Recombinant Clostridium botulinum Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase and what role does it play in bacterial physiology?

Queuine tRNA-ribosyltransferase (TGT) is an enzyme that catalyzes the exchange of guanine (G) with queuine (Q) at position 34 (the wobble position) in tRNAs, resulting in hypermodified transfer RNAs . This post-transcriptional modification is found in both eukaryotes and bacteria, including Clostridium botulinum, and plays an important role in translational efficiency and accuracy . TGT is part of the cellular machinery responsible for the queuosine (Q) salvage pathway.

The modification of tRNAs with queuosine affects codon recognition and may influence the translation of specific mRNAs. In bacterial physiology, this can impact various cellular processes including protein synthesis fidelity, stress responses, and potentially influence virulence factor expression. The modification specifically targets tRNAs with GUN anticodons, including tRNA^Asp^, tRNA^Asn^, tRNA^His^, and tRNA^Tyr^, which are central components of protein synthesis and cell signaling networks .

Research methodologies to study TGT's role in bacterial physiology include gene knockout studies, complementation assays, and comparative genomics approaches that examine conservation patterns across Clostridial species.

How is the tgt gene regulated in bacterial systems?

In many bacteria, genes involved in queuosine biosynthesis and salvage are regulated by preQ₁ riboswitches, which are RNA structural elements that bind to preQ₁ (a precursor of queuine) and regulate gene expression . These riboswitches have been described upstream of known Q synthesis genes and play a key role in the regulation of queuosine metabolism .

The RegPrecise database identifies several genes downstream of predicted preQ₁ riboswitches, including queC, queD, queE, queF, yhhQ, and genes previously associated with Q metabolism such as members of COG1957 (inosine-uridine nucleoside N-ribohydrolase; IunH), COG4708 (predicted membrane protein, QueT), and QrtT (substrate-specific component of queuosine-regulated ECF transporter) .

For Clostridium botulinum specifically, researchers would need to:

• Examine the genomic region surrounding the tgt gene to identify regulatory elements

• Perform RNA-seq under different growth conditions to determine expression patterns

• Use reporter gene assays to validate the activity of identified regulatory elements

• Apply genome-wide approaches like ChIP-seq to identify transcription factors involved in regulation

What techniques can be used to study the enzymatic activity of TGT?

Multiple complementary approaches can be employed to assess TGT enzymatic activity:

• Northern blot analysis with 3-(acrylamido)phenylboronic acid (APB) gels: This technique can detect Q-modified tRNAs, which migrate more slowly than unmodified tRNAs. After transfer to a nylon membrane, detection uses biotinylated probes specific for target tRNAs such as tRNA^Asp^GUC .

• In vitro base exchange assays: These assays measure the enzyme's ability to exchange guanine with queuine in tRNA substrates, typically using:

  • Radiolabeled substrates ([³H]-guanine or [³H]-queuine)

  • Purified or in vitro transcribed tRNA substrates

  • Appropriate buffer conditions with Mg²⁺ cofactor

• Mass spectrometry: LC-MS/MS analysis of enzymatically digested tRNAs can identify and quantify queuosine modifications with high sensitivity and specificity.

• HPLC analysis: Nucleoside analysis following enzymatic digestion of tRNAs provides quantitative measurement of modification levels.

When performing these assays, researchers should include appropriate controls such as:

• Reactions without enzyme

• Heat-inactivated enzyme controls

• Catalytically inactive TGT mutants

• tRNAs from organisms known to contain or lack Q modifications

What expression systems are most effective for producing recombinant Clostridium botulinum TGT?

Based on successful expression of related Clostridial enzymes, Escherichia coli represents an effective heterologous expression system for recombinant C. botulinum TGT . The following approach is recommended:

• Vector selection:

  • pGEX vectors for GST-fusion proteins, allowing affinity purification and potential thrombin cleavage

  • pET vectors with His-tags for nickel affinity purification

  • Vectors with inducible promoters (T7, tac) for controlled expression

• E. coli strain optimization:

  • BL21(DE3) and derivatives for high-level expression

  • Rosetta strains to address potential rare codon usage in C. botulinum genes

  • SHuffle strains if disulfide bonds are critical for proper folding

• Expression conditions:

  • Induction at OD₆₀₀ of 0.6-0.8

  • Lower temperature induction (16-20°C) to enhance solubility

  • Extended expression time (overnight) at reduced temperatures

  • Varying IPTG concentrations (0.1-1.0 mM) to optimize expression level

• Codon optimization:

  • Analyze the C. botulinum tgt gene for rare codons

  • Consider synthetic gene synthesis with E. coli-optimized codons

Evidence from the expression of CDTa from C. difficile demonstrates that Clostridial enzymes can be successfully expressed in E. coli as GST fusion proteins with retained activity after purification .

