Bacterial pathogens sense host-derived signals to control virulence, yet how these cues shape translation remains unclear. McShane et al. show that the host hormone norepinephrine rewires the tRNA epitranscriptome of enterohemorrhagic Escherichia coli (EHEC) O157:H7, altering wobble-position modifications and shifting decoding capacity toward A/U-ending codons (A. E. C. McShane, C.-K. Chan, R. Chen, M. S. DeMott, et al., mSystems 11:e01418-24, 2026, https://doi.org/10.1128/msystems.01418-25). This bias aligns with the codon architecture of the locus of enterocyte effacement (LEE), a major virulence locus, enabling selective translation of virulence genes. Multi-omic analyses link these changes in tRNA chemistry to proteome remodeling without major shifts in tRNA abundance. By coupling host hormone sensing to codon-biased translation, this work establishes the tRNA epitranscriptome as a dynamic regulator of bacterial virulence.

Bacterial defense systems rely on self/non-self discrimination to prevent autoimmunity, yet the role of orphan DNA methyltransferases in this process remains unclear. Here, we show that the orphan cytosine DNA methyltransferase VchM is genetically and functionally linked to a Menshen defense-associated system in Vibrio cholerae . We demonstrate that the growth defect of ∆ vchM mutants results from activation of the adjacent MenshenVc system rather than from an intrinsic requirement for DNA methylation. In the absence of VchM, MenshenVc disrupts 16S rRNA maturation and ribosome biogenesis, consistent with activation of an RNA-targeting effector. VchM-dependent methylation also suppresses MenshenVc toxicity in Escherichia coli , demonstrating that DNA methylation is sufficient to prevent system activation in a heterologous host. Although MenshenVc did not protect against the phages tested here, it reduced natural transformation with unmethylated donor DNA, suggesting a potential role in restricting horizontal gene transfer. Together, our findings identify DNA methylation as a mechanism preventing defense-system autoimmunity and establish orphan DNA methyltransferases as integral components of bacterial immune systems.

In eubacteria, decoding of isoleucine codon AUA requires a specialized tRNA (tRNAIle2) modified with lysidine (k2C) at the anticodon wobble position (C34), which switches decoding specificity from methionine (AUG) to isoleucine (AUA). Recently, aminovaleramide cytidine (ava2C) was discovered at the same tRNA position in several bacteria and plants and shown to support AUA decoding and Ile-specific aminoacylation. However, the enzyme catalyzing ava2C was unknown. Here, we report that tRNAIle-aminovaleramididine synthetase (AvaS) catalyzes ava2C biosynthesis in Pseudomonas aeruginosa PA14. AvaS converts k2C to ava2C through a pyridoxal-phosphate-dependent oxidative decarboxylation mechanism, supported by site-directed mutagenesis and in vitro enzymatic assays. Dual-reporter assays demonstrated that ava2C-modified tRNA exhibits lower AUA decoding efficiency than k2C-modified tRNA. Additionally, genome-wide screening revealed an unexpected link between ava2C levels and metabolic and stress response pathways influencing i6A/ms2i6A dynamics. Together, these findings define the molecular basis of ava2C biosynthesis and its broader cellular metabolic networks. Sun, Wu et al. identify AvaS as a pyridoxal-phosphate-dependent RNA-modifying enzyme, producing the tRNA modification ava2C from k2C in bacteria and plants and linking RNA chemistry to translational regulation of metabolic stress responses.

By integrating a literature review with transcriptomic, proteomic, and phenotypic data from two model bacteria, Escherichia coli and Vibrio cholerae, we put forward the hypothesis that defects in tRNA modification broadly impact processes that are evolutionarily tuned to be sensitive to translation speed. These include the translation of regulatory proteins associated with motility, iron homeostasis, and leader peptide-driven attenuation mechanisms. Some of these translation speed-dependent processes are influenced by the absence of a single modification, while others are affected by the absence of multiple modifications. Although further experiments are needed to clarify the mechanisms involved in each case, this work provides a foundational framework to guide future research.

