# tRNA-modifying enzymes in bacterial stress adaptation

Louna Fruchard, Claudia Salinas, Andre Carvalho, Zeynep Baharoglu

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Louna Fruchard, Claudia Salinas, Andre Carvalho, Zeynep Baharoglu. tRNA-modifying enzymes in bacterial stress adaptation. Open Biology, 2025, 15 (10), pp.250194. 10.1098/rsob.250194. pasteur-05347156

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Submitted on 4 Nov 2025

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**Cite this article:** Fruchard L, Salinas C, Carvalho A, Baharoglu Z. 2025 tRNA-modifying enzymes in bacterial stress adaptation. *Open Biol.* **15**: 250194.
[https://doi.org/10.1098/rsob.250194](https://doi.org/10.1098/rsob.250194)

Received: 3 June 2025

Accepted: 26 August 2025

**Subject Areas:**

microbiology, molecular biology

**Keywords:**

epitranscriptomics, tRNA modifications, bacterial stress responses, oxidative stress, modification tunable transcripts, transfer RNAs, tRNA-modifying enzymes, antibiotic stress, bacterial adaptive responses, selective translation

**Author for correspondence:**

Zeynep Baharoglu
e-mails: zeynep.baharoglu@pasteur.fr;
zeynep.baharoglu@ibpc.fr

# tRNA-modifying enzymes in bacterial stress adaptation

Louna Fruchard<fnref n="1" />,<fnref n="2" />, Claudia Salinas<fnref n="1" />,<fnref n="3" />, Andre Carvalho<fnref n="2" />,<fnref n="4" />,<fnref n="5" /> and Zeynep Baharoglu<fnref n="1" />

<figure><img src="image_5.png" /></figure>

ZB, 0000-0003-3477-2685

Transfer RNAs (tRNAs) and their modifications are central to bacterial translation and physiology, yet their roles in stress adaptation remain underexplored. While extensively studied in eukaryotes, and linked to diseases, bacterial tRNA modifications are only recently gaining attention. This review highlights emerging insights into how tRNA modifications and associated enzymes contribute to bacterial survival under oxidative and antibiotic stresses, both disrupting proteostasis. We examine the environmental and physiological stresses bacteria encounter, focussing on reactive oxygen species and sub-lethal antibiotic exposure. These stresses challenge proteome integrity and trigger adaptive responses involving key stress regulators. We explore the expanding field of bacterial epitranscriptomics, detailing the diversity, dynamics and structural impact of tRNA modifications, and how they influence selective translation. Central to this is the concept of modification tunable transcripts, linking specific codon usage patterns to stress-responsive translation reprogramming. Beyond their catalytic roles, tRNA-modifying enzymes also have additional functions. We discuss this dual functionality and its broader implications for bacterial adaptability. By integrating recent technological advances and conceptual models, this review underscores the potential of targeting tRNA modifications as a novel strategy to combat bacterial pathogenicity and antibiotic resistance. With many aspects still unresolved, the study of bacterial tRNA modifications promises rich opportunities for discovery and therapeutic innovation.

## 1. Introduction

Transfer RNAs (tRNAs) are vital for translation and bacterial function, with tRNA modifying enzymes crucially regulating key cellular processes. While extensively studied in humans, where tRNA modifications link to diseases like cancer, diabetes and intellectual disability, their roles in bacteria remain underexplored. Understanding bacterial tRNA modifications is critical amid the antimicrobial resistance crisis, as bacteria rapidly adapt through conserved mechanisms and serve as ideal models for research.

Prokaryotes thrive in diverse, often extreme environments, constantly facing abiotic stresses (temperature, pH, oxidative stress, nutrient limitation) and ecological competition, including antibiotics. Past reviews have detailed tRNA modification diversity and regulation [1–7], with growing bacterial-focussed research since 2000.

Reactive oxygen species (ROS) and sub-lethal antibiotic exposure are particularly important in this context, as they represent common and often

<footnote n="1">¹Epitranscriptomic and Translational Responses to Antibacterial Stress Team, Expression Génétique Microbienne, CNRS UMR8261, Institut Pasteur, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris, France</footnote>

<footnote n="2">²Sorbonne Université, Collège Doctoral, F-75005, Paris, France</footnote>

<footnote n="3">³Ecole Doctorale 474 FIRE, Université Paris Cite, Paris, France</footnote>

<footnote n="4">⁴Institut Pasteur, Université Paris Cité, Unité Plasticité du Génome Bactérien, 75015, Paris, France</footnote>

<footnote n="5">⁵Complutense University of Madrid Faculty of Veterinary, Madrid, Community of Madrid, Spain</footnote>
simultaneous challenges to bacterial survival. ROS can arise from environmental sources or as a by-product of metabolism, damaging DNA, proteins and lipids, while many antibiotics, including aminoglycosides, both induce ROS production and disrupt proteostasis. These overlapping stresses require coordinated responses that maintain translation fidelity, selectively adjust protein synthesis and stabilize the proteome under damage pressure.

tRNA modifications are increasingly recognized as key regulators in this adaptive process. Under stress, changes in tRNA modification patterns can bias translation towards proteins that promote survival, while limiting synthesis of non-essential or stress-sensitive proteins. This selective translation reprogramming provides a powerful, rapid and reversible means for bacteria to remodel their proteome in response to ROS and antibiotic stress.

A review centred on tRNA modifications in the context of ROS and antibiotic stress is timely because it links two major, interconnected stressors with an adaptive molecular system that is both highly conserved and functionally versatile. By focussing on how tRNA modifications and their enzymes influence bacterial responses to oxidative and antibiotic stress, this work aims to clarify their contribution to survival strategies, highlight potential vulnerabilities and point towards novel approaches for tackling antibiotic resistance.

After outlining the link between these stresses and bacterial physiology, we examine tRNA-modifying enzymes and their vital role in stress response and adaptation. Their diversity, specific impacts on proteins and pathways, and potential regulation by stress conditions make them compelling targets for further study.

