Recombinant Vibrio vulnificus Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (TGT) and what is its primary function in bacterial cells?

Queuine tRNA-ribosyltransferase (TGT) is an enzyme that catalyzes the exchange of guanine (G) at position 34 (the wobble position) of specific tRNAs with queuine or its precursors. Specifically, TGT targets tRNAs with GUN anticodons, including tRNA^Asn_GUU, tRNA^Asp_GUC, tRNA^His_GUG, and tRNA^Tyr_GUA . This post-transcriptional modification is critical for translational fidelity and efficiency.

In bacterial systems like Vibrio species, TGT functions by incorporating the precursor base 7-aminomethyl-deazaguanine (preQ₁) rather than queuine directly. This modification is part of a multi-step pathway that ultimately produces queuosine (Q), a hypermodified 7-deaza-guanosine analog . The presence of this modification has been shown to affect codon recognition, translational efficiency, and bacterial fitness under various stress conditions .

How can researchers express and purify recombinant Vibrio vulnificus TGT in a laboratory setting?

Expression System Selection:
For optimal expression of V. vulnificus TGT, E. coli BL21(DE3) or similar expression strains are recommended. Based on protocols established for related bacterial TGTs, the following methodology is effective:

• Clone the V. vulnificus tgt gene into an expression vector with an inducible promoter (e.g., pET system).

• Transform into expression hosts and culture in rich media (LB or 2×YT) until mid-log phase.

• Induce expression with IPTG (typically 0.5-1.0 mM) at reduced temperature (16-25°C) to enhance proper folding.

• Harvest cells after 4-6 hours of induction.

Purification Protocol:

• Lyse cells using sonication or pressure-based methods in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and 10 mM imidazole.

• If using a His-tagged construct, purify using IMAC (immobilized metal affinity chromatography).

• Further purify using size exclusion chromatography to obtain highly pure enzyme.

• Verify enzyme activity using appropriate tRNA substrates and analyze modification efficiency.

The approach mirrors protocols established for E. coli TGT, which serves as a model for other bacterial TGT enzymes .

What experimental methods can be used to detect and quantify queuosine modification in tRNAs?

Several methodologies have been developed to detect queuosine modification, each with specific advantages:

1. APB Northern Blotting:
This technique exploits the presence of a cis-diol in the Q modification, which interacts with N-acryloyl-3-aminophenylboronic acid (APB) in polyacrylamide gels. This interaction retards the migration of Q-modified tRNAs compared to unmodified variants, allowing visualization by Northern blotting with specific tRNA probes .

2. LC-MS/MS Analysis:
Chromatography-coupled tandem quadrupole mass spectrometry offers precise quantification of queuosine levels:

• Digest tRNAs with nuclease P1 and phosphatase.

• Separate nucleosides using HPLC (typically with a C18 column).

• Detect Q based on characteristic mass transition patterns (precursor m/z 410, products m/z 163 and 295) .

• Normalize Q signal against canonical ribonucleosides for quantification.

3. Direct RNA Sequencing with Nanopore Technology:
This emerging methodology allows direct detection of Q and its precursors in native tRNAs without prior modification or amplification steps .

4. Bisulfite Sequencing:
While primarily used for detecting cytosine methylation, this approach can also reveal Q-dependent methylation patterns, as queuine incorporation has been shown to enhance C38 methylation in the anticodon loop .

How does the tgt gene differ between Vibrio vulnificus and other bacterial species?

While specific sequence data for V. vulnificus tgt is not provided in the search results, comparative analysis based on related Vibrio species reveals important insights:

Vibrio species TGT enzymes share high sequence homology but display subtle differences in substrate specificity and catalytic efficiency compared to other bacterial TGTs. In Vibrio cholerae, TGT plays a critical role in optimal growth, particularly in the presence of aminoglycoside antibiotics .

Key differences observed in Vibrio TGT compared to E. coli counterparts include:

• Functional Impact: In V. cholerae, TGT deletion (Δtgt) has a more dramatic effect on aminoglycoside susceptibility compared to E. coli .

