonical nucleotides. Regarding the nitrogenous bases, adenine can be methylated at various positions (m1A, m2A, and m6A) as can guanine (m1G, m2G, and m7G), whereas uridine and cytosine are only modified at one position (m5U and m5C). Methylation also occurs on the ribose part of the four nucleotides, this process is known as a 2′-O-methylation (Am, Gm, Um, and Cm) [10,17,18].

Certain modifications are specific to a single type of nucleotide. For example, isomerization of the base to form pseudouridine (ψ) [19] or the hydrogenation to form dihydrouridine (D) [20] are specific to uridine. Moreover, deamination reaction to form inosine (I) [21] and the addition of a threonylcarbamoyl group (t6A) [22] are specific to adenosine while the complex modification into queuosine (Q) is specific to guanosine [23]. Similarly, the acylation reaction is specific to cytosine (ac4C) [10]. Other modifications involve the addition of a sulfur atom with the thiolation at position 2 for cytidine (s2C) [24] and at position 2 or 4 for uridine (s2U and s4U) [25].

Among this variety of modifications, some are sequential and require the completion of an initial modification prior to their occurrence. This is the case of the methyl thiolation of adenosine (ms2i6A) that necessitates the previous addition of an isopentyl group to form i6A [26]. Similarly, the acacp3U modification discovered in Vibrio cholerae (V. cholerae) is an acetylated form of the acp3U modification [27]. Other examples include the successive reactions that modify uridine to create mnm5s2U and then cmnm5s2U [28], or the hydroxylation at position 5 to form ho5U, followed by the methylation at the same position to produce mo5U, and finally the carboxylation to generate cmo5U [29]. Notably, certain modifications do not necessitate an initial modification to arise; however, the existence of specific modifications can facilitate their occurrence. For example, in Escherichia coli (E. coli), acp3U47 is stimulated by m7G46 and m5U54, as is ms2i6A37 by m5U54, and s4U8 by ψ55 and m5U54 [30].

Bacteria therefore exhibit a diversity of tRNA modifications, some of which are exclusive to their kingdom. Among the modifications cited above, m2A, s2C, s4U, ms2i6A, mnm5s2U, ho5U, mo5U, and cmo5U are specific to bacteria [7,10,18,31].

What achieves these modifications?

Specific enzymes catalyze the chemical reactions to modify tRNAs at precise sites (Table 1). The first tRNA-modifying enzyme was published in 1962 and 1963 by two groups, Borek in the USA and Svensson in Sweden [5]. This enzyme, known in bacteria as TrmA methylates the uridine at position 54 of almost all tRNAs to form m5U54 [34,35].

Improvements in the detection of tRNA modifications and next-generation sequencing [36–38], combined with comparative genomics and structural studies, have facilitated the comprehensive mapping of enzymes with the corresponding tRNA modifications in model bacteria such as E. coli and Bacillus subtilis (B. subtilis) [39]. Recently, the predicted landscape of tRNA modifications has been explored and partially validated in other bacteria, such as Pseudomonas aeruginosa (P. aeruginosa) [40], two Bartonella species [41], and Streptomyces albidoflavus [42]. Additionally, tRNA modifications have been identified in Staphylococcus aureus (S. aureus) [43].

Most enzymes involved in RNA modifications target a single type of RNAs, typically tRNAs or rRNAs, and less commonly other non-coding RNAs and mRNAs. However, there are a few notable exceptions where certain enzymes perform the same type of modification on multiple types of RNA. For example, RlmN in E. coli catalyzes m2A37 in tRNA and m2A2503 in 23S rRNA [44]; and RluA catalyzes the formation of pseudouridine at position 32 in tRNA and 746 in rRNA [39]. Another dual-specificity enzyme is the TadA enzyme that deaminates adenosine to inosine at position 34 of Arg-tRNA and also catalyzes the formation of inosine in bacterial mRNA [21,45]. Interestingly, certain enzymes act specifically on tRNAs but are responsible for two types of modifications. This is the case of TilS in V. cholerae, which catalyzes both the k2C34 and the ava2C34 modifications in tRNA-Ile [46,47].

These enzymes are rather conserved among bacteria and most of the modifications identified so far are shared between species. Nevertheless, a comparison of the tRNA modification landscape of E. coli and B. subtilis shows that the modifications of tRNAs from Gram-positive bacteria are less diverse than that from Gram-negative bacteria, and some modifications appear to be specific to one of the two species. For instance, ψ55 and Gm18 modifications are only detected in E. coli whereas m1A22 and ms2t6A37 modifications are specific to B. subtilis [39].

Which positions on the tRNA are subject to these modifications?

tRNAs are the most modified RNAs, harboring an average of 14 modifications per molecule, equivalent to one modification every three nucleotides [48]. Among the four major parts of tRNA secondary structure, while the D- and T-arms possess some modifications, the most modified region is the anticodon stem-loop (ASL) within which two residues are hypermodified, the first at position 34 (the wobble base, the third of the anticodon) and the second at position 37 (adjacent to the anticodon; Fig 1). For example, in E. coli, 16 distinct modifications have been identified at position 34, while eight have been found at position 37. In B. subtilis, 12 different modifications have been detected at position 34, and nine at position 37 [39].

thumbnail
Download:
Fig 1. Map of bacterial tRNA modifications and their impact on translation.

The secondary structure of tRNA is numbered each five positions and is composed of six parts. The acceptor-stem is colored in gray, the D-arm in purple, the variable loop in orange, the T-arm in pink, the anticodon stem-loop in blue, and the anticodon in green. The effects of the modifications on translation mentioned in this review and previously known are indicated as follows. The modification is in duck blue if it affects tRNA structure; is marked with an asterisk if it affects reading frame maintenance or readthrough; and is underlined if it affects codon decoding.

https://doi.org/10.1371/journal.ppat.1013600.g001

Certain modifications are specific to one region, such as dihydrouridine in the D-arm or inosine and queuosine that are only present at position 34. This indicates that each modification has a specific role and effect on tRNA activity; modifications present in the core body (elbows and arms) of the tRNA are implicated in the stability and flexibility of the structure whereas modifications in the ASL are involved in decoding aspects, as well as efficiency and fidelity of translation [49] (Fig 1). Furthermore, it is important to note that not all tRNAs exhibit all types of modifications, and most modifications are specific to one or a few different tRNAs [7].

Effects of tRNA modifications on the translation process

Translation, along with replication and transcription, is one of the three fundamental molecular processes essential for cell viability and is composed of four steps. Firstly, translation is initiated with the assembly of the ribosome complex on the produced mRNA at the start codon. Then, the elongation of the new peptide requires the correct decoding of the mRNA codons by tRNA molecules until a stop codon is reached. Termination factors then hydrolyze the ester bond of the peptidyl tRNA to produce the new protein and dissociate the complex for ribosome recycling [6]. A few regulatory systems can modulate the process. For example, some regulatory RNAs, ribosomal proteins, and riboswitches can prevent the formation of the ribosomal complex or induce specific mRNA structural changes to control the initiation step [4]. An additional regulatory mechanism involves tRNA modifications, as they affect tRNAs structure and stability, thereby impacting the fidelity and efficiency of translation, in which tRNAs play a central role [10].

How tRNA modifications impact tRNA structure?

The structural characteristics of tRNA, including its 2D cloverleaf and 3D L-shaped structure, are crucial for translation, as they allow interactions with mRNA, ribosomes, and aminoacyl-tRNA synthetases. To maintain these structural characteristics, tRNA modifications are required. For example, in mitochondrial tRNA-Lys, the presence of m1A9 modification disrupts Watson-Crick base pairing to allow formation of secondary and tertiary structures [50]. In bacteria, 2′O-methylation, thiolation, pseudouridylation [30], and m5U modifications [51] stabilize the structure of tRNA by inducing conformational rigidity through an improvement of base stacking interactions [52]. In contrast, dihydrouridine is the only known modification that promotes flexibility of tRNA structure [20]. Additionally, ψ32 and ψ39 are involved in the correct ASL structure of E. coli tRNAs [53].

Moreover, it has been demonstrated that certain modifying enzymes have a chaperone function that facilitates the folding of tRNAs such as TruB (ψ) and TrmA (m5U) enzymes in E. coli that contribute to the correct folding of tRNAs, even in the absence of their catalytic activity [54,55].