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q34 in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the overall bacterial proteome, and elucidated the molecular mechanisms underlying the effects of Q34 modification in antibiotic translational stress response. Using molecular reporters, we showed that Q34 impacts the efficiency of decoding at tyrosine TAT and TAC codons. Proteomics analyses revealed that the anti-SoxR factor RsxA is better translated in the absence of tgt. RsxA displays a codon bias toward tyrosine TAT and overabundance of RsxA leads to decreased expression of genes belonging to SoxR oxidative stress regulon. We also identified conditions that regulate tgt expression. We propose that regulation of Q34 modification in response to environmental cues leads to translational reprogramming of transcripts bearing a biased tyrosine codon usage. In silico analysis further identified candidate genes which could be subject to such translational regulation, among which DNA repair factors. Such transcripts, fitting the definition of modification tunable transcripts, are central in the bacterial response to antibiotics.

Aminoglycosides (AGs) are broad-spectrum antibiotics effective against Gram-negative bacteria, with uptake dependent on membrane potential. However, the mechanisms of AG entry remain incompletely understood. Here, we identify a previously undescribed uptake pathway via carbohydrate transporters in E. coli. By deleting or overexpressing 26 carbohydrate transporters, we found that 18 facilitated AG uptake, a mechanism conserved across several Gram-negative ESKAPEE pathogens. Using fluorescent-labeled AGs and flow cytometry, we quantified differential uptake. To enhance AG efficacy, we screened 198 carbon sources for their ability to induce transporter expression using a cmtA-gfp fusion. Uridine emerged as a strong inducer of cmtA and 12 additional AG-importing transporters. Coadministration of uridine considerably improved AG efficacy against clinical and resistant E. coli strains by enhancing drug uptake. This combination also improved outcomes in human blood ex vivo and in a murine urinary tract infection model. Given uridine's clinical safety, it holds promise as an adjuvant to potentiate AG treatment against multidrug-resistant infections.

Transfer RNAs (tRNAs) are central to protein synthesis, ensuring precise decoding of the genetic code by delivering aminoacids to the ribosome. Among all RNA species, tRNAs are the most heavily and diversely modified, with modifications playing critical roles in stability, folding, and function. Here, we present a comprehensive, isodecoder-level map of tRNA modifications in the human pathogen Vibrio cholerae. This map was generated by chemical-based sequencing methods, comparing wild-type and deletion strains. By assigning specific tRNA modifications to their cognate enzymes, we defined a comprehensive modification landscape in Vibrio cholerae and confirmed species-specific features, such as the presence of a functional TrmK enzyme, largely restricted to Gram-positive bacteria. Additionally, we detected a modification at U55 that occurs independently of TruB. To assess the biological significance of these modifications, we evaluated fitness under both standard conditions and subinhibitory antibiotic stress, and examined how modifications in the anticodon stem-loop region influence codon decoding efficiency and accuracy. Based on a comparative analysis of E. coli and V. cholerae, we discuss how species-specific differences in tRNA isodecoder gene repertoires may influence the functional impact and biological importance of tRNA modifications. This work provides the first experimentally validated, genome-wide map of tRNA modifications in V. cholerae, serving as a reference for future research into RNA modifications, translation regulation, and pathogen biology.

RNA modifications play a fundamental role in regulating essential cellular processes, including translation fidelity and stress adaptation. While these modifications are installed post-transcriptionally by specialized enzymes, their broader functional roles remain largely unexplored. Here, we uncover an unexpected function for the Vibrio cholerae tRNA dihydrouridine synthase B (VcDusB) beyond its canonical role in tRNA dihydrouridylation. We show that deletion of dusB severely compromises V. cholerae resistance to oxidative stress, not through the loss of tRNA modification, but via disruption of an intrinsic NADPH oxidase activity. Mutational analyses reveal that DusB redox function is essential for survival under oxidative stress. Proteomic and transposon insertion sequencing analysis further linked DusB to NADPH homeostasis and metabolic reprogramming during stress adaptation. These findings redefine DusB as a bifunctional enzyme coupling tRNA modification to redox regulation, expanding the functional repertoire of RNA-modifying enzymes in stress adaptation. More broadly, this work paves the way for exploring the evolutionary versatility of tRNA-modifying enzymes, suggesting that their functions extend far beyond RNA metabolism to direct integration of translational control with cellular redox state.

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.