• Regulatory Mechanisms: Expression of tgt in Vibrio species is influenced by stress conditions and regulatory factors like CRP (cAMP receptor protein) .

• Codon Bias Effect: The impact of TGT activity appears to be more pronounced for genes with specific codon usage patterns, particularly those biased toward TAT codons in Vibrio species .

These differences suggest that Vibrio vulnificus TGT likely has unique characteristics that reflect its adaptation to the specific ecological niche and physiological requirements of this pathogen.

How does queuosine modification affect translational reprogramming in Vibrio species?

Queuosine modification exhibits a codon-specific effect on translation in Vibrio species, particularly affecting genes with biased codon usage patterns. Research with V. cholerae has revealed several key mechanisms:

Codon-Specific Effects:

• In V. cholerae, the absence of Q modification (Δtgt) decreases misincorporation at TAT codons, suggesting better decoding of these codons without Q modification .

• Conversely, the presence of Q appears to enhance translation of transcripts biased in C-ending codons (TAC) .

Gene Expression Impact:
Experimental data from V. cholerae reveals that Q modification influences the expression of specific genes based on their tyrosine codon usage:

Translational Efficiency:
The Q modification acts as a post-transcriptional regulator of gene expression, creating a class of "modification tunable transcripts" (MoTTs) whose translation efficiency depends on the presence or absence of Q .

What is the relationship between TGT activity, queuosine modification, and antibiotic resistance in Vibrio species?

The relationship between TGT activity and antibiotic resistance in Vibrio species, particularly to aminoglycosides, reveals an intriguing connection between tRNA modification and stress response:

Aminoglycoside Sensitivity:

• In V. cholerae, deletion of the tgt gene (Δtgt) significantly increases sensitivity to tobramycin (TOB) and other aminoglycosides .

• This phenotype is specific to Vibrio species, as E. coli Δtgt does not show such dramatic effects with aminoglycosides .

Mechanistic Basis:
The increased sensitivity appears to be mediated through translational reprogramming of specific proteins:

• RsxA Overexpression: In the absence of Q modification (Δtgt), the oxidative stress regulator RsxA is overexpressed by approximately 13-fold despite unchanged transcript levels .

• Experimental evidence demonstrates that induced overexpression of RsxA in wild-type V. cholerae mimics the Δtgt phenotype, confirming its role in aminoglycoside sensitivity .

Oxidative Stress Connection:

• Aminoglycosides are known to trigger oxidative stress in V. cholerae .

• RsxA controls a regulon involved in oxidative stress response, including SoxR-regulated genes .

• The reduced fitness of Δtgt strains in the presence of aminoglycosides may result from impaired oxidative stress response due to dysregulated RsxA levels .

This relationship suggests that targeting TGT activity could potentially serve as an adjuvant strategy to enhance aminoglycoside efficacy against Vibrio pathogens.

How can structural modeling approaches be used to study V. vulnificus TGT and design potential inhibitors?

Homology Modeling Methodology:
For V. vulnificus TGT structural prediction, researchers can employ both template-based and ab initio modeling approaches:

• Template Selection: High-resolution crystal structures of bacterial TGTs serve as excellent templates, particularly the structure from Thermotoga maritima (PDB 2ASH) .

• Subunit Modeling: For organisms with heterodimeric TGT complexes, both the catalytic subunit (QTRT1) and non-catalytic subunit (QTRTD1) should be modeled separately .

• Complex Assembly: The complete functional TGT complex can be assembled by protein-protein docking using tools like HADDOCK or ClusPro.

Inhibitor Design Strategy:
Based on structural insights, rational design of V. vulnificus TGT inhibitors can follow these approaches:

• Active Site Targeting: Design compounds that compete with preQ₁ for binding at the active site.

• Allosteric Inhibition: Identify potential allosteric sites that could disrupt catalytic activity.