How tRNA modifications affect readthrough and reading frame maintenance?

During translation, recognition of stop codons and maintenance of the reading frame are critical to ensure the accurate protein production. Modifications in the ASL region are important to prevent readthrough. In E. coli, it has been reported that the absence of Q34 or m2A37 leads to an increase of readthrough at the UAG stop codon [44,56]. ASL modifications are also crucial for reading frame maintenance. In E. coli, the absence of m1G37 in tRNA-Pro results in an incorrect translation of proline, particularly for CCC and CCU codons as this modification prevents the ribosome from stalling on the mRNA, thereby avoiding a + 1 frameshift and premature termination [57,58]. In extraintestinal pathogenic Escherichia coli (ExPEC) strains, the amount of i6A37 modification catalyzed by the MiaA enzyme modulates −1 and +1 frameshifts; a deletion or over-expression of miaA increases frameshifts errors [59]. Another modification at residue 37, ms2i6A37, prevents frameshifting by stabilizing the interaction between the tRNA anticodon at position 34 and the mRNA codon at position 1 [60]. Similarly to ms2i6A37, the mnm5s2U34 modification avoids frameshifting by increasing the affinity of the codon-anticodon interaction [61]. In addition, the formation of queuosine (Q) or the thiolation of uridine (s2U) at position 34 participates in reading frame maintenance [60,62].

How tRNA modifications influence decoding and codon usage bias?

The degeneracy of the genetic code is linked to crucial interactions between the wobble base of the mRNA codon and the anticodon first base at residue 34. These interactions are influenced by tRNA modifications within or near the anticodon, which are essential for ensuring the fidelity of translation by affecting decoding aspects. A notable example in bacteria is the queuosine modification which replaces guanosine at position 34 of tRNA-GUN that decodes NAC and NAU codons (Asp-GAC/GAU, Asn-AAC/AAU, Tyr-UAC/UAU, and His-CAC/CAU). In E. coli, G34-tRNAs preferentially decode NAC over NAU codons while Q34-tRNAs exhibit an enhancement of translational efficiency for NAU codons. Thus, queuosine has the capacity to suppress the initial codon usage bias, exhibiting a 20% decrease in NAU decoding in the absence of Q34 and a 34% increase in over-modified tRNAs [63]. However, the opposite has been demonstrated in V. cholerae, particularly for UAU codons of tyrosine that are more efficiently decoded compared to UAC codons in absence of queuosine [64]. Among the other examples, the absence of the MiaA enzyme, responsible of i6A37 modification, alters the decoding of UXX and UUA codons in Streptomyces albus (S. albus) [65] and its presence is required for the correct decoding of Leu-UUX codons in E. coli [66]. Still in E. coli, the s2U34 modification enhances the binding affinity of the tRNA-Gln for CAA and CAG codons [62], and the mnm5s2U34 modification on tRNA-Lys(UUU) stabilizes non-canonical base pairing [67]. In Mycobacterium strains, the increase of cmo5U-modified tRNA-Thr(UGC) induces a preferential translation of ACG over ACA and ACU codons [68]. Moreover, it has been demonstrated that a gidA (now named mnmG) mutant of P. aeruginosa, deficient for cmnm5U34 modification, shows a decrease of translation efficiency for Arg-AGA and Leu-UUA codons that are rare codons [69].

One way for tRNA modifications to enhance decoding efficiency, without favoring specific codons, is by increasing codon degeneracy. For instance, the TadA enzyme catalyzes the deamination of adenosine at position 34 of tRNA-Arg(ACG) into inosine, and this modified nucleotide is then read as a guanosine in sequencing reactions. The presence of inosine facilitates the recognition of CGA, CGU, and CGC codons, and induces a more efficient translation of arginine codons [70]. Furthermore, this extension of codon degeneracy allows the bacteria to reduce the requirement of tRNA-genes [71].

It is worth noting that while tRNA modifications predominantly have a positive influence on translation by affecting structure, fidelity, and efficiency, certain modifications have the potential to exert undesirable effects. In E. coli, mnm5s2U and ψ modifications respectively induce misreading of aspartate and histidine codons, creating mistranslation errors [72,73].

How modifications provide a quality control for tRNAs?

Given that RNA modifications are essential for the formation and stabilization of tRNA molecules, the status and extent of these modifications can serve as a quality control mechanism for tRNAs. In eukaryotes, the recognition and elimination of hypo- or hyper-modified tRNAs is facilitated by specific RNA degradation systems, including the nuclear surveillance pathway and the rapid tRNA decay pathway. For example, the absence of certain tRNA modifications act as a signal molecule for these two systems in eukaryotes [30]. In Vibrio cholerae, the absence of s4U modification in the elbow of tRNAs results in the decay of hypo-modified tRNAs by the RNA degradosome system [74]. Another way to ensure tRNAs quality control has been described in E. coli where the presence of m5U54 contributes to tRNA maturation as its absence is associated with an altered modifications pattern and impaired ribosome translocation [75].

Impact of tRNA modifications on bacterial virulence

Bacteria are well known for adapting their lifestyle to the environment. Specifically, when considering pathogenic bacteria, their ability to respond to stress and switch to a state of virulence is key for manipulating, killing, and/or surviving within theirs hosts [1,2]. These aspects involve numerous proteins and effectors that must be finely regulated to ensure that each virulence factors are expressed at the right place and time during infection. As described above, one way of regulating gene expression is at the translation level. Additionally, the modulation of translation fidelity is imperative under stress exposure and during host interaction as, under optimal conditions, amino acid misincorporation is around 0.01% and the readthrough frequency around 0.5%; whereas under stressful conditions, the misincorporation rate increases to 1% and readthrough errors to 10% [76].

Considering the significant and varied impacts of tRNA modifications on the translation process and the translation of specific codons, their influence on the production of stress responses and virulence factors in pathogenic bacteria is of particular interest.

Are tRNA modifications essential?

Most enzymes responsible for tRNA modifications are not essential under optimal growth laboratory conditions and a significant number of phenotypes linked to tRNA modifications have been discovered under stress conditions, suggesting a strong implication in cellular fitness despite their non-essential nature [49] (Fig 2 and S1 Table).

thumbnail
Download:
Fig 2. tRNA modifications drive bacterial virulence and stress adaptation.

The effects of the tRNA modifications on bacterial physiology mentioned in this review are listed under the six categories described. The modifying enzymes are listed with their modifications in parentheses, and the related bibliographic references are indicated with superscripts. The illustrations in this figure were created using BioRender: Moreau, K. (2025) https://BioRender.com/3ohwa91.

https://doi.org/10.1371/journal.ppat.1013600.g002

Nevertheless, there are tRNA-modifying enzymes that are essential in the model bacteria E. coli and B. subtilis. They mostly modify the ASL: TilS (k2C34), TsaBCDE (t6A37), and TrmD (m1G37) [39]. Enzymes can also be essential only for some species such as TadA (I34) that is essential for E. coli and B. subtilis but not for S. aureus [21,77].

Moreover, certain tRNA modifications have synergistic effects as double knockout mutants showed an exacerbation of the phenotype observed in single knockout mutants. For example, in E. coli, five pairs of modifications were identified as essential, as their double knockout mutants exhibited no growth. Among these five pairs, which represents 1% of the tested combinations, four include ASL-modifying enzymes. Notably, TruA is the most frequent, catalyzing the ψ39 and ψ40 modifications that serve as quality control of tRNAs. More generally, it is important to note that the lethality of the tested pair depends on the growth conditions [78]. The requirement for tRNA modifications depends on conditions such as culture medium, temperature, and substrate. For instance, levels of queuosine modification in Streptococcus mutans (S. mutans) are influenced by medium composition with the micronutrients content [79].

How do tRNA modifications alter growth and metabolism?