# 2. Bacterial life is stressful: stress in the environment

## 2.1. Oxidative challenges throughout the bacterial life cycle

### 2.1.1. Oxygen: sustainer and threat

Although current concerns focus on carbon dioxide (CO₂), the major challenge facing organisms two billion years ago was dioxygen (O₂) [8]. While many bacteria today rely on oxygen for energy production and for the synthesis of various essential metabolites, excessive oxygen levels are toxic. ROS are generated through spontaneous or enzyme-mediated reduction of molecular oxygen, producing superoxide (O₂•), hydrogen peroxide (H₂O₂), hydroxyl radical (HO•) and hydroxyl anion (OH•) (figure 1A). Their reactivity follows the order: HO• > ′O₂ > O₂• > H₂O₂ [10]. To survive, organisms have evolved mechanisms to maintain redox balance and mitigate oxygen-induced damage [11]. Redox balance, the equilibrium between oxidants and antioxidants, is essential for cell function. When ROS exceed antioxidant capacity, oxidative stress (OS) occurs, a concept introduced by Sies in the 1990s [12]. Figure 1B illustrates various sources of ROS that bacteria encounter throughout their lifecycle.

### 2.1.2. Respiration

Dioxygen is a potent electron acceptor that diffuses freely across membranes, preventing cells from lowering internal O₂ levels below ambient concentrations. Studies in bacteria show endogenous ROS production; for example, *Escherichia coli* generates 10–15 µM H₂O₂ per second under air-saturated conditions [13].

### 2.1.3. The aquatic environment

Natural water contains 20–1400 nM H₂O₂ [14], primarily from solar radiation, with concentrations fluctuating in sync with the day–night UV cycle [15]. ROS levels are higher on sunlit surfaces due to photochemical production. Rainfall can increase ROS over 10-fold, especially at the ocean surface and down to 50 m. Anthropogenic pollution further elevates H₂O₂, while cyanobacterial reservoirs contribute to oxidative stress through photosynthesis.

### 2.1.4. The host

During infections, various host immune cells undergo an ‘oxidative burst’ or ‘respiratory burst’ [16]. This pathogen-fighting process relies on the release of ROS [17], enabling macrophages to oxidatively damage and degrade engulfed bacteria, key to innate immunity. Supporting this, and as an example, stool samples [18] and duodenal biopsies [19] from cholera patients show elevated ROS levels compared to healthy individuals, indicating that *Vibrio cholerae* encounters toxic ROS in the host.

### 2.1.5. Other reactive oxygen species-generating factors

Microorganisms can produce and secrete ROS to eliminate competitors in nutrient-limited environments, gaining a competitive advantage [20]. This may result from long-term co-evolution between closely related species.

Finally, and as detailed below, antibiotic exposure can lead to ROS accumulation.

# 2.2. Antibiotic challenge in bacterial environments

## 2.2.1. Antibiotics in the environment

Antibiotics, whether natural or synthetic, inhibit bacterial growth (bacteriostatic) or kill bacteria (bactericidal), targeting pathogens with minimal host damage. Since the discovery of the first antibiotic, it became clear that many microbes produce
A

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B

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**Figure 1.** (A) Sequential one-electron and direct four-electron reduction of O₂ to water. The successive reduction of O₂ by single electrons is composed of four reduction steps resulting in intermediate ROS (in red). H₂O₂ reduction to HO⁺ occurs via the Fenton reaction, necessitating an iron (Fe) catalyst, Fe²⁺, which will be oxidized into Fe³⁺. The final step is the addition of an electron and a H⁺ to HO⁺ to obtain water. HO⁺, O₂⁻, and H₂O₂, classified as ROS, are more likely to interact with biomolecules than O₂. Singlet molecular oxygen (¹O₂), the last of the four major ROS (not represented here), is highly toxic to organisms, as it also reacts with most organic molecules, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, lipids and thus alters cellular functions [9]. SOD stands for superoxide dismutase. (B) The diverse ROS-generating conditions surrounding bacteria throughout their lifecycle.

such compounds to inhibit competitors, making their isolation vital for treating infections. New classes and synthetic derivatives revolutionized twentieth-century medicine. Antibiotics were also used as agricultural growth promoters [21] until banned in Europe in 2006 [22]. Residues from farming and human use enter the environment through food, urine and wastewater, where treatment plants often fail to fully remove them [23]. These environmental antibiotic gradients [21,23–25] drive the emergence and spread of resistant strains [21,26,27] (figure 2).

### 2.2.2. The underestimated importance of low doses of antibiotics

Sub-MIC (sub-inhibitory concentrations) of antibiotics alter bacterial transcriptomes and proteomes [29,33], affecting both targeted and unrelated pathways (for a review [28]), sometimes with effects opposite to those at high doses [34]. They can also trigger ROS accumulation and oxidative stress [35,36]. Understanding these responses is essential for addressing resistance.

## 3. Epitranscriptomic adaptation to stress from a tRNA perspective

### 3.1. The tRNA function, structure and biogenesis

tRNAs are short non-coding RNAs (approx. 70–100 nucleotides) essential for translation, delivering amino acids to the ribosome to ensure proteins match their genes (for a review: [37]). They fold into a conserved cloverleaf secondary structure and three-dimensional L-shape (figure 3), with key regions including the acceptor stem (amino acid carrier), D-arm, anticodon arm (with the anticodon triplet at positions 34–36), variable loop and TψC-arm. The D- and TψC-arms interact to form the hydrophobic elbow. The anticodon arm consists of a five-base pair stem and the anticodon loop (ACL), containing the triplet anticodon that pairs with mRNA codons. The anticodon’s wobble position (nucleotide 34) allows flexible base pairing.

tRNAs are often transcribed as polycistronic RNAs and processed into mature forms by cleavage, trimming and modifications [39]. The final step is aminoacylation, attaching the correct amino acid via aminoacyl-tRNA synthetases.