What purification strategy yields the highest activity for recombinant TGT?

A multi-step purification strategy is recommended for obtaining highly active recombinant TGT:

• Initial capture:

  • Affinity chromatography using GST-fusion approach with glutathione-sepharose columns

  • Alternative: Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

• Tag removal:

  • Site-specific protease treatment (thrombin for GST-tag, TEV protease for His-tag)

  • Optimization of cleavage conditions to prevent non-specific degradation

• Secondary purification:

  • Ion exchange chromatography to separate the cleaved protein from contaminants

  • Size exclusion chromatography as a final polishing step

• Buffer optimization for activity preservation:

  • Include reducing agents (5-10 mM DTT or 2-5 mM β-mercaptoethanol)

  • Add stabilizing agents (10-20% glycerol)

  • Maintain appropriate pH (typically 7.5-8.0)

  • Include Mg²⁺ (1-5 mM) if required for enzyme stability

Purification progress should be monitored by SDS-PAGE, and activity assays should be performed at each purification step to track activity yield. Enzyme preparations should be stored in small aliquots at -80°C to avoid repeated freeze-thaw cycles, which can diminish activity.

The purity and identity of the final preparation should be confirmed by mass spectrometry and western blotting with specific antibodies if available .

How should researchers address solubility issues with recombinant TGT?

Solubility challenges are common when expressing bacterial proteins in heterologous systems. For C. botulinum TGT, the following strategies can address solubility issues:

• Fusion partner approach:

  • GST tag has been successful for related Clostridial enzymes

  • Alternative solubility-enhancing tags include MBP (maltose-binding protein), SUMO, and Trx (thioredoxin)

  • Systematic comparison of different fusion partners for optimal solubility

• Expression condition optimization:

  • Reduce induction temperature to 16-20°C

  • Decrease inducer concentration (0.1-0.5 mM IPTG)

  • Use auto-induction media for gradual protein expression

  • Optimize growth media components (additives like sorbitol, betaine)

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Co-express with potential binding partners if known

• Protein engineering approaches:

  • Express individual domains if the full-length protein is insoluble

  • Identify and remove aggregation-prone regions

  • Introduce solubility-enhancing mutations based on homology modeling

• Refolding from inclusion bodies:

  • Solubilize inclusion bodies in 6-8 M urea or guanidine hydrochloride

  • Employ stepwise dialysis for refolding

  • Screen refolding buffer conditions (pH, salt, additives)

Successful solubilization should be verified by comparing specific activity of the soluble protein with refolded protein to ensure proper folding and function.

What are the critical catalytic residues in TGT and how can they be identified?

Identification of critical catalytic residues in C. botulinum TGT requires a systematic approach combining sequence analysis and experimental validation:

• Initial identification through bioinformatics:

  • Multiple sequence alignment with well-characterized TGT enzymes

  • Identification of highly conserved residues across bacterial species

  • Structural homology modeling to predict active site architecture

• Experimental validation through site-directed mutagenesis:

  • Design primers containing desired mutations targeting conserved residues

  • Perform PCR with high-fidelity polymerase using wild-type plasmid template

  • Transform into E. coli and confirm mutations by sequencing

  • Express and purify mutant proteins following established protocols

• Potential residues to target:

  • Aspartic acid residues involved in catalysis or substrate binding

  • Histidine residues that might function in acid-base catalysis

  • Conserved aromatic residues for base stacking with queuine/guanine substrates

  • Residues involved in tRNA recognition and binding

• Functional characterization of mutants:

  • Kinetic analysis to determine changes in K_m and k_cat values

  • Binding studies to distinguish between catalytic vs. binding defects

  • Thermal stability assays to ensure mutations don't disrupt protein folding

This approach has been successfully applied to related enzymes, as demonstrated in search result , which describes the systematic mutagenesis of a Clostridial enzyme to identify functional residues.