• Protein-Protein Interface Disruption: For heterodimeric TGTs, target the interface between subunits.

Virtual Screening Pipeline:

• Structure-based pharmacophore modeling based on key active site residues.

• Virtual screening of compound libraries against the model.

• Molecular dynamics simulations to evaluate binding stability.

• In vitro validation of top candidates using enzymatic assays.

This approach has proven successful for developing inhibitors against bacterial enzymes with similar structural characteristics .

What is the relationship between queuosine modification levels and bacterial stress response pathways?

Research indicates a complex relationship between queuosine modification and stress response in bacteria:

Oxidative Stress Response:

• In V. cholerae, Q modification influences the expression of oxidative stress response genes, particularly through regulation of the RsxA-SoxR pathway .

• The absence of Q leads to overexpression of RsxA, which negatively impacts the SoxR regulon involved in oxidative stress management .

• This connection between Q and oxidative stress has been observed in both prokaryotic and eukaryotic organisms .

Dynamic Regulation of Q Levels:

• Q modification levels are not static but dynamically regulated in response to environmental conditions.

• In V. cholerae, CRP (cAMP receptor protein) regulates tgt expression, with a Δcrp strain showing a significant 1.6-fold increase in Q levels .

• Exposure to sub-inhibitory concentrations of antibiotics can alter Q modification levels, though the changes may be subtle .

DNA Repair Connection:
Genomic analysis reveals that DNA repair genes in V. cholerae tend to be biased toward TAT codons, suggesting their expression may be particularly sensitive to Q modification status . This creates a potential regulatory mechanism linking translational control to genome maintenance systems during stress conditions.

What methodological approaches can be used to study the impact of TGT on bacterial virulence in infection models?

In Vitro Cellular Models:

• Adhesion and Invasion Assays:

  • Compare wild-type and Δtgt V. vulnificus for adherence and invasion of intestinal epithelial cell lines.

  • Quantify bacterial attachment and internalization using gentamicin protection assays.

• Cytotoxicity Measurements:

  • Assess the production of cytotoxins by measuring lactate dehydrogenase (LDH) release from host cells.

  • Compare cytotoxicity kinetics between wild-type and Δtgt strains.

Animal Infection Models:

• Mouse Infection Model:

  • Utilize established mouse models of V. vulnificus infection (oral, intraperitoneal, or wound infection).

  • Compare survival rates, bacterial burdens, and inflammatory markers between wild-type and Δtgt infections.

• Zebrafish Model:

  • Transparent zebrafish larvae allow real-time visualization of infection progression.

  • Compare dissemination patterns and neutrophil recruitment in response to wild-type versus Δtgt strains.

Molecular Approaches:

• RNA-Seq Analysis:

  • Perform comparative transcriptomics of wild-type and Δtgt strains under infection-relevant conditions.

  • Identify virulence-associated genes whose expression depends on Q modification.

• Proteomics Analysis:

  • Use quantitative proteomics to identify proteins differentially expressed in Δtgt mutants.

  • Focus on virulence factors, especially those with biased codon usage toward TAT or TAC.

• Reporter Constructs:

  • Develop transcriptional and translational GFP fusions to monitor expression of key virulence factors in wild-type and Δtgt backgrounds .

  • Use these to assess virulence gene expression during infection.

Complementation Strategies:
Genetic complementation using plasmid-expressed tgt can confirm phenotypes are specifically due to the absence of Q modification rather than secondary mutations.

What controls should be included when studying the phenotypic effects of tgt deletion in Vibrio vulnificus?

Essential Controls for Δtgt Phenotype Studies:

• Genetic Complementation:

  • Include a complemented strain where the tgt gene is reintroduced on a plasmid under native or inducible promoter control.

  • This confirms that observed phenotypes are specifically due to tgt deletion rather than polar effects or secondary mutations.

• Related Modification Pathway Controls:

  • Include mutants in other modification enzymes that affect the same tRNAs but through different mechanisms.