Bacterial adaptability stems from genetic variation through mutation, horizontal gene transfer and epigenetics, driving phenotypic diversity and survival under stress. Gene expression is regulated at multiple levels, with RNA modification enzymes playing a key role in shaping the proteome. The next section explores tRNA modification genes in bacterial adaptation.

### 3.2. tRNA modifications

#### 3.2.1. Diversity and roles

Before the mid-twentieth century, nucleic acids were thought to contain only four bases (A, T/U, G, C). It is now known that RNAs are highly modified molecules [40]. Over 170 RNA modifications have been identified across all three domains of life and viruses [38], with more than 110 found in tRNAs, making them the most modified RNA class. In bacteria, 2–15% of tRNA bases are modified, with a median of eight modifications per molecule [38].

Most tRNA modifications occur in the anticodon loop (ACL), D-loop and TψC-loop, with positions 34 and 37 showing the highest diversity [38] (see review on tRNA modification circuits in the anticodon loop [41]). These modifications, part of the epitranscriptome, are added by specific enzymes and range from simple (e.g. methylation, thiolation) to complex (e.g. queuosine). They can affect the ribose or nucleobase, influencing tRNA structure [42] and degradation [43], from subtle to
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**Figure 2.** Environmental dissemination and impact of antibiotics on bacterial stress and adaptation (adapted from [28]). Circulation of antibiotics from major anthropogenic sources into natural ecosystems lead to their accumulation in soil, freshwater, and marine environments. This widespread sublethal pressure imposes continuous selective and physiological pressures on bacteria, promote adaptive responses and thus contribute to AMR's emergence and spread. Antibiotic stress adaptation has been described to be through transcriptional [29–32] as well as post-transcriptional regulation (reviewed here).

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**Figure 3.** tRNA modifications in bacteria. In the schematic representation of the tRNA molecule, each nucleotide position is labelled with the modification(s) identified in bacteria (source: MODOMICS [38]). Next to each modification, the associated catalysing enzyme is indicated. In cases where the modifying enzyme has not been identified yet, 'unknown' is written. We note that methods employed for modified residues identification do not detect all modifications, and therefore, the list provided is not exhaustive. For instance, 2-thiouridine (s2U), among others, is not included in this representation. (Included species: *Azospirillum lipoferum*, *Bacillus subtilis*, *Escherichia coli*, *Geobacillus stearothermophilus*, *Lactococcus lactis*, *Mycobacterium smegmatis*, *Mycoplasma capricolum*, *M. mycoides*, *Rhodospirillum rubrum*, *Salmonella typhimurium*, *Spiroplasma citri*, *Staphylococcus epidermidis*, *Streptomyces coelicolor A3(2)*, *S. griseus*, *Synechococcus elongatus PCC 6301*, *Sy. sp*. PCC 7002, *Synechocystis* sp., *Thermus aquaticus*, *T. thermophilus*.)

major conformational changes [44] (figure 3). The MODOMICS database [38] catalogues these modifications, their enzymes and biosynthetic pathways. The (non-exhaustive) diversity of tRNA modifications identified in bacteria is shown in figure 3.

To date, the roles of tRNA modifications appear to depend largely on their specific location within the molecule [7]. Modifications at the anticodon loop (ACL) have been reported to influence aminoacylation rate [45–47] and reliability [48], codon decoding, translation speed, accuracy [49], reading-frame maintenance [50] and tRNA fragment (tRF) biogenesis [51]. Modifications outside the ACL are generally thought to have minimal direct impact on codon-anticodon pairing. However, they can influence various aspects of tRNA function, such as ribosome affinity, structural stability and maturation, ultimately affecting protein synthesis [52,53].

### 3.2.2. tRNA modifications are dynamic

tRNA modifications were once seen as static, but are now known to be dynamically regulated. Bacterial tRNA modification profiles can change rapidly in response to internal or external cues, such as growth phase [54], rate [55], stress [56,57], nutrient
levels [58], oxygen [56,59] and temperature [60,61]. tRNA modification levels can also respond to phage infection, boosting phage gene expression [62]. This is achieved through shifts in expression of modifying enzymes, tRNA synthesis or degradation and demodifying enzyme activity. Together, these mechanisms allow for dynamic epitranscriptomic regulation. Technological advances have helped track such changes under stress [63–65], but the specific roles of dynamic tRNA modifications in bacterial adaptation are still not fully understood.

Beyond RNA-modifying enzymes (*writers*), cells also use *erasers* (which remove modifications) [66,67] and *readers* (which recognize modifications), playing a crucial role in interpreting them and facilitating information transmission within the cell; reviewed in [68]. Without erasers, bacteria would rely on slow tRNA turnover to shift modification profiles, an impractical delay, given tRNAs' high *in vivo* stability [69]. Methylations are the most studied reversible RNA modifications, especially in eukaryotes, where several demethylases have been characterized [70–75]. In contrast, few bacterial tRNA demodifying enzymes have been identified. The only known examples are Rmd enzymes, which remove N¹-methyladenosine in *Streptomyces venezuelae* [67], and RudS enzymes, proposed to reverse 4-thiouridine [66]. These findings suggest demodifying activity also exists in bacteria and more such enzymes remain to be discovered.

Notably, some tRNA modifications depend on the presence of others. While certain enzymes act directly on unmodified tRNAs, others require prior modifications to function. These interdependencies form ‘modification circuits’, especially in the anticodon loop [41,76,77], coordinating the timing and structure of tRNA decoration.

### 3.2.3. Modifications can impact tRNA structure and stability

Post-transcriptional tRNA modifications are essential for forming the three-dimensional L-shape through D- and T-arm interactions. Pseudouridine (Ψ), the most abundant RNA modification, stabilizes the T-arm and enhances D–T arm binding (a review [78]). TruB-mediated Ψ55 is crucial for bacterial adaptation to temperature shifts, aiding *E. coli* in heat shock survival [79] and supporting cold-shock growth in *Thermus thermophilus* [80].