How does queuine recognition and binding occur in bacterial TGT enzymes?

Understanding queuine recognition and binding in bacterial TGT enzymes involves multiple experimental approaches:

• Structural studies:

  • X-ray crystallography of TGT in complex with queuine or analogs

  • Molecular dynamics simulations to model binding pocket interactions

  • NMR studies to investigate protein-ligand interactions in solution

• Binding pocket analysis:

  • Hydrogen-deuterium exchange mass spectrometry to identify regions protected upon substrate binding

  • Site-directed mutagenesis of predicted binding pocket residues

  • Thermodynamic characterization of binding using isothermal titration calorimetry (ITC)

• Substrate analog studies:

  • Synthesis of queuine analogs with systematic modifications

  • Competitive binding assays with natural substrate

  • Structure-activity relationship analysis

• Species-specific differences:

  • Comparative analysis of binding sites across bacterial TGTs

  • Creation of chimeric enzymes to identify regions responsible for substrate specificity

  • Computational docking studies with substrates across different TGT orthologs

Research has shown that TGT from Chlamydia trachomatis (TGT Ct) can use queuine as a substrate, suggesting a conserved mechanism for substrate recognition . This knowledge can be applied to investigations of C. botulinum TGT's substrate recognition features.

What is the relationship between TGT structure and tRNA substrate specificity?

The relationship between TGT structure and tRNA substrate specificity can be investigated through the following methodological approaches:

• tRNA recognition elements:

  • In vitro modification assays with natural and synthetic tRNAs

  • Mutational analysis of tRNA features (anticodon loop, D-arm, T-arm)

  • SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify preferred tRNA sequences

• Protein-RNA interaction mapping:

  • RNA footprinting to identify protected regions upon TGT binding

  • Crosslinking studies to capture direct protein-RNA contacts

  • Cryo-EM structure determination of TGT-tRNA complexes

• Domian mapping of TGT:

  • Limited proteolysis to identify stable domains

  • Expression of individual domains to assess tRNA binding capability

  • Domain swapping experiments between TGTs with different specificities

• Experimental validation:

  • Measure modification efficiency across different tRNA substrates

  • Compare kinetic parameters for various tRNA isoacceptors

  • Assess competition between different tRNAs for modification

TGT typically modifies tRNAs containing GUN anticodons (tRNA^Asp^, tRNA^Asn^, tRNA^His^, and tRNA^Tyr^), but species-specific differences in substrate preference may exist . Understanding these preferences requires systematic comparison of modification efficiency across different tRNA substrates.

How can recombinant TGT be used to study bacterial tRNA modification pathways?

Recombinant C. botulinum TGT serves as a valuable tool for investigating tRNA modification pathways through several research applications:

• Pathway reconstruction:

  • In vitro reconstitution of complete modification pathways

  • Identification of intermediate modifications and processing order

  • Determination of rate-limiting steps in the pathway

• Metabolic labeling studies:

  • Using recombinant TGT with isotopically labeled substrates

  • Tracking the fate of modified nucleosides in cellular RNA

  • Quantifying flux through modification pathways

• Comparative analysis of bacterial pathways:

  • Characterization of species-specific differences in Q-modification

  • Identification of alternative salvage routes in different bacteria

  • Study of the evolutionary conservation of modification enzymes

• Methodological approaches:

  • Northern blot analysis with 3-(acrylamido)phenylboronic acid (APB) gels to detect Q-modified tRNAs

  • Mass spectrometry to quantify modification levels and identify intermediates

  • Next-generation sequencing approaches to map modifications transcriptome-wide

Research has identified multiple salvage pathways for queuosine in pathogenic bacteria , and recombinant TGT can help elucidate whether similar pathways exist in C. botulinum and how they compare with the well-characterized de novo Q biosynthesis pathway.

What potential connections exist between TGT activity and bacterial pathogenesis?