Metabolism is a key determinant of virulence as it allows bacteria to adapt and persist within its host. In ExPEC strains, the MiaA (i6A37) enzyme is finely regulated as it modulates frameshift level to acquire metabolic flexibility in vivo. This regulatory process creates a fitness advantage during gut colonization, urinary tract infections, and bloodstream infections [59]. MiaA is also involved in morphogenesis of certain bacteria as its inactivation leads to delayed hyphal development and spore formation in Streptomyces [65]. Moreover, in Streptomyces ghanaensis (S. ghanaensis), ms2i6A37 modification by MiaB, which occurs following MiaA modification, modulates morphology development by increasing translation efficiency of rare codon Leu-UAA in life cycle genes [80]. Queuosine (Q34) modification is also implicated in metabolism, as overexpression of queF confers a fitness advantage for P. putida over E. coli [63] and its absence reduces the viability of E. coli during the stationary phase [81].

How do tRNA modifications allow bacteria to adapt to temperature?

During growth or infection, bacteria may encounter varying temperatures, thus it is crucial for them to respond effectively to these temperature shifts. Most of the tRNA modifications involved in this aspect are in the arms of tRNAs and have an impact on structure maintenance. Ψ55 modification made by the TruB enzyme is critical for the survival of E. coli at temperatures over 37 °C. Deficiency in this modification alters significantly the proteome, particularly affecting outer-|membrane proteins [82]. To better understand the role of tRNA modifications in temperature adaptation, various studies have been conducted on nonpathogenic thermophilic species. In Thermus thermophilus (T. thermophilus), TruB is essential for adaptation to low temperatures, as the ΔtruB mutant presents an impaired growth at 55 °C. Moreover, TruB regulates the amount of other tRNA modifications at 55 °C such as Gm18, m5s2U54, and m1A58, therefore impacting tRNA structure and melting temperature [83]. Certain of these modifications contribute also to the survival of E. coli at high temperatures including Gm18 by the TrmH enzyme and m5U54 by the TrmA enzyme [84].

Thermophilic bacteria require appropriate cellular systems for growth in hot environments. RNA modifications play a role in this resistance and, interestingly, some of these modifications are even specific to thermophilic species. For instance, the m5s2U54 modification is exclusively found on the tRNAs of thermophiles and is essential for protein production at high temperature by increasing the melting temperature of tRNAs [85]. Its content is finely regulated to ensure a balance to adapt growth to different temperature gradients. At low temperatures, there is a higher proportion of m5U54 compared to m5s2U54, while at high temperatures, the opposite is found [86]. Other modifications, not specific to thermophilic bacteria, have been also described as essential for T. thermophilus growth at high temperatures such as m1A58 by the TrmI enzyme at temperatures above 80 °C [87] and m7G46 by the TrmB enzyme at temperatures above 70 °C [88].

A comparison of RNA modifications presents in psychrophiles, mesophiles, and thermophiles belonging to the Bacillales at minimal, optimal, and maximal temperatures reveals distinct profiles. In thermophiles, the amount of s4U8 and Ψ55 modifications increase under optimal growth temperature to confer more rigidity to the tRNA structure. In contrast, at minimal growth temperature for mesophiles and psychrophiles, only an increase of dihydrouridine modifications is observed which allows a more flexible tRNA structure to survive in cold environment [89].

How do tRNA modifications modulate the virulence levels of bacteria?

Bacterial virulence is illustrated by numerous aspects, such as infection capacity, cytotoxicity, and survival in animal models. GidA (MnmG), that catalyzes multiple tRNA modifications, has been identified as a key enzyme involved in the regulation of overall virulence in various pathogenic bacteria. For instance, in Streptococcus pyogenes (S. pyogenes), its inactivation reduces protease expression, affecting the global proteome and decreases the translation of virulence factors, thus reducing virulence in the murine ulcer model [90]. In Salmonella enterica (S. enterica), a similar impact was observed as the lack of the GidA (MnmG) enzyme leads to pleiotropic phenotypes ultimately illustrated by a decrease of cytotoxicity and lower intracellular survival in the murine model [91]. GidA (MnmG) also promotes virulence via directly enhancing translation of bacterial toxins such as cytotoxic necrotizing factor 1 (CNF1) in E. coli [92] and Act in the food pathogen Aeromonas hydrophola (A. hydrophola) [93]. Several studies have also demonstrated the implication of the GidA (MnmG) enzyme in the virulence of P. aeruginosa, in vitro and in vivo. Firstly, Gupta and colleagues observed in 2009 that GidA (MnmG) impacts quorum-sensing gene expression by regulating the translation efficiency of RhlR, one of the two major quorum-sensing systems [94]. Then, Srimahaeak and colleagues reported in 2023 that GidA (MnmG) is required for the full bacterial virulence in macrophages in vitro and in Caenorhabditis elegans in vivo [95]. Lastly, Krueger and colleagues reported in 2024, that the absence of GidA (MnmG) results in an avirulent P. aeruginosa strain in the Galleria mellonella model and exhibiting a fitness disadvantage during mouse infection due to a reduced cytotoxicity against macrophages [69].

While the role of GidA (MnmG) in bacterial virulence is evident, it is not the only one. Several other enzymes also play crucial roles in modulating bacterial infection. For instance, in Shigella flexneri (S. flexneri), MiaA and Tgt enzymes favor translation of the major regulator VirF to increase pathogenicity [96] and the absence of TruB enzyme reduces the expression of several virulence genes [84]. Furthermore, the presence of s2U34 modification, made by the MnmA enzyme, in Mycobacterium tuberculosis (M. tuberculosis) enhances intracellular growth in macrophages. Interestingly, this benefit was not observed under optimal growth conditions, indicating that this modification is only required for growth in infectious contexts [97]. Another example is the TrmB (m7G46) enzyme that promotes Acinetobacter baumannii (A. baumannii) adaptation, as its absence results in decreased virulence in mice and reduced replication within macrophages [98].

Interestingly, bacteria can also use regulation of tRNA modifications to prevent phage infection. It has been described for the first time that the lack of m6A modification in E. coli reduces the infection efficiency of T5 phage [99].

How do tRNA modifications alter the formation of biofilm and bacterial motility?

Biofilm is a strategy employed by bacteria to survive in hostile environments, by adhering to a surface and aggregating within an extracellular matrix. This process enables the bacterial population to evade the immune system and antimicrobial agents, making it a crucial aspect of bacterial virulence [100]. As a key component of bacterial pathogenicity, biofilm is also impacted by tRNA modifications. For instance, biofilm formation is stimulated by Q34 modification that increases bacterial aggregation in E. coli and teichoic acid production in B. subtilis [63]. The inactivation of GidA (MnmG) or MnmE enzymes in S. mutans also reduces glucose-dependent biofilm biomass by 50% due to impaired initial surface attachment [101]. Similarly, GidA (MnmG) influences biofilm formation in P. aeruginosa, where the absence of cmnm5U34 modification alters biofilm structure, leading to more dead bacteria at the base and an overall reduction in the amount of biofilm [69,95].

Motility is another advantage during infection as it allows bacteria to disseminate within the host. As for biofilm formation, the GidA (MnmG) enzyme stimulates bacterial motility by improving translation efficiency of flagella proteins of P. aeruginosa [69,95]. Likewise, GidA (MnmG) inactivation decreases S. enterica motility by a factor of two [91,102]. Two other tRNA-modifying enzymes, TadA and TrmH, are implicated in bacterial motility regulation. TadA usually modifies A34 to I34; however, in the rice pathogen Xanthomonas oryzae, tadA induction after H2O2 stress exposure, increases S128C mutation in FliC, which modifies flagellar structure and increases motility [103]. In contrast, absence of Gm18 modification made by TrmH in E. coli enhances expression of motility genes, associated with an increase of swarming [104].

How do tRNA modifications impact stress responses?

Bacteria are exposed to different stress during infection such as oxidative and osmotic stresses, variation of pH, metal starvation or excess, and antibiotics [105]. They have developed many strategies to adapt their gene expression in response to these exposures, with tRNA modifications being a notable example. In E. coli, MiaA (i6A37), TrmL (Cm34 and Um34), and TusA (s2U34) enzymes allow the translation of the major regulator of stress response, the sigma factor RpoS. Indeed, RpoS possess a bias toward the Leu-UUX codon, necessitating Cm/Um34 and s2U modifications for efficient and accurate translation. As Cm34/Um34 only occur when i6A37 is present on tRNAs, all three modifications are required for the translation of the sigma factor [66,106]. In a similar manner, i6A37 supports the correct translation of another major regulator, Hfq chaperon protein [106]. MiaA in ExPEC strains is also involved in various stress responses as the presence of the enzyme stimulates growth under nitrosative, oxidative, hyper- and hypo-osmotic conditions [59], possibly due to its influence on the translation of RpoS and Hfq.