Dihydouridine (D), produced by dihydrouridine synthases (Dus), is found mainly in the D-loop and varies by tRNA, species, and environment. In *E. coli*, DusA, DusB and DusC generate D at positions 16, 17, 20–20a. While once thought to stabilize the D-stem-loop structure, D is now known to increase flexibility and conformational dynamics [81].

These modifications fine-tune tRNA stability and act as quality control signals. Hypomodification, especially outside the ACL, can trigger targeted tRNA decay and reduce aminoacylation [43,82]. They also regulate tRNA cleavage [83].

### 3.2.4. Codon-anticodon base pairing: the wobble pairing hypothesis

In 1966, Francis Crick proposed the wobble hypothesis, suggesting that the anticodon's 34th nucleotide (wobble position) can form non-canonical base pairs with the third codon base [84]. Unlike strict Watson-Crick-Franklin pairing at positions one and two, wobble allows flexible pairing, such as modified U pairing with G and inosine pairing with U, C or A [85]. These modifications enable one tRNA anticodon to recognize multiple codons, explaining genetic code degeneracy and why organisms like *E. coli* have fewer tRNAs: 35, than codons: 61. Only methionine and tryptophan have unique codons, and 30-40% of decoding relies on wobble pairing [85]. Certain base modifications can expand the codon recognition range of a specific anticodon [86–89], while others can inhibit or restrict wobble pairing [90,91].

In 1982, Yarus introduced the extended anticodon concept, emphasizing the role of nucleotides around the anticodon and their modifications in codon recognition [92]. Position 37, a frequently modified purine adjacent to the anticodon, stabilizes codon-anticodon pairing, prevents frameshifts and maintains the anticodon loop structure [93–96].

In summary, tRNA modifications generate diversity and ensure proper folding, especially of the anticodon loop, critical for translation efficiency and translocation [97].

### 3.2.5. The emerging relevance of epitranscriptomics

Initially a niche field due to the limited number of described modified residues and a lack of understanding of their cellular roles, RNA modification research has advanced significantly with recent technological breakthroughs. While systematic studies of modification diversity and dynamics have been conducted in only a few species, next-generation sequencing (NGS), large-scale epitranscriptome mapping, and the identification of new RNA-modifying enzymes via reverse genetics and mass spectrometry have revolutionized the field [98,99].

The conservation and remarkable diversity of RNA-modifying genes across species, which exceed the essential requirements for canonical base synthesis as mentioned above, highlight their crucial role in biological processes.

## 3.3. tRNA modifications for bacterial phenotypic adaptation

### 3.3.1. The concept of modification tunable transcripts

Translation efficiency depends on the balance between tRNA supply and the cell's translational demand [100]. When aminoacyl-tRNAs meet demand, translation proceeds efficiently and accurately. Under stress, rapid synthesis of key proteins is crucial for adaptation. One proposed model suggests that bacteria reprogramme tRNA modifications to modulate translation, adjusting tRNA abundance and codon decoding to selectively express stress response proteins [57,101,102].
These proteins are encoded by modification tunable transcripts (MoTTs), transcripts enriched in codons decoded by dynamically modified tRNAs. Endres et al. [101] defined MoTTs as transcripts that (i) encode stress-response proteins, (ii) exhibit specific codon biases and (iii) are translationally regulated by wobble base modifications. MoTTs often contain ‘rare’ codons, typically associated with low-abundance tRNAs that increase under stress, enabling selective translation.

In fact, codon usage, though influenced by species GC content, frequently clusters in genes with related functions [103]. Codon patterns vary between genes and may include enrichment of specific codons [104], codon stretches [94,105] or codon pair/triplet combinations [106]. Over evolutionary time, codon usage and tRNA modification profiles may co-adapt to optimize translation efficiency and conserve energy during protein synthesis [107].

The role of tRNA modifications in codon decoding has been demonstrated across various modifications, codons and organisms. This post-transcriptional layer of regulation adds complexity beyond classical transcriptional control.

### 3.3.2. Modification tunable transcripts and adaptation to antibiotics and fluctuating oxygen concentrations

To survive antibiotics, bacteria have developed diverse strategies [108]. While resistance, growth at high antibiotic concentrations, is generally well characterized, treatment failure also stems from non-resistant phenotypes like persistence and tolerance [109]. Tolerant cells, although susceptible, die more slowly due to reduced drug uptake, target activity or stress-induced metabolic slowdown [110]. Unlike certain rRNA modifications, which are directly linked to resistance [111], tRNA modifications are increasingly associated with tolerance.

In V. cholerae, high-throughput screens identified RNA modification genes involved in responses to various antibiotics [112], though mechanisms, MoTT-related or not, remain unclear. One key step in tolerance is antibiotic uptake, reliant on proper expression of outer membrane (OM) proteins. In E. coli and Salmonella, OM proteins like LolB, OmpA and TolC depend on TrmD-mediated m¹G37 tRNA methylation, which enhances translation of proline-rich codons under stress [113]. TrmD, essential in bacteria, regulates m¹G37 levels affecting tRNAs for Pro, Leu and Arg; loss of this modification leads to uncharged tRNA accumulation and ribosome stalling [114], especially in Pro-rich cold-shock transcripts. Similarly, TusB-dependent tRNA modification promotes doxycycline tolerance in Yersinia pseudotuberculosis [115].

As previously noted, oxidative and antibiotic stresses are closely linked. In V. cholerae, Tgt-mediated queuosine (Q) modification of tRNA<sup>Tyr</sup> is critical for aminoglycoside tolerance [87,112]. Induced by sub-MIC tobramycin, Tgt enhances oxidative stress responses by modulating SoxR regulon expression via codon-dependent translation of its repressor, the Rsx complex [87]. Bioinformatics also links Tgt to MoTT-regulated genes involved in biofilm formation in E. coli [103] and metal homeostasis [116].

