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tRNA modifications as regulators of bacterial virulence and stress responses [1]

['Chloé Teixeira', 'Ciri', 'Centre International De Recherche En Infectiologie', 'Université De Lyon', 'Inserm', 'Université Claude Bernard Lyon', 'Cnrs', 'Ens De Lyon', 'Lyon', 'François Vandenesch']

Date: 2025-11 

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.

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.

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.

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.

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 ].

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 ].

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.

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 ].

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 m 1 A22 and ms 2 t 6 A37 modifications are specific to B. subtilis [ 39 ].

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 m 2 A37 in tRNA and m 2 A2503 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 k 2 C34 and the ava 2 C34 modifications in tRNA-Ile [ 46 , 47 ].

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 ].

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 m 5 U54 [ 34 , 35 ].

Bacteria therefore exhibit a diversity of tRNA modifications, some of which are exclusive to their kingdom. Among the modifications cited above, m 2 A, s 2 C, s 4 U, ms 2 i 6 A, mnm 5 s 2 U, ho 5 U, mo 5 U, and cmo 5 U are specific to bacteria [ 7 , 10 , 18 , 31 ].

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 (ms 2 i 6 A) that necessitates the previous addition of an isopentyl group to form i 6 A [ 26 ]. Similarly, the acacp 3 U modification discovered in Vibrio cholerae (V. cholerae) is an acetylated form of the acp 3 U modification [ 27 ]. Other examples include the successive reactions that modify uridine to create mnm 5 s 2 U and then cmnm 5 s 2 U [ 28 ], or the hydroxylation at position 5 to form ho 5 U, followed by the methylation at the same position to produce mo 5 U, and finally the carboxylation to generate cmo 5 U [ 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), acp 3 U47 is stimulated by m 7 G46 and m 5 U54, as is ms 2 i 6 A37 by m 5 U54, and s 4 U8 by ψ55 and m 5 U54 [ 30 ].

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 (t 6 A) [ 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 (ac 4 C) [ 10 ]. Other modifications involve the addition of a sulfur atom with the thiolation at position 2 for cytidine (s 2 C) [ 24 ] and at position 2 or 4 for uridine (s 2 U and s 4 U) [ 25 ].

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 (m 1 A, m 2 A, and m 6 A) as can guanine (m 1 G, m 2 G, and m 7 G), whereas uridine and cytosine are only modified at one position (m 5 U and m 5 C). 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 ].

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 ).

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.

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 ].

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 s 4 U 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 m 5 U54 contributes to tRNA maturation as its absence is associated with an altered modifications pattern and impaired ribosome translocation [ 75 ].

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, mnm 5 s 2 U and ψ modifications respectively induce misreading of aspartate and histidine codons, creating mistranslation errors [ 72 , 73 ].

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 ].

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 i 6 A37 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 s 2 U34 modification enhances the binding affinity of the tRNA-Gln for CAA and CAG codons [ 62 ], and the mnm 5 s 2 U34 modification on tRNA-Lys(UUU) stabilizes non-canonical base pairing [ 67 ]. In Mycobacterium strains, the increase of cmo 5 U-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 cmnm 5 U34 modification, shows a decrease of translation efficiency for Arg-AGA and Leu-UUA codons that are rare codons [ 69 ].

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 m 2 A37 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 m 1 G37 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 i 6 A37 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, ms 2 i 6 A37, prevents frameshifting by stabilizing the interaction between the tRNA anticodon at position 34 and the mRNA codon at position 1 [ 60 ]. Similarly to ms 2 i 6 A37, the mnm 5 s 2 U34 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 (s 2 U) at position 34 participates in reading frame maintenance [ 60 , 62 ].

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 (m 5 U) enzymes in E. coli that contribute to the correct folding of tRNAs, even in the absence of their catalytic activity [ 54 , 55 ].

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 m 1 A9 modification disrupts Watson-Crick base pairing to allow formation of secondary and tertiary structures [ 50 ]. In bacteria, 2′O-methylation, thiolation, pseudouridylation [ 30 ], and m 5 U 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 ].

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 ].

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). PPT PowerPoint slide

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TIFF original image 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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].

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