Reactive oxygen species (ROS) are the products of oxidative stress and alters biomolecules to impact metabolism, causing significant cellular damage [107]. Bacteria use diverse systems to counteract ROS, and RNA is recognized as a central signaling molecule [108]. Several modifying enzymes, such as TrmJ (Am32, Cm32, and Um32) and TtcA (s2C32), have been described to favor P. aeruginosa resistance to H2O2 exposure by up-regulating expression and activity of catalase. Furthermore, ttcA is upregulated under oxidative stress [109,110]. TrmB is also essential for bacterial survival under oxidative stress as its modification, m7G46, is raised following H2O2 exposure, leading to the increase of translation efficiency of specific codons present in the catalases KatA and KatB [111]. A comparable effect on the translation of these catalases has been observed for GidA (MnmG) and mnm5U34 modification [95]. Another example can be found in A. baumannii, in which a decrease in m7G46 modification impairs growth under oxidative stress by affecting translation. This modification enhances the translation of ion transport and metabolic proteins, while its absence promotes the translation of transcription and replication proteins to compensate for altered protein production [98]. More generally, all-but-three RNA modifications decrease after oxidative stress exposure in E. coli. One of these three, m5C49, rises under stress, accompanied by an upregulation of its modifying enzyme RsmF, enhancing bacterial resistance to oxidative stress [112].

Regarding metal stress, mnm5s2U34 modification catalyzed by the TusA enzyme in E. coli promotes translation of the Fur regulator that represses expression of siderophore genes and activates those involved in iron storage [113]. Additionally, Q34 modification is responsible for pleiotropic effects regarding metal stress as its absence enhances resistance to nickel and cobalt as well as sensitivity to cadmium [114]. Under osmotic and acidic stress inactivation of the GidA (MnmG) enzyme reduces growth of S. mutans [101]. The same outcome under acidic stress has been observed in the absence of TrmB as the lack of the enzyme leads do a reduction of A. baumannii proliferation at pH5, associated with decrease in replication in macrophages [98]. Another type of stress encountered during infection is hypoxia. For Mycobacterium bovis (M. bovis), the major regulator of hypoxia, DosR, is enriched in codons ACG that need cmo5U modification on tRNA-Thr(UGU) to be efficiently translated. Intriguingly, the amount of cmo5U and tRNA-Thr(UGU) increases under hypoxia [68].

How do tRNA modifications influence antibiotic susceptibility?

Antibiotic resistance is a major problem in healthcare. Pathogens are rapidly developing new resistance mechanisms, which outpace the development of new drugs, making infections more difficult to treat. In V. cholerae and, to a lesser extent in E. coli, tRNA and rRNA modifications impact antibiotic resistance and growth under sub-inhibitory concentrations of various antibiotic classes. However, the effects observed vary according to the antibiotic, the enzyme, and the species [115]. Queuosine is one of the modifications involved in aminoglycoside tolerance in V. cholerae, as tgt inactivation results in a growth defect under sub-minimal inhibitory concentrations of tobramycin, and tgt is overexpressed under similar stress conditions [64]. RsxA is a stress response inhibitor that reduces bacterial survival and is enriched in Tyr-TAT codons (83% vs. 53% on average) and, conversely, housekeeping genes are more prone to a TAC bias, which require Q34 for an efficient translation. Therefore, Tgt and Q34 promote bacterial survival under tobramycin stress by reducing the translation of RsxA and increasing that of housekeeping proteins [64]. In addition, Q34 modification has been shown to favor resistance to aminoglycosides in E. coli [114].

In Enterococcus faecalis (E. faecalis), the RlmN enzyme, which modifies both tRNA and 23S rRNA, plays a role in antibiotic resistance as a ROS sensor and immediately regulates the translation of stress response proteins. Overexpression of rlmN increases sensitivity to ampicillin and ciprofloxacin, while its absence leads to resistance to chloramphenicol [116].

Another modification involved in response to antibiotic exposure is m1G37 made by TrmD, especially in E. coli and S. enterica [117]. This methylation modulates translation by promoting the decoding of CCC and CCU proline codons. In the absence of modification, ribosomes stall on mRNA, inducing a frameshift or premature termination, which can result in cellular death. Membrane-associated proteins in Gram-negative bacteria are enriched in these proline codons due to the structure requirement of the transmembrane [57,58]. A decrease in m1G37 has been shown to impair the outer membrane structure and envelope integrity in E. coli and Salmonella, thus making the bacteria more susceptible to various classes of antibiotics, including beta-lactams, aminoglycosides, and quinolones [117].

Conclusion and perspectives

Organisms from the three domains of life harbor tRNA modifications, and some of these are more conserved than others. The core body of tRNAs, i.e., the elbows and arms, exhibit a high degree of conservation in modification functions, attributable to their impact in tRNAs structural rigidity and stability. In contrast, ASL modifications have a lower degree of conservation among organisms, suggesting that these modifications may play a role in adaptation [49]. Together, all tRNA modifications modulate translation process by influencing tRNA structure, reading frame maintenance, stop codon readthrough, as well as the accuracy and efficiency of codon decoding. However, despite many studies reviewed here reporting the impact of tRNA modifications on virulence and stress responses, the majority do not investigate the molecular mechanism underlying these phenotypes. Most studies rely on mutants lacking a single tRNA-modifying enzyme and demonstrate that its absence results in specific phenotypes, without establishing the precise link between the modification, translation alteration, and the phenotype. This gap remains a key challenge in the field and should be addressed by future studies to clarify how tRNA modifications mediate their effects.

Despite the identification of the first RNA modification in yeast 70 years ago [9], it is only recently, with advancements in detection methods, that the first mapping of tRNA modifications has been achieved in model bacteria [5,39]. The improvement of sequencing methods, bioinformatics tools, and analytical techniques such as mass spectrometry coupled with liquid chromatography or single-cell RNA sequencing, now enable the precise study of dynamic regulation, as is the case for RNA modifications [86]. As mentioned in the 2024 NASEM report, the comprehensive mapping of RNA modifications in bacteria is still in its early stages [118].

While the primary function of tRNA-modifying enzymes is catalytic, these enzymes can also play additional roles within the cell. For example, some modifying enzymes act as chaperones [30]. Therefore, deletion of such enzymes may not only affect tRNA modification but may also impact other cellular functions. This highlights the potential link between tRNA modifications and cellular processes, suggesting that perturbation of these enzymes could have far-reaching effects beyond their primary role.

As demonstrated in the present review, tRNA modifications are essential for environmental adaptation and virulence without requiring protein synthesis [85]. The regulatory mechanism involving tRNA modifications can occur in two distinct ways: either directly, by modulating the expression of virulence factors, or indirectly, by targeting a regulator upstream of the virulence factor. Additionally, some modifications have pleiotropic effects by affecting a global regulator or causing protein aggregation through misfolding [10]. Considering the significant and varied impacts of tRNA modifications on the translation process, and given that modulating translation fidelity is crucial for bacterial stress responses and host interactions [76], these modifications need to be finely tuned for precise regulation; an aspect that has yet to be fully explored. One strategy to achieve this is the turnover of modified tRNAs through degradation and de novo synthesis. However, another quicker solution relies on the notion of reversibility of tRNA modifications. Therefore, as modifying enzymes exist (known as writers), de-modifying enzymes also exist (known as erasers) to regulate modification levels. These enzymes are responsible for specific de-modification on tRNA by converting the modified nucleotide into its initial form. Erasers are primarily described in eukaryotes [119], but two examples have also been described in bacteria. Firstly, the RudS de-modifying enzyme catalyzes the conversion of s4U8 to uridine. Several RudS orthologs from P. putida, S. enterica and different strains of E. coli have been tested, and all reduced the level of s4U8 modification in E. coli [120]. Secondly, the two erasers RMD1 and RMD2, have been identified in Streptomyces venezulae in which they regulate the amount of m1A58 modification in function of environmental conditions [121]. Nevertheless, with only these two examples reported in the literature to date, the concept of erasers and the role of de-modifying enzymes in bacterial adaptation remain largely unexplored. It is essential to first identify new eraser candidates and then to investigate their impact on bacterial virulence and stress responses.