In Pseudomonas aeruginosa, oxidative stress induces TrmB, increasing m⁷G modification on tRNA<sup>Phe</sup> and tRNA<sup>Asp</sup>. This boosts translation of catalase genes (katA, katB) enriched in phenylalanine and aspartic acid codons, enhancing oxidative stress resistance [104]. The MoTT feedback loop is clear: stress → TrmB induction → m⁷G increase tRNAs Asp and Phe → selective translation of catalase transcripts → restored homeostasis. Similar codon enrichment is observed in E. coli catalases (katE, katG), and ΔtrmB strain show growth defects under peroxide stress [117]. The exact mechanism by which position 46 m⁷G affects decoding remains unclear.

In colistin-resistant Acinetobacter baumannii, ΔtrmB mutants lacking m⁷G on specific tRNAs show heightened oxidative stress sensitivity, reduced macrophage replication and lower infectivity [118]. This is partly due to failure to induce siderophore biosynthesis and uptake proteins, though the codon bias underlying this effect remains untested. TrmB is also induced by tigecycline [119], alongside an uncharacterized tRNA methyltransferase [120], though neither has been directly linked to tigecycline resistance [118].

In M. bovis BCG, a complete MoTT loop has been described under hypoxia: CmoM-mediated cmo⁵U modification of tRNA<sup>Thr</sup> enhances translation of ACG-enriched transcripts, including DosR, a master regulator of hypoxic persistence [59].

The general stress sigma factor RpoS is a confirmed MoTT in E. coli, helping to limit ROS accumulation during antibiotic stress [35]. Its stability depends on IraP in E. coli [121], and both rpoS and iraP require efficient decoding of leucine codons mediated by the MiaA-catalysed ms²i⁶A³⁷ tRNA modification [96,122], along with contributions from TrmL and TusA [123]. Though the specific stress signals enhancing leucine codon decoding remain unknown, miaA itself is post-transcriptionally regulated by small RNAs [124]. MiaA also affects codon decoding in other contexts, including the RNA chaperone Hfq in E. coli²⁴¹ and secondary metabolism genes in S. albus, a producer of bioactive compounds [125].

Stress resistance is tightly linked to host colonization. Oxidative stress-sensitive bacteria, for instance, show reduced infectivity due to macrophage-induced ROS. In M. tuberculosis, MnmA-mediated uridine sulfuration supports intracellular growth, likely by enhancing oxidative stress responses [126], also observed in E. coli [127]. Like RpoS [122], MiaA-dependent ms²i⁶A³⁷ and Tgt-mediated Q modifications are required for translating leucine-rich virulence genes such as virF in Shigella flexneri [128]. Similarly, mnm⁵s²U³⁴ synthesis by GidA-MnmE supports virulence in E. coli and Salmonella [129], while GidA affects P. aeruginosa pathogenicity by modulating decoding of rare codons (e.g. AGA, GGA, UUA [130]). Some tRNA modifications even influence host responses, e.g. stress-regulated TrmH-mediated 2'-O-methylation of tRNA<sup>Tyr</sup> in E. coli may help evade immune defences [131,132].

While most examples above involve stress-induced upregulation of RNA modification enzymes, some act as direct sensors. In E. coli, MiaB, an iron-sulfur enzyme, modifies codons in the uof-fur operon and is activated by iron, allowing post-transcriptional regulation of the iron stress response [133]. Similarly, in Enterococcus faecalis, RlmN, a dual tRNA/rRNA methyltransferase, senses ROS via inactivation of its iron-sulphur cluster, triggering superoxide dismutase expression and reducing virulence [134]. In Escherichia coli, DusA/B/C also respond to oxidative stress by sensing NADPH levels, adjusting dihydrouridine modification activity accordingly [135]. These enzymes illustrate how RNA modification machinery can act as metabolic or redox sensors, linking environmental cues to translational control.
Though codon dependencies remain poorly characterized, several studies link specific tRNA modifications to the expression of stress response genes. In *S. typhimurium*, deletion of *GidA*, which installs the mnm⁵s²U³⁴ modification, leads to downregulation of oxidative stress proteins like *YghA* and *Tpx* [136]. In *P. aeruginosa*, *TrmJ*-mediated 2'-O-methylation at position 32 (when A, C or U) is essential for *OxyR* expression and its regulon [137]. In uropathogenic *E. coli*, *TusBCD/MnmA*-driven tRNA sulfuration is required for synthesizing virulence factors such as flagella and fimbriae [138]. Loss of tRNA modification enzymes frequently affects motility-related traits [139], underscoring their role in virulence and stress adaptation.

### 3.3.3. Non-modification tunable transcripts responses and general antibiotic phenotypes

Beyond MoTT-based mechanisms (figure 4), stress adaptation can also result from tRNA structural changes mediated by modifications. In thermophilic archaea, such modifications stabilize tRNA against heat-induced destabilization, preserving translation efficiency [60]. Similar roles are likely in other organisms' heat shock responses [61].

Under oxidative stress in *E. coli*, *Tgt* has been shown to damage its substrate tRNAs by introducing abasic sites at the anticodon, potentially reducing translation during stress [140]. Additionally, a recent study identified 5-methylcytidine (m⁵C) at position 49 of *E. coli* tRNA<sup>Tyr</sup>, catalysed by RsmF, an rRNA methyltransferase induced under oxidative and heat stress [141]. Although ΔrsmF mutants are ROS-sensitive, the precise role of m⁵C in stress resilience remains unclear. This finding is particularly interesting because it was previously thought that m5C was not found on bacterial tRNAs.

UV irradiation, another oxidative stressor, also alters RNA modification profiles [142]. In *E. coli*, UV-induced crosslinking of 4-thiouridine at position 8 with cytidine at position 13 in tRNA<sup>Phe</sup> and tRNA<sup>Pro</sup> disrupts aminoacylation, halts translation, and temporarily inhibits growth [143], likely a reversible protective mechanism against UV damage.