  • For example, include a ΔrluF strain to control for potential indirect effects, as demonstrated in V. cholerae studies .

• Trans-species Complementation:

  • Test whether tgt genes from related species (E. coli, V. cholerae) can complement V. vulnificus Δtgt.

  • This reveals species-specific functions versus conserved roles.

• Temporal Controls:

  • Monitor phenotypes across growth phases, as TGT activity and Q levels may vary temporally.

  • Compare exponential versus stationary phase cultures.

• Environmental Stress Controls:

  • Include conditions that mimic relevant host environments (low pH, oxidative stress, nutrient limitation).

  • Test multiple antibiotic classes to determine specificity of any resistance phenotypes.

How can researchers accurately quantify the levels of queuosine and its precursors in Vibrio vulnificus?

Comprehensive Quantification Approach:

For accurate quantification of queuosine and its precursors in V. vulnificus, researchers should employ a multi-method strategy:

LC-MS/MS Protocol:

• Extract total tRNA using acidic phenol extraction to preserve modifications.

• Enzymatically digest tRNA to individual nucleosides using nuclease P1 and alkaline phosphatase.

• Analyze using liquid chromatography-tandem mass spectrometry with the following parameters:

  • Column: Hypersil aQ GOLD (2.1 mm × 100 mm; 1.9-μm particle size)

  • Mobile phase: 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B)

  • Gradient elution: 0-12 min (0% B); 12-15.3 min (0-1% B); 15.3-18.7 min (1-6% B); 18.7-20 min (6% B); 20-24 min (6-100% B); 24-27.3 min (100% B); 27.3-36 min (100-0% B); 36-41 min (0% B)

  • Flow rate: 0.3 mL/min at 25°C

  • Detection parameters: Triple quadrupole MS with Q transitions (precursor m/z 410, products m/z 163 and 295)

• Normalize Q signal against canonical ribonucleosides (U, C, A, G) for quantification.

Nanopore Sequencing Approach:
Direct RNA sequencing using nanopore technology provides an alternative method that can detect Q modification in intact tRNAs .

APB Gel Electrophoresis:
This technique provides a relative measure of Q modification levels across different growth conditions or mutant strains .

For comprehensive analysis, researchers should compare results across multiple methodologies and include appropriate standards and controls.

How can researchers interpret conflicting results between in vitro and in vivo studies of TGT function?

Systematic Approach to Resolving Discrepancies:

When faced with conflicting results between in vitro enzymatic assays and in vivo phenotypic observations of TGT function, consider these potential explanations and resolution strategies:

Potential Sources of Discrepancy:

• Substrate Availability:

  • In vitro assays typically use excess substrate concentrations that may not reflect physiological conditions.

  • Solution: Perform in vitro assays with varying substrate concentrations that mirror cellular levels.

• Regulation of TGT Activity:

  • TGT activity in vivo may be regulated by factors absent in purified systems.

  • Evidence indicates that Q modification levels are dynamic and influenced by stress and regulatory proteins like CRP .

  • Solution: Include potential regulatory factors in in vitro assays or examine TGT activity in cellular extracts.

• Indirect Effects vs. Direct Function:

  • Phenotypes attributed to TGT may result from downstream effects rather than direct enzymatic activity.

  • For example, in V. cholerae, aminoglycoside sensitivity in Δtgt strains appears to be mediated through RsxA overexpression .

  • Solution: Design experiments that can distinguish direct from indirect effects, such as suppressor mutations or targeted gene expression studies.

• Strain-Specific Factors:

  • Genetic background may influence TGT phenotypes.

  • Solution: Test multiple clinical and laboratory strains of V. vulnificus.

Resolution Strategies:

What are the common technical challenges when working with recombinant TGT and how can they be addressed?

Technical Challenges and Solutions:

• Protein Solubility Issues:

  • Challenge: Recombinant TGT often forms inclusion bodies when overexpressed.