As tRNA modifications play a role in the development of antibiotic resistance [57,58,64,115,117], it is important to exploit this aspect to develop new drugs targeting RNA-modifying enzymes. For instance, a peptide was designed to mimic the α-helix structure required for YeaZ dimer formation, a protein involved in t6A37 modification synthesis. The initial 14-amino acid peptide (PMP1) was optimized into PMP3 by adding a cell-penetrating peptide. PMP3 was able to enter P. aeruginosa cells and slightly reduce growth. However, in E. coli, the effect was much weaker, likely due to structural differences in YeaZ [122]. This result highlights the species specificity of protein-protein interaction (PPI) modulators and suggests that broad-spectrum applications would require targeting conserved motifs or developing species-specific compounds [122]. Another illustration of this application is the development of TrmD (m1G37) inhibitors as this modifying enzyme is conserved and essential in various bacterial pathogens while its human homologue presents a different structure. Thanks to a high-throughput screening method, a library of 116,350 small molecules was tested for their inhibitory action against the methyltransferase activity of TrmD. Five molecules were identified as being active against Gram-positive bacteria and causing a growth inhibition of M. bovis, Mycobacterium smegmatis, S. aureus, Streptococcus pneumoniae or E. faecalis, with one demonstrating a more global antibacterial activity against all of these species [123]. In a subsequent study, the same authors aimed to elucidate the mechanism of action of these compounds and to expand their activity towards Gram-negative bacteria before antibiotic development. They synthetized different thienopyrimidinone derivatives, guided by the findings of their first study and the TrmD crystal structure of P. aeruginosa and M. tuberculosis [124]. Some of these derivatives interfered with TrmD enzymatic activity by preventing the binding of the cofactor or the tRNA substrate. Three of the identified compounds exhibited antibacterial activity not only against Gram-positive but also against Gram-negative bacteria by inhibiting growth of A. baumannii and Salmonella enteritidis [124]. In addition, small molecules inhibiting Tgt (Q34) have also been produced, but their antibacterial activity remains to be tested. For example, lin-benzoguanine derivatives were synthesized and their inhibitory activity against Tgt was evaluated. These molecules act as nucleobase analogs, competing with tRNA for binding to Tgt by occupying the PreQ1 pocket [125]. Nevertheless, extensive testing is still required for these three examples to be further developed as antibiotics. Specifically, investigations of in vivo stability, human cell toxicity, and the potential emergence of resistance must be conducted. It is also essential to ensure that these molecules do not target human RNA-modifying enzymes.

Another clinical application of tRNA modifications could be to detect bacterial infection by quantifying the amount of tRNA modifications specific to bacteria such as m2A modification. For example, m2A levels increase after antibiotic treatment or immune cells exposure, indicating that this modification is released after bacterial lysis. Furthermore, m2A is detected in mouse urine after m2A injection or E. coli infection, and thus m2A could be used as a signaling molecule to detect bacterial infection in urine samples [31]. Although the m2A tRNA modification is not specific to pathogenic bacteria, a rise in amount may be an alert for further investigation.

In conclusion, although the study of tRNA modifications in bacteria is still in its infancy, their central role in adaptation, virulence, and antibiotic resistance highlights an exciting field, where future discoveries could lead to fine-tuned regulatory mechanisms and therapeutic applications.

Supporting information

S1 Table. Summary of the impacts of tRNA modifications on bacterial virulence and stress adaptation.

The table summarizes the data discussed throughout the study and illustrated in Fig 2. The tRNA modifications and the associated enzymes, the species studied, and the impacts observed in a mutant lacking the enzyme are listed.

https://doi.org/10.1371/journal.ppat.1013600.s001

(DOCX)

Acknowledgments

We thank Philip Robinson (DRS, Hospices Civils de Lyon, Lyon, France) for help in manuscript preparation.