The relationship between TGT activity and bacterial pathogenesis represents an emerging area of research with several potential connections:

• Translation fidelity during infection:

  • Q-modifications may enhance translational accuracy under stress conditions encountered during host infection

  • Improved codon reading could affect expression of virulence factors

• Adaptation to host environments:

  • TGT activity might influence bacterial adaptation to different niches within the host

  • Modification levels could change in response to host-derived signals

• Metabolic integration:

  • Q-salvage pathways might provide metabolic advantages during infection

  • Connection to other cellular processes critical for pathogenesis

• Experimental approaches to investigate these connections:

  • Gene deletion studies to assess virulence in animal models

  • Transcriptome and proteome analysis of TGT-deficient strains

  • Measurement of tRNA modification levels during different stages of infection

  • Assessment of stress response and antibiotic tolerance in TGT mutants

For C. botulinum specifically, TGT activity could potentially influence toxin production, which is the primary virulence mechanism of this pathogen . The botulinum toxin relies on precise protein synthesis, which could be affected by altered tRNA modification status.

How might TGT serve as a target for antimicrobial development?

TGT presents several characteristics that make it a potential target for antimicrobial development:

• Target validation approaches:

  • Essentiality studies through gene deletion or silencing

  • Chemical biology approaches using small molecule inhibitors

  • Assessment of growth inhibition and virulence attenuation

• Inhibitor development strategies:

  • Structure-based design targeting the active site

  • Fragment-based screening for novel scaffolds

  • Nucleoside analog development to compete with natural substrates

  • High-throughput screening of compound libraries

• Therapeutic potential evaluation:

  • In vitro assessment of antimicrobial activity

  • Analysis of resistance development frequency

  • Synergy with existing antibiotics

  • Animal model efficacy studies

• Methodological considerations:

  • Development of robust activity assays for screening

  • Cellular penetration assessment of potential inhibitors

  • Selectivity profiling against human enzymes

  • Pharmacokinetic and toxicity evaluation

The role of tRNA modifications in bacterial adaptation and stress response suggests that targeting TGT could potentially interfere with bacterial survival during infection. Additionally, if TGT activity influences toxin production in C. botulinum, inhibitors could potentially reduce toxin-mediated pathogenesis .

How do environmental conditions affect TGT expression and activity in Clostridium botulinum?

Environmental regulation of TGT expression and activity represents an important aspect of understanding its physiological role:

• Transcriptional regulation:

  • RNA-seq analysis under various growth conditions

  • Reporter gene assays to monitor promoter activity

  • ChIP-seq to identify transcription factors involved in regulation

  • Riboswitch activity assessment in response to metabolite fluctuations

• Post-transcriptional control:

  • mRNA stability analysis

  • Assessment of small RNA regulation

  • Translation efficiency measurements

  • Protein stability under different conditions

• Enzymatic activity modulation:

  • Influence of pH, temperature, and ionic conditions

  • Metabolite availability (e.g., queuine, preQ₁)

  • Cofactor requirements and availability

  • Post-translational modifications affecting activity

• Physiologically relevant conditions to test:

  • Oxygen tension (aerobic vs. anaerobic growth)

  • Nutrient limitation (carbon, nitrogen sources)

  • Growth phase dependence

  • Host-relevant environments (temperature, pH)

Experimental design should incorporate physiologically relevant conditions that C. botulinum encounters in its natural environments. The presence of preQ₁ riboswitches upstream of genes involved in queuosine metabolism suggests that availability of pathway intermediates plays a regulatory role .

What are the challenges in distinguishing the functions of TGT from different bacterial species in complex communities?

Studying species-specific TGT functions in mixed microbial communities presents several methodological challenges:

• Sequence and functional similarity challenges:

  • TGT enzymes from different bacterial species likely share sequence homology

  • Functional redundancy may mask species-specific effects

  • Cross-reactivity of antibodies or probes

• Methodological solutions:

  • Develop species-specific antibodies targeting unique epitopes

  • Design PCR primers for species-specific detection

  • Create activity-based probes with selectivity for C. botulinum TGT

  • Apply CRISPR-Cas9 for species-specific gene targeting

• Experimental strategies:

  • Single-cell approaches to correlate TGT activity with specific bacteria

  • Metaproteomics to identify and quantify TGT proteins from different species

  • Competitive inhibition studies using species-specific inhibitors

  • Stable isotope probing to track species-specific tRNA modifications

• Data analysis approaches:

  • Computational methods to deconvolute species-specific signals

  • Machine learning for pattern recognition in modification profiles

  • Network analysis to infer species interactions based on modification patterns

These approaches would help researchers distinguish the specific contributions of C. botulinum TGT in complex communities such as the human gut microbiome or environmental samples.