  • Solutions:

    • Reduce induction temperature to 16-18°C

    • Use solubility tags (MBP, SUMO, or TrxA)

    • Express as a fusion with its natural binding partner (QTRTD1 for eukaryotic-like TGTs)

    • Optimize buffer conditions with stabilizing agents (glycerol, arginine)

• Enzymatic Activity Preservation:

  • Challenge: TGT activity can be lost during purification or storage.

  • Solutions:

    • Include reducing agents (DTT or β-mercaptoethanol) in all buffers

    • Add glycerol (10-20%) to storage buffer

    • Maintain protein at high concentration (>1 mg/mL)

    • Store as flash-frozen aliquots at -80°C; avoid freeze-thaw cycles

• Substrate Preparation:

  • Challenge: Obtaining properly folded tRNA substrates.

  • Solutions:

    • In vitro transcription followed by proper refolding protocols

    • Include Mg²⁺ in refolding buffer for correct tRNA structure

    • Verify tRNA folding using thermal denaturation profiles

• Assay Sensitivity:

  • Challenge: Detecting queuosine incorporation accurately.

  • Solutions:

    • Use radiolabeled substrates for highest sensitivity

    • Employ APB gel electrophoresis for modified/unmodified discrimination

    • Develop LC-MS/MS methods with proper controls

• Enzyme Kinetics Analysis:

  • Challenge: Accounting for multiple steps in the modification process.

  • Solutions:

    • Use simplified model substrates for initial rate determinations

    • Employ global fitting approaches for complex kinetic models

    • Include proper controls for product inhibition effects

By addressing these technical challenges systematically, researchers can enhance the reliability and reproducibility of experiments with recombinant V. vulnificus TGT.

How should researchers interpret changes in queuosine modification levels in response to different growth conditions?

Analytical Framework for Interpreting Q Modification Changes:

When evaluating changes in queuosine modification levels under varying growth conditions, researchers should consider the following analytical approaches:

Normalization Considerations:

• For LC-MS/MS data, normalize Q signal against total canonical nucleosides (U, C, A, G) rather than using absolute values .

• For gel-based methods, compare the ratio of modified to unmodified tRNA species rather than band intensity alone .

• Include multiple biological replicates (minimum 3, preferably 12 as in published protocols) .

Statistical Analysis:

• Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric).

• Consider the magnitude of change in biological context - even small changes (1.6-fold) can be biologically significant, as seen with CRP regulation of Q levels .

Interpretation Framework:

Control Experiments:

• Include measurements of TGT expression levels alongside Q modification measurements.

• Assess availability of Q precursors, particularly for auxotrophic organisms.

• Examine modification of all Q-target tRNAs, as effects may be tRNA-specific.

This structured approach enables meaningful interpretation of changes in Q modification levels and connects them to physiological responses in V. vulnificus.

What emerging technologies could advance the study of tRNA modifications in Vibrio species?

Cutting-Edge Technologies for tRNA Modification Research:

• Nanopore Direct RNA Sequencing:
  This technology enables direct detection of tRNA modifications without prior conversion or amplification, providing a comprehensive view of the tRNA epitranscriptome . For V. vulnificus, this approach could:

  • Allow simultaneous detection of Q and other modifications

  • Reveal modification stoichiometry across different tRNA species

  • Enable time-course studies of modification dynamics during infection

• CRISPR-Based Modification Systems:
  Engineered CRISPR systems could enable:

  • Precise manipulation of tgt and related genes

  • Creation of conditional knockouts for essential modification genes

  • Development of biosensors for Q modification levels in vivo

• Single-Cell Analysis of tRNA Modifications:
  Emerging methods could enable:

  • Detection of cell-to-cell variation in Q modification levels

  • Correlation of modification states with cellular phenotypes

  • Identification of bacterial subpopulations with distinct modification profiles

• Chemoenzymatic Labeling Approaches:
  These methods could allow:

  • Selective tagging of Q-modified tRNAs for pulldown and analysis

  • Fluorescent visualization of Q distribution in bacterial cells

  • Enrichment of Q-modified tRNAs for subsequent analysis

• Cryo-EM Studies of Ribosome-tRNA Interactions:
  High-resolution structural studies could:

  • Reveal how Q-modified tRNAs interact with the ribosome

  • Provide insights into the structural basis for Q-dependent translational reprogramming

  • Guide the development of inhibitors targeting Q-dependent translation

These technologies would significantly advance our understanding of how tRNA modifications influence Vibrio pathogenesis and stress responses.