References

  1. 1. Dastgheyb SS, Otto M. Staphylococcal adaptation to diverse physiologic niches: an overview of transcriptomic and phenotypic changes in different biological environments. Future Microbiol. 2015;10(12):1981–95. pmid:26584249
  2. 2. Patel V, Matange N. Adaptation and compensation in a bacterial gene regulatory network evolving under antibiotic selection. eLife. 2021;10:e70931.
  3. 3. Lee J-W. Bacterial regulatory mechanisms for the control of cellular processes: simple organisms’ complex regulation. J Microbiol. 2023;61(3):273–6. pmid:37010794
  4. 4. Duval M, Simonetti A, Caldelari I, Marzi S. Multiple ways to regulate translation initiation in bacteria: mechanisms, regulatory circuits, dynamics. Biochimie. 2015;114:18–29. pmid:25792421
  5. 5. Grosjean H. Modification and editing of RNA: historical overview and important facts to remember. Topics in Current Genetics. Springer Berlin Heidelberg. 2005. p. 1–22.
  6. 6. Rodnina MV. Translation in Prokaryotes. Cold Spring Harb Perspect Biol. 2018;10(9):a032664. pmid:29661790
  7. 7. Cappannini A, Ray A, Purta E, Mukherjee S, Boccaletto P, Moafinejad SN. MODOMICS: a database of RNA modifications and related information. 2023 update. Nucleic Acids Res. 2024;52(D1):D239-44.
  8. 8. Wyatt GR. Occurrence of 5-methylcytosine in nucleic acids. Nature. 1950;166(4214):237–8. pmid:15439258
  9. 9. DaviS FF, Allen FW. Ribonucleic acids from yeast which contain a fifth nucleotide. J Biol Chem. 1957;227(2):907–15. pmid:13463012
  10. 10. de Crécy-Lagard V, Jaroch M. Functions of bacterial tRNA modifications: from ubiquity to diversity. Trends Microbiol. 2021;29(1):41–53. pmid:32718697
  11. 11. Leimkühler S. 2-thiouridine formation in Escherichia coli: a critical review. J Bacteriol. 2024;207(1):e00420-24.
  12. 12. de Crécy-Lagard V, Baharoglu Z, Yuan Y, Boël G, Babor J, Bacusmo JM. Are bacterial processes dependent on global ribosome pausing affected by tRNA modification defects?. J Mol Biol. 2025;437(16):169107.
  13. 13. Kompatscher M, Gonnella I, Erlacher M. Studying the function of tRNA modifications: experimental challenges and opportunities. J Mol Biol. 2025;437(16):168934.
  14. 14. McCown PJ, Ruszkowska A, Kunkler CN, Breger K, Hulewicz JP, Wang MC, et al. Naturally occurring modified ribonucleosides. Wiley Interdiscip Rev RNA. 2020;11(5):e1595. pmid:32301288
  15. 15. Antoine L, Bahena-Ceron R, Devi Bunwaree H, Gobry M, Loegler V, Romby P. RNA modifications in pathogenic bacteria: impact on host adaptation and virulence. Genes. 2021;12(8):1125.
  16. 16. Limbach PA, Crain PF, McCloskey JA. Summary: the modified nucleosides of RNA. Nucleic Acids Res. 1994;22(12):2183–96. pmid:7518580
  17. 17. Roovers M, Kaminska KH, Tkaczuk KL, Gigot D, Droogmans L, Bujnicki JM. The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK). Nucleic Acids Res. 2008;36(10):3252–62. pmid:18420655
  18. 18. Grobe S, Doberenz S, Ferreira K, Krueger J, Brönstrup M, Kaever V, et al. Identification and quantification of (t)RNA modifications in Pseudomonas aeruginosa by liquid chromatography-tandem mass spectrometry. ChemBioChem. 2019;20(11):1430–7.
  19. 19. Charette M, Gray MW. Pseudouridine in RNA: what, where, how, and why. IUBMB Life. 2000;49(5):341–51. pmid:10902565
  20. 20. Dalluge JJ, Hashizume T, Sopchik AE, McCloskey JA, Davis DR. Conformational flexibility in RNA: the role of dihydrouridine. Nucleic Acids Res. 1996;24(6):1073–9. pmid:8604341
  21. 21. Wolf J, Gerber AP, Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. EMBO J. 2002;21(14):3841–51. pmid:12110595
  22. 22. Deutsch C, El Yacoubi B, de Crécy-Lagard V, Iwata-Reuyl D. Biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside. J Biol Chem. 2012;287(17):13666–73. pmid:22378793
  23. 23. Harada F, Nishimura S. Possible anticodon sequences of tRNA His, tRNA Asm, and tRNA Asp from Escherichia coli B. Universal presence of nucleoside Q in the first postion of the anticondons of these transfer ribonucleic acids. Biochemistry. 1972;11(2):301–8.
  24. 24. Carbon J, David H, Studier MH. Thiobases in Escherchia coli transfer RNA: 2-thiocytosine and 5-methylaminomethyl-2-thiouracil. Science. 1968;161(3846):1146–7. pmid:17812290
  25. 25. Mueller EG, Buck CJ, Palenchar PM, Barnhart LE, Paulson JL. Identification of a gene involved in the generation of 4-thiouridine in tRNA. Nucleic Acids Res. 1998;26(11):2606–10. pmid:9592144
  26. 26. Leipuviene R, Qian Q, Björk GR. Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways. J Bacteriol. 2004;186(3):758–66. pmid:14729702
  27. 27. Kimura S, Dedon PC, Waldor MK. Comparative tRNA sequencing and RNA mass spectrometry for surveying tRNA modifications. Nat Chem Biol. 2020;16(9):964–72. pmid:32514182
  28. 28. Armengod M-E, Moukadiri I, Prado S, Ruiz-Partida R, Benítez-Páez A, Villarroya M, et al. Enzymology of tRNA modification in the bacterial MnmEG pathway. Biochimie. 2012;94(7):1510–20. pmid:22386868
  29. 29. Ryu H, Grove TL, Almo SC, Kim J. Identification of a novel tRNA wobble uridine modifying activity in the biosynthesis of 5-methoxyuridine. Nucleic Acids Res. 2018;46(17):9160–9.
  30. 30. Yared M-J, Marcelot A, Barraud P. Beyond the anticodon: tRNA core modifications and their impact on structure, translation and stress adaptation. Genes (Basel). 2024;15(3):374. pmid:38540433
  31. 31. Yamamura R, Nagayoshi Y, Nishiguchi K, Kaneko H, Yamamoto K, Matsushita K. Bacteria-specific modified nucleoside is released and elevated in urine of patients with bacterial infections. mBio. 2024;0(0):e03124-24.
  32. 32. Zallot R, Ross R, Chen W-H, Bruner SD, Limbach PA, de Crécy-Lagard V. Identification of a novel epoxyqueuosine reductase family by comparative genomics. ACS Chem Biol. 2017;12(3):844–51. pmid:28128549
  33. 33. Jaroch M, Sun G, Tsui HCT, Reed C, Sun J, Jörg M. Alternate routes to mnm5s2U synthesis in Gram-positive bacteria. J Bacteriol. 2024;206(4):e0045223.
  34. 34. Fleissner E, Borek E. A new enzyme of RNA synthesis: RNA methylase. Proc Natl Acad Sci U S A. 1962;48(7):1199–203.
  35. 35. Svensson I, Boman HG, Eriksson KG, Kjellin K. Studies on microbial RNA. I. Transfer of methyl groups from methionine to soluble rna from Escherichia coli. J Mol Biol. 1963;7:254–71. pmid:14065310
  36. 36. Marchand V, Blanloeil-Oillo F, Helm M, Motorin Y. Illumina-based RiboMethSeq approach for mapping of 2’-O-Me residues in RNA. Nucleic Acids Res. 2016;44(16):e135. pmid:27302133
  37. 37. Lucas MC, Pryszcz LP, Medina R, Milenkovic I, Camacho N, Marchand V, et al. Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing. Nat Biotechnol. 2024;42(1):72–86. pmid:37024678
  38. 38. Pichot F, Marchand V, Helm M, Motorin Y. Data analysis pipeline for detection and quantification of pseudouridine (ψ) in RNA by HydraPsiSeq. Methods Mol Biol. 2023;2624:207–23. pmid:36723818
  39. 39. de Crécy-Lagard V, Ross RL, Jaroch M, Marchand V, Eisenhart C, Brégeon D, et al. Survey and validation of tRNA modifications and their corresponding genes in Bacillus subtilis sp subtilis strain 168. Biomolecules. 2020;10(7):977. pmid:32629984
  40. 40. Mandler MD, Maligireddy SS, Guiblet WM, Fitzsimmons CM, McDonald KS, Warrell DL. The modification landscape of P. aeruginosa tRNAs. RNA N Y N. 2024.
  41. 41. Quaiyum S, Sun J, Marchand V, Sun G, Reed CJ, Motorin Y, et al. Mapping the tRNA modification landscape of Bartonella henselae Houston I and Bartonella quintana Toulouse. Front Microbiol. 2024;15:1369018. pmid:38544857
  42. 42. Koshla O, Vogt L-M, Rydkin O, Sehin Y, Ostash I, Helm M, et al. Landscape of post-transcriptional tRNA modifications in Streptomyces albidoflavus J1074 as portrayed by mass spectrometry and genomic data mining. J Bacteriol. 2023;205(1):e0029422. pmid:36468867
  43. 43. Antoine L, Wolff P, Westhof E, Romby P, Marzi S. Mapping post-transcriptional modifications in Staphylococcus aureus tRNAs by nanoLC/MSMS. Biochimie. 2019;164:60–9. pmid:31295507