On a different note, characterizing redox proteins, such as metal cofactor enzymes and flavoproteins, can shed light on their roles in managing or exacerbating oxidative stress. Notably, several tRNA-modifying enzymes, like the flavoprotein dihydrouridine synthases *DusA*, *DusB* and *DusC*, participate in redox reactions [144,145] and modulate their activity in response to the intracellular redox state [135]. Under oxidative stress, their reduced activity lowers dihydrouridine (D) levels on tRNAs, raising the question of whether D contributes to stress adaptation.

tRNA-modifying genes impact translation, regulation and stress responses, making them potential targets to combat antibiotic resistance [3] and bacterial virulence [130,146]. As proof-of-concept, the efficacy of fluoropyrimidines like 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) partly stems from their disruption of U-modifications (e.g. m⁵U and Ψ) in tRNAs, leading to tRNA destabilization [147]. Pre-existing tRNA hypomodification amplifies these effects, suggesting RNA-modifying enzymes could be leveraged to boost the potency of such treatments.

In some cases, tRNAs undergo post-transcriptional cleavage to generate tRNA-derived fragments (tRFs), including halves and shorter pieces (13–20 nt) [148]. First identified in human tumours [149] and phage-infected *E. coli* [150], tRF formation is conserved across all domains of life [151]. In bacteria, tRFs regulate diverse processes, such as gene expression [152] and host cell survival in *M. tuberculosis* [153]. While direct links between tRFs and RNA modifications are well-established in eukaryotes [154–156], this connection remains to be fully explored in bacteria.

tRNAs also participate in non-translational functions [37,148,157]. In *Streptomyces*, tRNA-delivered amino acids serve as precursors for antibiotic biosynthesis [158]. tRNAs can also donate amino acids for peptidoglycan synthesis [159], membrane lipid aminoacylation and N-terminal protein tagging, affecting protein stability and degradation [160].

Given the broad roles of tRNA modifications, their loss often causes pleiotropic effects, making it difficult to separate the impact of the modification from that of the enzyme. For instance, while *E. coli* pseudouridine synthases (e.g. *pus* genes) are dispensable under optimal growth, a Δ*truB* mutant is outcompeted by wild-type at 37°C, independently of *TruB*'s enzymatic activity [161]. This introduces the next topic: the roles of tRNA-modifying enzymes beyond their catalytic functions.

# 4. Beyond modification: other roles for tRNA-modifying enzymes

This section highlights that phenotypic effects may stem from either the modifications or the enzymes responsible for catalysing them, which can have distinct roles.

## 4.1. Dual function tRNA modification enzymes

Some tRNA-modifying enzymes have dual functions, either modifying multiple RNA species or performing additional, non-catalytic roles. For instance, *RlmN* [162], *RluF* [163] and *RluA* [164] modify both tRNAs and rRNAs. Notably, *RluA* was recently shown to pseudouridylate bacterial mRNAs [165], specifically targeting structured motifs in hairpin loops.

Beyond catalytic duality, several enzymes moonlight in non-modification roles. MiaB, for example, influences type III secretion in *P. aeruginosa* independently of its role in tRNA thiolation. This regulatory function may involve direct interaction with transcription factors, as it co-purifies with the global regulator *Vfr* [166]. Further studies are needed to elucidate the underlying mechanism.

Another example comes from a recent study showing that antimicrobial peptides increase the expression of *QueE* in *E. coli*. *QueE* is a key enzyme in queuosine biosynthesis for tRNAs (Asp, Asn, His, Tyr), and was recently shown to block cell division at the septal ring in response to antimicrobial peptides, a role unrelated to tRNA modification [167]. This moonlighting function appears conserved in *E. coli*, *S. typhimurium* and *K. pneumoniae*, but not in *P. aeruginosa* or *B. subtilis*, suggesting species-specific adaptation.
<figure><img src="image_10.png" /><figcaption>**Figure 4.** Modulation of bacterial translation under stress through tRNA modification and codon bias. Environmental and intracellular stresses can induce specific tRNA-modifying enzymes, altering tRNA modifications profiles. These epitranscriptomics changes modulate the decoding capacity and structural properties of tRNAs, influencing translation of mRNAs enriched in specific codons, referred to as modification tunable transcriptome (MoTT). This mechanism enables the selective synthesis of stress-responsive proteins, contributing to bacterial adaptation and survival under adverse conditions.</figcaption></figure>

Another striking case is TilS, essential for modifying tRNA<sup>de</sup> to ensure translational fidelity. In *Burkholderia cenocepacia* and *E. coli*, mutations in *tilS* that disrupt its catalytic function confer growth advantages under redox-imbalanced conditions. This suggests a trade-off between translation accuracy (via modified tRNA<sup>de</sup>) and metabolic fitness under redox-imbalanced conditions, via catalytically inactive TilS, pointing to a secondary role of TilS in redox homoeostasis [168].

These examples highlight that tRNA-modifying enzymes are not only central to translation, but also contribute to broader cellular functions such as stress response, cell division and virulence, often through mechanisms beyond their canonical enzymatic activity.

## 4.2. tRNA modifying enzymes as tRNA chaperones

tRNA structure and stability are essential for proper function. For example, efficient aminoacylation depends on correct folding. While tRNA processing is generally well-coordinated [169], proper folding into the native L-shaped three-dimensional structure is a critical maturation step, since aberrant, non-functional conformations can be energetically favourable and rapidly adopted [170,171]. Misfolding arises primarily from: (i) incorrect base pairing that is hard to reverse, and (ii) difficulties in achieving productive folding over energetically stable misfolded states. As a result, RNAs, including tRNAs, often become kinetically trapped in non-productive conformations [172,173], and only a minority reach their active state [174]. This contributes to longer folding timescales compared to proteins [175].