How might the study of TGT and queuosine modification contribute to new antimicrobial strategies?

TGT-Based Antimicrobial Approaches:

The relationship between queuosine modification and bacterial fitness offers several promising avenues for antimicrobial development:

• TGT Inhibitors as Antibiotic Adjuvants:

  • Research in V. cholerae demonstrates that Δtgt mutants show increased sensitivity to aminoglycosides .

  • Targeted inhibition of TGT could potentially sensitize Vibrio species to existing antibiotics.

  • Structure-based design of TGT inhibitors could yield compounds that enhance aminoglycoside efficacy.

• Targeting Q-Dependent Stress Responses:

  • Q modification influences oxidative stress responses through regulation of proteins like RsxA .

  • Compounds that interfere with Q-dependent translation of stress response proteins could render bacteria more susceptible to host immune defenses and antibiotics.

• Species-Specific Targeting:

  • The differential impact of TGT deletion between Vibrio species and E. coli suggests species-specific roles .

  • This offers the potential for narrow-spectrum antimicrobial approaches that selectively target Vibrio pathogens while sparing commensal bacteria.

• Combination Therapy Approaches:

  • Potential synergies between TGT inhibitors and:

    • Aminoglycosides (demonstrated sensitivity in Δtgt)

    • Oxidative stress-inducing agents

    • Host defense peptides

• Virulence Attenuation:

  • If Q modification regulates virulence factor expression in V. vulnificus as suggested for other proteins in V. cholerae, TGT inhibition could attenuate virulence without direct bactericidal effects.

  • This "anti-virulence" approach might reduce selection pressure for resistance development.

These strategies represent promising approaches to leverage our understanding of tRNA modification biology for addressing infections caused by Vibrio vulnificus and related pathogens.

What are the potential applications of structural comparisons between TGT enzymes from different bacterial species?

Comparative Structural Biology Applications:

Structural comparisons between TGT enzymes from different bacterial species, including V. vulnificus, offer numerous valuable applications:

• Structure-Based Inhibitor Design:

  • Identification of conserved catalytic residues across bacterial TGTs provides targets for broad-spectrum inhibitors.

  • Species-specific structural features enable selective targeting of particular pathogens.

  • Modeling approaches using templates like Thermotoga maritima TGT (PDB 2ASH) can guide rational drug design .

• Evolution of Substrate Specificity:

  • Structural comparisons can reveal how different bacterial TGTs have evolved distinct substrate preferences.

  • For instance, some bacterial TGTs prefer preQ₁, while others may incorporate preQ₀ .

  • These insights could explain species-specific phenotypes associated with Q modification.

• Protein Engineering Applications:

  • Understanding structural determinants of substrate specificity enables engineering TGTs with novel activities.

  • Potential applications include:

    • Creation of TGTs that incorporate synthetic nucleobase analogs

    • Development of TGT variants with altered tRNA specificity

    • Design of chimeric enzymes with combined functions

• Mechanistic Insights:

  • Structural comparisons can reveal conserved and divergent aspects of the catalytic mechanism.

  • This information aids in understanding how TGT achieves the base-exchange reaction and how this process might be inhibited.

• Co-Evolution with tRNA Substrates:

  • Comparing TGT structures alongside their cognate tRNA substrates across species can reveal co-evolutionary patterns.

  • These insights help explain how modification systems adapt to changes in the translational apparatus during bacterial evolution.

Structural biology approaches thus provide a foundation for both fundamental understanding and applied research into bacterial tRNA modification systems.