  44. 44. Benítez-Páez A, Villarroya M, Armengod M-E. The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy. RNA. 2012;18(10):1783–95. pmid:22891362
  45. 45. Bar-Yaacov D, Mordret E, Towers R, Biniashvili T, Soyris C, Schwartz S, et al. RNA editing in bacteria recodes multiple proteins and regulates an evolutionarily conserved toxin-antitoxin system. Genome Res. 2017;27(10):1696–703. pmid:28864459
  46. 46. Ikeuchi Y, Soma A, Ote T, Kato J, Sekine Y, Suzuki T. molecular mechanism of lysidine synthesis that determines tRNA identity and codon recognition. Mol Cell. 2005;19(2):235–46. pmid:16039592
  47. 47. Miyauchi K, Kimura S, Akiyama N, Inoue K, Ishiguro K, Vu T-S, et al. A tRNA modification with aminovaleramide facilitates AUA decoding in protein synthesis. Nat Chem Biol. 2025;21(4):522–31. pmid:39300229
  48. 48. Pan X, Bruch A, Blango MG. Past, present, and future of RNA modifications in infectious disease research. ACS Infect Dis. 2024;10(12):4017–29. pmid:39569943
  49. 49. Schultz SK, Kothe U. RNA modifying enzymes shape tRNA biogenesis and function. J Biol Chem. 2024;300(8):107488. pmid:38908752
  50. 50. Helm M, Giegé R, Florentz C. A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys. Biochemistry. 1999;38(40):13338–46. pmid:10529209
  51. 51. Sowers LC, Shaw BR, Sedwick WD. Base stacking and molecular polarizability: effect of a methyl group in the 5-position of pyrimidines. Biochem Biophys Res Commun. 1987;148(2):790–4.
  52. 52. Kawai G, Ue H, Yasuda M, Sakamoto K, Hashizume T, McCloskey JA, et al. Relation between functions and conformational characteristics of modified nucleosides found in tRNAs. Nucleic Acids Symp Ser. 1991;(25):49–50. pmid:1726811
  53. 53. Tworowska I, Nikonowicz EP. Base pairing within the psi32,psi39-modified anticodon arm of Escherichia coli tRNA(Phe). J Am Chem Soc. 2006;128(49):15570–1. pmid:17147349
  54. 54. Keffer-Wilkes LC, Veerareddygari GR, Kothe U. RNA modification enzyme TruB is a tRNA chaperone. Proc Natl Acad Sci U S A. 2016;113(50):14306–11. pmid:27849601
  55. 55. Keffer-Wilkes LC, Soon EF, Kothe U. The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain. Nucleic Acids Res. 2020;48(14):7981–90.
  56. 56. Frey B, Jänel G, Michelsen U, Kersten H. Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr. J Bacteriol. 1989;171(3):1524–30. pmid:2537821
  57. 57. Gamper HB, Masuda I, Frenkel-Morgenstern M, Hou Y-M. Maintenance of protein synthesis reading frame by EF-P and m(1)G37-tRNA. Nat Commun. 2015;6:7226. pmid:26009254
  58. 58. Gamper HB, Masuda I, Frenkel-Morgenstern M, Hou Y-M. The UGG isoacceptor of tRNAPro is naturally prone to frameshifts. Int J Mol Sci. 2015;16(7):14866–83. pmid:26140378
  59. 59. Fleming BA, Blango MG, Rousek AA, Kincannon WM, Tran A, Lewis AJ. A tRNA modifying enzyme as a tunable regulatory nexus for bacterial stress responses and virulence. Nucleic Acids Res. 2022.
  60. 60. Urbonavicius J, Qian Q, Durand JM, Hagervall TG, Björk GR. Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J. 2001 Sep 3;20(17):4863–73.
  61. 61. Brégeon D, Colot V, Radman M, Taddei F. Translational misreading: a tRNA modification counteracts a +2 ribosomal frameshift. Genes Dev. 2001;15(17):2295–306. pmid:11544186
  62. 62. Rodriguez-Hernandez A, Spears JL, Gaston KW, Limbach PA, Gamper H, Hou Y-M, et al. Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position. J Mol Biol. 2013;425(20):3888–906. pmid:23727144
  63. 63. Díaz-Rullo J, González-Pastor JE. tRNA queuosine modification is involved in biofilm formation and virulence in bacteria. Nucleic Acids Res. 2023;51(18):9821–37. pmid:37638766
  64. 64. Fruchard L, Babosan A, Carvalho A, Lang M, Li B, Duchateau M. Aminoglycoside tolerance in Vibrio cholerae engages translational reprogramming associated with queuosine tRNA modification. eLife. 2025;13:RP96317.
  65. 65. Koshla O, Yushchuk O, Ostash I, Dacyuk Y, Myronovskyi M, Jäger G, et al. Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces. Mol Microbiol. 2019;112(1):249–65. pmid:31017319
  66. 66. Aubee JI, Olu M, Thompson KM. The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation. RNA. 2016;22(5):729–42. pmid:26979278
  67. 67. Rozov A, Demeshkina N, Khusainov I, Westhof E, Yusupov M, Yusupova G. Novel base-pairing interactions at the tRNA wobble position crucial for accurate reading of the genetic code. Nat Commun. 2016;7:10457. pmid:26791911
  68. 68. Chionh YH, McBee M, Babu IR, Hia F, Lin W, Zhao W, et al. tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence. Nat Commun. 2016;7:13302. pmid:27834374
  69. 69. Krueger J, Preusse M, Oswaldo Gomez N, Frommeyer YN, Doberenz S, Lorenz A, et al. tRNA epitranscriptome determines pathogenicity of the opportunistic pathogen Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2024;121(11):e2312874121. pmid:38451943
  70. 70. Curran JF. Decoding with the A:I wobble pair is inefficient. Nucleic Acids Res. 1995;23(4):683–8. pmid:7534909
  71. 71. Yokobori S, Kitamura A, Grosjean H, Bessho Y. Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes. Nucleic Acids Res. 2013;41(13):6531–43. pmid:23658230
  72. 72. Hagervall TG, Pomerantz SC, McCloskey JA. Reduced misreading of asparagine codons by Escherichia coli tRNALys with hypomodified derivatives of 5-methylaminomethyl-2-thiouridine in the wobble position. J Mol Biol. 1998;284(1):33–42. pmid:9811540
  73. 73. Parker J. Specific mistranslation in hisT mutants of Escherichia coli. Mol Gen Genet. 1982;187(3):405–9. pmid:6757659
  74. 74. Kimura S, Waldor MK. The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA. Proc Natl Acad Sci U S A. 2019;116(4):1394–403.
  75. 75. Jones JD, Franco MK, Giles RN, Eyler DE, Tardu M, Smith TJ, et al. Conserved 5-methyluridine tRNA modification modulates ribosome translocation. Proc Natl Acad Sci U S A. 2024;121(35):e2401743121. pmid:39159370
  76. 76. Lyu Z, Wilson C, Ling J. Translational fidelity during bacterial stresses and host interactions. Pathogens. 2023;12(3):383. pmid:36986305
  77. 77. Losey HC, Ruthenburg AJ, Verdine GL. Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA. Nat Struct Mol Biol. 2006;13(2):153–9. pmid:16415880
  78. 78. Bacusmo JM, Babor J, Hu J, Cao B, Kellner S, Szkrybalo S. Synergistic effects of tRNA modification defects in Escherichia coli K12. BioRxiv Prepr Serv Biol. 2024.
  79. 79. Jaroch M, Savage K, Kuipers P, Bacusmo JM, Hu J, Sun J, et al. Environmental control of queuosine levels in Streptococcus mutans tRNAs. Mol Microbiol. 2025;123(1):48–59. pmid:39719891
  80. 80. Sehin Y, Koshla O, Dacyuk Y, Zhao R, Ross R, Myronovskyi M, et al. Gene ssfg_01967 (miaB) for tRNA modification influences morphogenesis and moenomycin biosynthesis in Streptomyces ghanaensis ATCC14672. Microbiology (Reading). 2019;165(2):233–45. pmid:30543507
  81. 81. Noguchi S, Nishimura Y, Hirota Y, Nishimura S. Isolation and characterization of an Escherichia coli mutant lacking tRNA-guanine transglycosylase. Function and biosynthesis of queuosine in tRNA. J Biol Chem. 1982;257(11):6544–50.
  82. 82. Kinghorn SM, O’Byrne CP, Booth IR, Stansfield I. Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance. Microbiology (Reading). 2002;148(Pt 11):3511–20. pmid:12427942
  83. 83. Ishida K, Kunibayashi T, Tomikawa C, Ochi A, Kanai T, Hirata A, et al. Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus. Nucleic Acids Res. 2011;39(6):2304–18. pmid:21097467
  84. 84. Urbonavicius J, Durand JMB, Björk GR. Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri. J Bacteriol. 2002;184(19):5348–57. pmid:12218021
  85. 85. Hori H. Regulatory factors for tRNA modifications in extreme-thermophilic bacterium Thermus thermophilus. Front Genet. 2019;10:204. pmid:30906314
  86. 86. Ohira T, Suzuki T. Transfer RNA modifications and cellular thermotolerance. Mol Cell. 2024;84(1):94–106. pmid:38181765
  87. 87. Droogmans L, Roovers M, Bujnicki JM, Tricot C, Hartsch T, Stalon V, et al. Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures. Nucleic Acids Res. 2003;31(8):2148–56. pmid:12682365