To overcome the challenges of RNA misfolding, organisms rely on RNA chaperones, proteins that assist RNA in reaching its correct conformation. They destabilize incorrect base-pairing, helping RNAs escape kinetic traps through a process known as iterative annealing [171,176] (figure 5). Unlike protein chaperones, RNA chaperones act in an energy-independent manner, relying on thermodynamic differences between native and misfolded conformations. To date, very few tRNA chaperones have been identified [178]. The best-characterized is the human La protein, which binds pre-tRNAs with high affinity to aid maturation and protect them from exonucleolytic decay [179]. In bacteria, only two tRNA chaperones are known so far, remarkably, these are the RNA-modifying enzymes TruB and TrmA [53,180,181].

Verifying chaperone activity in tRNA-modifying enzymes is challenging due to functional redundancy, as loss of one enzyme often has minimal impact [180]. Additionally, when tRNA destabilization is observed in knockout strains, it is hard to separate the effects of lost modification from the loss of chaperone activity.

In *E. coli*, ΔtruB causes moderate growth defects under normal conditions, which become more pronounced under stress [79,161,180]. However, catalytically inactive TruB variants that still bind tRNA can rescue the fitness defects of ΔtruB strains. TruB acts as a tRNA chaperone by transiently binding both folded and misfolded tRNAs, disrupting tertiary interactions in the elbow region (D- and T-arms), and promoting proper folding for aminoacylation, all independently of ATP or its Ψ55 synthase activity [180]. This chaperone role is essential for fitness under stress, as shown in co-culture assays [161,180]. Nonetheless, Ψ55 itself also aids survival during heat and cold shock [79,80].

Similarly, TrmA, a SAM-dependent methyltransferase that installs m⁵U at position 54, shows binding-dependent, catalysis-independent chaperone activity *in vitro* [181]. Like TruB, it targets the tRNA elbow, locally unfolding the T-loop to access its substrate [182]. However, dissecting TrmA's chaperone role is harder since m⁵U is essential for growth. Recent studies using catalytic mutants show that m⁵U54 contributes to resistance against translation elongation inhibitors like hygromycin B and ensures full modification under stress [183].

Might all RNA-modifiers moonlight as chaperones? Although RNA chaperones are often considered non-specific RNA binders [184], tRNA chaperones stand out by their selectivity: they may bind all tRNAs but do not interact with other RNA types. This mirrors the specificity of protein chaperones, which typically assist certain protein classes rather than all peptide chains [185]. Given that many tRNA-modifying enzymes promote structural rearrangements [182,186–188], including local unwinding or base-flipping, to access their target sites, their chaperone-like activity seems intuitive. The Kothe lab proposed that such remodelling of tRNA structure, particularly in the elbow region critical for three-dimensional folding [53,180], may underpin the chaperone functions of modification enzymes [53]. This highlights the close link between tRNA folding,
<figure><img src="image_11.png" /><figcaption>**Figure 5.** RNA folding energy landscape and kinetic traps (adapted from [177], generated using ChatGPT). This schematic represents a theoretical three-dimensional RNA folding energy landscape, where colour gradients correspond to energy levels (red: high energy; dark blue: low energy). Unfolded RNA is associated with high-energy (red zone), while folding proceeds downhill towards energy minima. Light blue regions represent *local minima* associated with incorrect pairings and non-productive conformations, also named *kinetic traps*. In contrast, the global minima, represented in dark blue, corresponds to the native, productive structure. RNA chaperones facilitate correct folding by helping misfolded RNAs to escape from kinetic traps. Theoretical RNA structures are depicted along the folding trajectory for illustrative purposes.</figcaption></figure>

maturation and modification. A similar phenomenon is observed in ribosomal RNA, where certain modifying enzymes, like the methyltransferase RsmC, also exhibit chaperone activity [189].

# 5. Future directions

## 5.1. Expanding knowledge of tRNA modifications across species and environments

To date, the complete landscape of tRNA modifications and their corresponding enzymes has been characterized in only a few bacterial species, including *E. coli* [190], *Mycoplasma capricolum* [191] and *Bacillus subtilis* [192]. More recently, in 2024, comprehensive tRNA modification profiles have also been mapped in *Pseudomonas aeruginosa* [193] and *Bartonella* [194]. Partial modification maps also exist for *Staphylococcus aureus* [195], *Methanocaldococcus jannaschii* [196], *Streptomyces albus* [197] and thermophilic aerobic bacilli [198], although the corresponding enzymes remain unidentified in many cases.

New tRNA modifications and enzymes continue to be discovered, such as aminovaleramide [199], *V. cholerae*-specific cytidine-to-pseudouridine editing [200], a flavin-dependent methyltransferase [201] and several others [202–204]. High-throughput techniques are increasingly used to study regulatory networks governing tRNA modifications [205–207], including responses to heat stress [208], temperature shifts [207,209], nutrient availability [210,211], phage infection [62,212] and phage-encoded tRNAs [213].

Transcriptomics and genomic studies across various environments and species have been conducted over many years, providing valuable data on the expression and fitness of different tRNA modification genes. A differential gene expression profile between *in vivo* (patient) and *in vitro* (urine) conditions in uropathogenic *E. coli* (UPEC), revealed that genes involved in tRNA modification, including *queA*, *thiI*, *miaB*, *tsaB*, *dusC*, *tadA*, *truC*, *tcdA*, *dusB*, *tusA* and *trmA*, were among those significantly upregulated during urinary tract infection (UTI) [214]. Supporting this, MiaA was also found to be required for maximal fitness in mouse UTI and systemic infection models of extraintestinal pathogenic *E. coli* (ExPEC) [215]. Shea *et al*. further identified the tRNA modification genes *thdF* and *gidA* as important fitness factors for *E. coli* CFT073 during UTI, necessary for bacterial growth in human urine and effective urinary tract colonization [216]. Similarly, deletion of the *tusDCB* sulfur relay system in UPEC impaired the production of virulence factors such as type 1 fimbriae and flagella, and reduced bacterial aggregation in bladder epithelial cells [138]. Beyond *E. coli*, other pathogens also rely on tRNA modifications for successful infection. In *P. aeruginosa*, *GidA* [130], *TrmA* [217] and *TrhPO* [218] were shown to be essential for pathogenicity by facilitating translation of virulence genes involved in motility, cytotoxicity, biofilm formation and overall fitness. In *V. cholerae*, a transposon insertion sequencing screen in neonatal rabbits identified several tRNA modification genes, including *mnmE*, *gidA* and *thiI*, as critical for intestinal colonization [219]. A separate high-coverage Tn-seq analysis in *V. cholerae* E7946 also found *thiI*, *truA*, *queA* and *tgt* to be essential or strongly required for both intestinal colonization and survival in pond water [220]. During *Shigella* infection an organoid model uncovered the role of MnmE and *GidA* on host colonization [221]. In plant pathogens, a genome-wide screen during *Xanthomonas hortorum pv. vitians* infection in lettuce showed that disruption of *tgt*, severely compromised bacterial fitness in plants [222]. As a result, post-transcriptional tRNA modifications are increasingly recognized for having a role in bacterial fitness and virulence across diverse infection models.
## 5.2. Modification networks