How do tRNA modifications by TGT influence translation dynamics during stress conditions?

The impact of TGT-mediated tRNA modifications on translation dynamics during stress represents a frontier in understanding bacterial adaptation:

• Translation efficiency analysis:

  • Ribosome profiling in wild-type vs. TGT-deficient strains

  • Translation rate measurements using reporter systems

  • Polysome profiling under various stress conditions

  • tRNA charging levels and aminoacylation status assessment

• Codon-specific effects:

  • Codon occupancy measurements

  • Pause site analysis during translation

  • Readthrough frequency at stop codons

  • Frameshifting rates at specific mRNA sequences

• Stress response integration:

  • Analysis of stress response protein synthesis

  • Mistranslation rates under different stressors

  • Protein aggregation and quality control

  • Persister cell formation and antibiotic tolerance

• Methodological approaches:

  • SILAC-based proteomic comparison under stress

  • Fluorescent reporters for translation fidelity

  • Single-molecule techniques to monitor translation kinetics

  • Computational modeling of translation dynamics

The Q modification at the wobble position influences codon recognition and may affect translation of specific stress response proteins . This could provide a mechanism by which bacteria fine-tune their proteome in response to environmental challenges.

What are common pitfalls in TGT activity assays and how can they be addressed?

Researchers often encounter several challenges when assessing TGT enzymatic activity:

• Substrate quality issues:

  • tRNA substrate degradation or contamination

  • Variability in in vitro transcribed tRNAs

  • Insufficient tRNA folding or misfolding

• Enzyme stability problems:

  • Activity loss during storage or assay setup

  • Protein aggregation during the reaction

  • Cofactor requirements not fully met

• Detection limitations:

  • Sensitivity issues in detecting modifications

  • Background signals in radioactive assays

  • Interference in colorimetric or fluorescent readouts

• Methodological solutions:

  • tRNA quality control: Verify integrity by gel electrophoresis before use

  • Proper tRNA folding: Include heating and cooling steps before the assay

  • Enzyme stability: Add stabilizers (glycerol, BSA) to reaction buffers

  • Reduce oxidation: Include reducing agents in all buffers

  • Standardization: Include positive controls (known active TGT) in each assay

  • Method validation: Cross-verify results using orthogonal detection methods

• Northern blot optimization:

  • When using APB gels to detect Q-modified tRNAs :

    • Carefully control polyacrylamide percentages

    • Optimize transfer conditions to nylon membranes

    • Test multiple probe designs for specificity

    • Include well-defined positive and negative controls

These approaches will help ensure reliable and reproducible activity measurements, crucial for accurate characterization of recombinant TGT enzymes.

What controls are essential when studying the impact of TGT-mediated modifications on translation?

When investigating how TGT-mediated tRNA modifications affect translation, several critical controls must be included:

• Genetic controls:

  • Wild-type strain (positive control)

  • TGT knockout strain (negative control)

  • Complemented strain (rescued phenotype)

  • Catalytically inactive TGT mutant (separation of enzymatic vs. structural roles)

• Modification status verification:

  • Direct measurement of Q-modification levels in tRNAs

  • Northern blot analysis with APB gels

  • Mass spectrometry confirmation of modification status

  • Correlation of modification levels with observed phenotypes

• Translation-specific controls:

  • Reporter systems with codons dependent vs. independent of Q-modified tRNAs

  • Frameshift and readthrough controls

  • mRNA level measurements to distinguish transcription from translation effects

  • In vitro translation assays with defined components

• Environmental variables control:

  • Standardized growth conditions

  • Precise timing of sample collection

  • Consistent stress application protocols

  • Matched growth phases between compared samples

• Data analysis considerations:

  • Biological and technical replicates

  • Appropriate statistical analysis

  • Normalization strategies for comparative studies

  • Careful interpretation of indirect effects