  88. 88. Tomikawa C, Yokogawa T, Kanai T, Hori H. N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network. Nucl Acids Res. 2010;38(3):942–57.
  89. 89. Hoffmann A, Lorenz C, Fallmann J, Wolff P, Lechner A, Betat H. Temperature-dependent tRNA modifications in Bacillales. Int J Mol Sci. 2024;25(16):8823.
  90. 90. Cho KH, Caparon MG. tRNA modification by GidA/MnmE is necessary for Streptococcus pyogenes virulence: a new strategy to make live attenuated strains. Infect Immun. 2008;76(7):3176–86. pmid:18426891
  91. 91. Shippy DC, Eakley NM, Bochsler PN, Chopra AK, Fadl AA. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene. Microb Pathog. 2011;50(6):303–13. pmid:21320585
  92. 92. Yu H, Kim KS. mRNA context dependent regulation of cytotoxic necrotizing factor 1 translation by GidA, a tRNA modification enzyme in Escherichia coli. Gene. 2012;491(2):116–22. pmid:22020226
  93. 93. Sha J, Kozlova EV, Fadl AA, Olano JP, Houston CW, Peterson JW, et al. Molecular characterization of a glucose-inhibited division gene, gidA, that regulates cytotoxic enterotoxin of Aeromonas hydrophila. Infect Immun. 2004;72(2):1084–95. pmid:14742556
  94. 94. Gupta R, Gobble TR, Schuster M. GidA posttranscriptionally regulates rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol. 2009;191(18):5785–92. pmid:19592591
  95. 95. Srimahaeak T, Thongdee N, Chittrakanwong J, Atichartpongkul S, Jaroensuk J, Phatinuwat K, et al. Pseudomonas aeruginosa GidA modulates the expression of catalases at the posttranscriptional level and plays a role in virulence. Front Microbiol. 2023;13:1079710. pmid:36726575
  96. 96. Durand JM, Dagberg B, Uhlin BE, Björk GR. Transfer RNA modification, temperature and DNA superhelicity have a common target in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene. Mol Microbiol. 2000;35(4):924–35. pmid:10692168
  97. 97. Tomasi FG, Kimura S, Rubin EJ, Waldor MK. A tRNA modification in Mycobacterium tuberculosis facilitates optimal intracellular growth. eLife. 2023;12.
  98. 98. McGuffey JC, Jackson-Litteken CD, Di Venanzio G, Zimmer AA, Lewis JM, Distel JS, et al. The tRNA methyltransferase TrmB is critical for Acinetobacter baumannii stress responses and pulmonary infection. mBio. 2023;14(5):e0141623. pmid:37589464
  99. 99. Saikia B, Riquelme-Barrios S, Carell T, Brameyer S, Jung K. Depletion of m6A-RNA in Escherichia coli reduces the infectious potential of T5 bacteriophage. Microbiol Spectr. 2024;12(12):e0112424. pmid:39422505
  100. 100. Sionov RV, Steinberg D. Targeting the holy triangle of quorum sensing, biofilm formation, and antibiotic resistance in pathogenic bacteria. Microorganisms. 2022;10(6):1239. pmid:35744757
  101. 101. Li D, Shibata Y, Takeshita T, Yamashita Y. A novel gene involved in the survival of Streptococcus mutans under stress conditions. Appl Environ Microbiol. 2014;80(1):97–103. pmid:24123744
  102. 102. Shippy DC, Eakley NM, Lauhon CT, Bochsler PN, Fadl AA. Virulence characteristics of Salmonella following deletion of genes encoding the tRNA modification enzymes GidA and MnmE. Microb Pathog. 2013;57:1–9. pmid:23375888
  103. 103. Nie W, Wang S, He R, Xu Q, Wang P, Wu Y, et al. A-to-I RNA editing in bacteria increases pathogenicity and tolerance to oxidative stress. PLoS Pathog. 2020;16(8):e1008740. pmid:32822429
  104. 104. Galvanin A, Vogt LM, Grober A, Freund I, Ayadi L, Bourguignon-Igel V. Bacterial tRNA 2’-O-methylation is dynamically regulated under stress conditions and modulates innate immune response. Nucleic Acids Res. 2020;48(22):12833–44.
  105. 105. Fang FC, Frawley ER, Tapscott T, Vázquez-Torres A. Bacterial stress responses during host infection. Cell Host Microbe. 2016;20(2):133–43. pmid:27512901
  106. 106. Aubee JI, Olu M, Thompson KM. TrmL and TusA are necessary for rpoS and MiaA is required for hfq expression in Escherichia coli. Biomolecules. 2017;7(2):39. pmid:28471404
  107. 107. Fasnacht M, Polacek N. Oxidative stress in bacteria and the central dogma of molecular biology. Front Mol Biosci. 2021;8:671037. pmid:34041267
  108. 108. Buchser R, Sweet P, Anantharaman A, Contreras L. RNAs as sensors of oxidative stress in bacteria. Annu Rev Chem Biomol Eng. 2023;14:265–81. pmid:37289561
  109. 109. Jaroensuk J, Atichartpongkul S, Chionh YH, Wong YH, Liew CW, McBee ME. Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa. Nucleic Acids Res. 2016;44(22):10834–48.
  110. 110. Romsang A, Duang-Nkern J, Khemsom K, Wongsaroj L, Saninjuk K, Fuangthong M, et al. Pseudomonas aeruginosa ttcA encoding tRNA-thiolating protein requires an iron-sulfur cluster to participate in hydrogen peroxide-mediated stress protection and pathogenicity. Sci Rep. 2018;8(1):11882. pmid:30089777
  111. 111. Thongdee N, Jaroensuk J, Atichartpongkul S, Chittrakanwong J, Chooyoung K, Srimahaeak T, et al. TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa. Nucleic Acids Res. 2019;47(17):9271–81.
  112. 112. Valesyan S, Jora M, Addepalli B, Limbach PA. Stress-induced modification of Escherichia coli tRNA generates 5-methylcytidine in the variable loop. Proc Natl Acad Sci U S A. 2024;121(46):e2317857121. pmid:39495928
  113. 113. Olivieri P, Zupok A, Yildiz T, Oltmanns J, Lehmann A, Sokolowska E, et al. TusA influences Fe-S cluster assembly and iron homeostasis in E. coli by reducing the translation efficiency of Fur. Microbiol Spectr. 2024;12(8):e0055624. pmid:38916309
  114. 114. Pollo-Oliveira L, Davis NK, Hossain I, Ho P, Yuan Y, Salguero García P, et al. The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12. Metallomics. 2022;14(9):mfac065. pmid:36066904
  115. 115. Babosan A, Fruchard L, Krin E, Carvalho A, Mazel D, Baharoglu Z. Nonessential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress. Microlife. 2022;3:uqac019. pmid:37223353
  116. 116. Lee WL, Sinha A, Lam LN, Loo HL, Liang J, Ho P, et al. An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis. Nat Commun. 2023;14(1):4093. pmid:37433804
  117. 117. Masuda I, Matsubara R, Christian T, Rojas ER, Yadavalli SS, Zhang L. tRNA methylation is a global determinant of bacterial multi-drug resistance. Cell Syst. 2019;8(4):302-314.e8.
  118. 118. Charting a future for sequencing RNA and its modifications: a new era for biology and medicine. Washington, D.C.: National Academies Press. 2020.
    • 119. Zhang W, Foo M, Eren AM, Pan T. tRNA modification dynamics from individual organisms to metaepitranscriptomics of microbiomes. Mol Cell. 2022;82(5):891–906. pmid:35032425
    • 120. Jamontas R, Laurynėnas A, Povilaitytė D, Meškys R, Aučynaitė A. RudS: bacterial desulfidase responsible for tRNA 4-thiouridine de-modification. Nucleic Acids Res. 2024;52(17):10543–62. pmid:39166491
    • 121. Foo M, Frietze LR, Enghiad B, Yuan Y, Katanski CD, Zhao H, et al. Prokaryotic RNA N1-methyladenosine erasers maintain tRNA m1A modification levels in Streptomyces venezuelae. ACS Chem Biol. 2024;19(7):1616–25. pmid:38912606
    • 122. Bollati M, Fasola E, Pieraccini S, Freddi F, Cocomazzi P, Oliva F, et al. Impairing protein-protein interactions in an essential tRNA modification complex: an innovative antimicrobial strategy against Pseudomonas aeruginosa. J Pept Sci Off Publ Eur Pept Soc. 2024;:e3658.
    • 123. Zhong W, Koay A, Ngo A, Li Y, Nah Q, Wong YH. Targeting the bacterial epitranscriptome for antibiotic development: discovery of novel tRNA-(N1G37) methyltransferase (TrmD) inhibitors. ACS Infect Dis. 2019;5(3):326–35.
    • 124. Zhong W, Pasunooti KK, Balamkundu S, Wong YH, Nah Q, Gadi V, et al. Thienopyrimidinone derivatives that inhibit bacterial tRNA (Guanine37-N1)-methyltransferase (TrmD) by restructuring the active site with a tyrosine-flipping mechanism. J Med Chem. 2019;62(17):7788–805. pmid:31442049
    • 125. Hörtner SR, Ritschel T, Stengl B, Kramer C, Schweizer WB, Wagner B, et al. Potent inhibitors of tRNA-guanine transglycosylase, an enzyme linked to the pathogenicity of the Shigella bacterium: charge-assisted hydrogen bonding. Angew Chem Int Ed Engl. 2007;46(43):8266–9. pmid:17902085