The emergence of new resistant bacterial strains is a worldwide challenge. A resistant bacterial population can emerge from a single cell that acquires resistance or persistence. Hence, new ways of tackling the mechanism of antibiotic response, such as single cell studies are required. It is necessary to see what happens at the single cell level, in order to understand what happens at the population level. To date, linking the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics remains challenging due to the trade-offs between the resolution and the field of view. Here we present a platform that measures the ability of individual E. coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidic chip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome’s peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature’s preference for utilizing reduced flavin in the reduction of uridines and their derivatives.

BACKGROUND:Vibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium. RESULTS:In this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens. CONCLUSION:We thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae.

AbstractThe possible active entry of aminoglycosides in bacterial cells has been debated since the development of this antibiotic family. Here we report the identification of their active transport mechanism inVibriospecies. We combined genome-wide transcriptional analysis and fitness screens to identify alterations driven by treatment ofV. choleraewith sub-minimum inhibitory concentrations (sub-MIC) of the aminoglycoside tobramycin. RNA-seq data showed downregulation of the small non-coding RNAncRNA586during such treatment, while Tn-seq revealed that inactivation of this sRNA was associated with improved fitness in the presence of tobramycin. This sRNA is located near sugar transport genes and previous work on a homologous region inVibrio tasmaniensissuggested that this sRNA stabilizes gene transcripts for carbohydrate transport and utilization, as well as phage receptors. The role forncRNA586, hereafter namedctrR, in the transport of both carbohydrates and aminoglycosides, was further investigated. Flow cytometry on cells treated with a fluorescent aminoglycoside confirmed the role ofctrRand of carbohydrate transporters in differential aminoglycoside entry. Despite sequence diversity,ctrRshowed functional conservation across the Vibrionales. This system in directly modulated by carbon sources, suggesting regulation by carbon catabolite repression, a widely conserved mechanism in Gram-negative bacteria, priming future research on aminoglycoside uptake by sugar transporters in other bacterial species.

The possible active entry of aminoglycosides in bacterial cells has been debated since the development of this antibiotic family. Here we report the identification of their active transport mechanism in Vibrio species. We combined genome-wide transcriptional analysis and fitness screens to identify alterations driven by treatment of V. cholerae with sub-minimum inhibitory concentrations (sub-MIC) of the aminoglycoside tobramycin. RNA-seq data showed downregulation of the small non-coding RNA ncRNA586 during such treatment, while Tn-seq revealed that inactivation of this sRNA was associated with improved fitness in the presence of tobramycin. This sRNA is located near sugar transport genes and previous work on a homologous region in Vibrio tasmaniensis suggested that this sRNA stabilizes gene transcripts for carbohydrate transport and utilization, as well as phage receptors. The role for ncRNA586, hereafter named ctrR, in the transport of both carbohydrates and aminoglycosides, was further investigated. Flow cytometry on cells treated with a fluorescent aminoglycoside confirmed the role of ctrR and of carbohydrate transporters in differential aminoglycoside entry. Despite sequence diversity, ctrR showed functional conservation across the Vibrionales. This system in directly modulated by carbon sources, suggesting regulation by carbon catabolite repression, a widely conserved mechanism in Gram-negative bacteria, priming future research on aminoglycoside uptake by sugar transporters in other bacterial species.

ABSTRACT RavA-ViaA were reported to play a role in aminoglycoside (AG) sensitivity, but the mechanisms remain elusive. Here, we performed competition and survival experiments to confirm that deletion of ravA-viaA increases tolerance of the Gram-negative pathogen Vibrio cholerae to low and high AG concentrations during aerobic growth. Using high-throughput strategies in this species, we identify Cpx and Zra2 two-component systems as new partners of RavA-ViaA. We show that the AG tolerance of ∆ravvia requires the presence of these membrane stress sensing two-component systems. We propose that deletion of the RavA-ViaA function facilitates the response AGs because of a pre-activated state of Cpx and Zra2 membrane stress response systems. We also find an impact of these genes on vancomycin resistance, and we show that simultaneous inactivation of ravvia function together with envelope stress response systems leads to outer membrane permeabilization. Vancomycin is mostly used for Gram-positive because of its low efficiency for crossing the Gram-negative outer membrane. Targeting of the ravA-viaA operon for inactivation could be a future strategy to allow uptake of vancomycin into multidrug-resistant Gram-negative bacteria. IMPORTANCE The RavA-ViaA complex was previously found to sensitize Escherichia coli to aminoglycosides (AGs) in anaerobic conditions, but the mechanism is unknown. AGs are antibiotics known for their high efficiency against Gram-negative bacteria. In order to elucidate how the expression of the ravA-viaA genes increases bacterial susceptibility to aminoglycosides, we aimed at identifying partner functions necessary for increased tolerance in the absence of RavA-ViaA, in Vibrio cholerae. We show that membrane stress response systems Cpx and Zra2 are required in the absence of RavA-ViaA, for the tolerance to AGs and for outer membrane integrity. In the absence of these systems, the ∆ravvia strain’s membrane becomes permeable to external agents such as the antibiotic vancomycin.