Many unknowns remain about modification networks, including the precise order of modifications and their mutual influence [76]. Few studies have systematically examined combinations of modification deficient mutants [223]. While single gene deletions rarely cause growth defects, combined mutations may reveal compensatory interactions.

## 5.3. New challenges

Despite the constraints, new approaches continue to emerge [224]. For instance, nanopore technology now enables direct analysis of aminoacylation on native tRNAs [225], facilitating studies on how individual modifications affect charging efficiency. The concept of metaepitranscriptomics, the study of tRNA modifications within microbiomes and natural communities, has also been proposed [206], though such studies have yet to be reported and practical applications are still in early stages.

It is important to note that, in addition to tRNA modifications, ribosomal RNA (rRNA) modifications have also been shown to play roles in cellular adaptation to stress. In particular, certain rRNA-modifying enzymes respond dynamically to environmental conditions, contributing to ribosome heterogeneity and functional reprogramming under stress [226–228]. Given the mechanistic parallels between rRNA and tRNA modification systems, including overlapping enzymes and shared regulatory pathways, approaches and technologies developed for the study of rRNA modifications, such as high-throughput sequencing, structure-probing techniques or real-time modification mapping, could be adapted or repurposed to advance our understanding of tRNA modifications. As such, cross-applications of these methodologies may offer promising avenues for future research into the regulatory dynamics of the tRNA epitranscriptome.

## 6. Conclusion

A recent NASEM report highlights the importance of mapping RNA modifications in various species and conditions [229]. In line with this, this review addresses a critical and timely topic, from a bacterial stress-response perspective. While our focus is on tRNA modifications, recent reviews on rRNA modifications also emphasize the role of modification-driven ribosome heterogeneity and dynamics [226,227].

The field of RNA modifications in bacteria has long been underexplored. While tRNA and rRNA modifications have been extensively studied in eukaryotes, often in the context of human disease, their roles in bacterial stress response and adaptation remain poorly characterized. This gap is partly due to the lack of clear phenotypes in unstressed cells when many RNA modification genes are deleted, despite some being essential [230]. Additionally, technical challenges have limited the detection of these modifications.

Interest in bacterial RNA modifications is growing, driven by recent studies linking tRNA modifications to stress responses. Research on SARS-CoV-2 has further boosted attention to RNA modifications, revealing their roles in viral infection and host immunity [231]. Notably, the success of mRNA vaccines against SARS-CoV-2 is partly due to the methyl-pseudouridine (m¹Ψ) modification, which improves translation efficiency [232].

In recent years, Illumina-based chemical detection methods have been developed to identify a broad range of tRNA and rRNA modifications [233–237]. While these techniques have enabled important discoveries using simple RNA extracts, they have notable limitations. Short read lengths make it difficult to study multiple modifications on a single RNA molecule or distinguish closely related RNA species, such as similar rRNA operons. Low-abundance RNAs may also go undetected, and the technical complexity limits their use to a few specialized laboratories. Importantly, newly identified modifications require validation through multiple approaches, particularly when the responsible enzyme is unknown and cannot be disrupted to confirm loss of the modification. At the same time, advances in nanopore sequencing are rapidly improving RNA modification detection, offering promising avenues for future research.

tRNA modifications in bacteria offer an exciting frontier for novel therapeutic strategies [238–241]. Although often non-essential under standard conditions, these modifications play key roles in bacterial physiology and pathogenicity. Targeting tRNA-modifying enzymes or pathways can disrupt processes such as virulence, motility, host colonization and the expression of antibiotic-resistance genes [129,146,242]. Some modifications enhance the translation of resistance factors, stress regulators or efflux pumps, interfering with them could weaken bacterial fitness or increase antibiotic sensitivity without imposing strong selective pressure.

Altogether, the study of tRNA modifications holds great promise, offering new insights into bacterial physiology and potential therapeutic avenues, with many questions still unanswered and exciting discoveries ahead.

**Ethics.** This work did not require ethical approval from a human subject or animal welfare committee.

**Data accessibility.** This article has no additional data.

**Declaration of AI use.** While preparing this work, the author(s) used ChatGPT to refine a few sentences for clarity and conciseness. All content was subsequently reviewed and edited by the authors, who take full responsibility for the final version of the publication.

**Authors' contributions.** L.F.: formal analysis, investigation, methodology, writing—original draft, writing—review and editing; C.S.: investigation, methodology, writing—original draft; A.C.: investigation, methodology, writing—original draft; Z.B.: conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, writing—original draft, writing—review and editing.
All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration. We declare we have no competing interests.

Funding. Research in the lab was funded, in whole or in part, by the Institut Pasteur, the Centre National de la Recherche Scientifique, ANR ModRNAntibio (ANR-21-CE35-0012), ANR EpiRNA (ANR-24-CE12-7224) and the Fondation pour la Recherche Médicale (FRM EQU202103012569).

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