RavA-ViaA were reported to play a role in aminoglycoside sensitivity but the mechanisms remain elusive. Here, we performed competition and survival experiments to confirm that deletion of ravA-viaA increases tolerance of the Gram-negative pathogen Vibrio cholerae to low and high aminoglycoside concentrations, during aerobic growth. Using high throughput strategies in this species, we identify Cpx and Zra2 two-component systems as new partners of RavA-ViaA. We show that the aminoglycoside tolerance of Δravvia requires the presence of these membrane stress sensing two-component systems. We propose that deletion of the RavA-ViaA function facilitates the response aminoglycosides because of a pre-activated state of Cpx and Zra2 membrane stress response systems. We also find an impact of these genes on polymyxin B sensitivity and vancomycin resistance, and we show that simultaneous inactivation of ravvia function together with envelope stress response systems leads to outer membrane permeabilization. Vancomycin is mostly used for Gram-positive because of its low efficiency for crossing the Gram-negative outer membrane. Targeting of the ravA-viaA operon for inactivation could be a future strategy to allow uptake of vancomycin into multidrug resistant Gram-negative bacteria. ### Competing Interest Statement The authors have declared no competing interest.

Given the emergence of antimicrobial drug resistance, it is critical to understand the heterogeneity of response to an antibiotic within a population of cells. Since the drug can exert a selection pressure that leads to the emergence of resistant phenotypes. To date, neither bulk nor single-cell methods are able to link the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics. Here we present a platform that measures the ability of individual E. coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidic chip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms.

SUMMARY Aminoglycosides (AGs) are long-known molecules successfully used against Gram-negative pathogens. While their use declined with the discovery of new antibiotics, they are now classified as critically important molecules because of their effectiveness against multidrug-resistant bacteria. While they can efficiently cross the Gram-negative envelope, the mechanism of AG entry is still incompletely understood, although this comprehension is essential for the development of new therapies in the face of the alarming increase in antibiotic resistance. Increasing antibiotic uptake in bacteria is one strategy to enhance effective treatments. This review aims, first, to consolidate old and recent knowledge about AG uptake; second, to explore the connection between AG-dependent bacterial stress and drug uptake; and finally, to present new strategies of potentiation of AG uptake for more efficient antibiotic therapies. In particular, we emphasize on the connection between sugar transport and AG potentiation.

Vibrio cholerae N16961 genome encodes 18 type II Toxin/Antitoxin (TA) systems, all but one located inside gene cassettes of its chromosomal superintegron (SI). This study aims to investigate additional TA systems in this genome. We screened for all two-genes operons of uncharacterized function by analyzing previous RNAseq data. Assays on nine candidates, revealed one additional functional type II TA encoded by the VCA0497-0498 operon, carried inside a SI cassette. We showed that VCA0498 antitoxin alone and in complex with VCA0497 represses its own operon promoter. VCA0497-0498 is the second element of the recently identified dhiT/dhiA superfamily uncharacterized type II TA system. RNAseq analysis revealed that another SI cassette encodes a novel type I TA system: VCA0495 gene and its two associated antisense non-coding RNAs, ncRNA495 and ncRNA496. Silencing of both antisense ncRNAs lead to cell death, demonstrating the type I TA function. Both VCA0497 and VCA0495 toxins do not show any homology to functionally characterized toxins, however our preliminary data suggest that their activity may end up in mRNA degradation, directly or indirectly. Our findings increase the TA systems number carried in this SI to 19, preferentially located in its distal end, confirming their importance in this large cassette array.