Recombinant Edwardsiella ictaluri Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase and what is its function in E. ictaluri?

Queuine tRNA-ribosyltransferase (tgt) is an enzyme responsible for the incorporation of queuosine (Q), a modified nucleoside, into specific tRNAs. In Edwardsiella ictaluri, this enzyme catalyzes the exchange of guanine at position 34 (the wobble position) in the anticodon of specific tRNAs (tRNAAsn, tRNAAsp, tRNAHis, and tRNATyr) with queuine, forming queuosine-modified tRNAs . This modification is believed to enhance translational accuracy and efficiency. In bacterial systems like E. ictaluri, the tgt enzyme is part of the queuosine biosynthetic pathway, where bacteria can synthesize queuosine de novo through the conversion of GTP to preQ0 and preQ1 precursors .

How does E. ictaluri tgt relate to the pathogen's virulence mechanisms?

While the provided search results don't directly link tgt to E. ictaluri virulence, we can infer potential relationships based on known bacterial pathogenesis mechanisms. E. ictaluri utilizes sophisticated type III secretion systems (T3SS) for virulence in channel catfish, which are regulated by the EsrAB two-component regulatory system . tRNA modifications can influence protein synthesis efficiency, potentially affecting the expression of virulence factors. The precise role of queuosine modifications in E. ictaluri pathogenesis remains an area for investigation, particularly whether tgt enzyme activity is modulated during infection or whether it influences the expression of virulence-associated proteins regulated by the EsrAB-EsrC pathway .

What is the genomic context of the tgt gene in E. ictaluri?

The tgt gene in E. ictaluri is likely part of the core genome rather than being located within the pathogenicity islands that encode virulence factors. The T3SS of E. ictaluri is encoded within a dedicated pathogenicity island, with various regulatory elements including EsrA, EsrB, and EsrC controlling the expression of virulence genes . Unlike these specialized virulence systems, tgt is typically a housekeeping gene involved in tRNA modification across many bacterial species. Researchers interested in the genomic context should examine the E. ictaluri genome annotation for the precise location and neighboring genes of the tgt locus.

What are the optimal expression systems for producing recombinant E. ictaluri tgt?

For recombinant expression of E. ictaluri tgt, Escherichia coli expression systems are commonly used due to their efficiency and ease of manipulation. When designing expression constructs, researchers should consider:

• Vector selection: pET vectors with T7 promoters offer high-level, inducible expression

• E. coli strains: BL21(DE3) or Rosetta strains are suitable for tgt expression, with the latter providing additional tRNAs for rare codons

• Fusion tags: His6-tag or Flag-tag can facilitate purification and detection, similar to the strategy used for other E. ictaluri proteins

• Expression conditions: Induction at lower temperatures (16-25°C) may enhance solubility
  The expression construct design can follow principles similar to those used for Flag epitope fusion in E. ictaluri T3SS proteins, where specific restriction sites (SacI, BglII, SphI) facilitated in-frame fusion of the tag to the target gene .

What purification challenges are specific to E. ictaluri tgt and how can they be addressed?

Purification of recombinant E. ictaluri tgt may present several challenges:

How can I verify the proper folding and activity of purified recombinant E. ictaluri tgt?

Verification of properly folded and active recombinant E. ictaluri tgt can be achieved through several complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assays to determine protein stability

  • Size exclusion chromatography to confirm monomeric/oligomeric state

• Enzymatic activity assays:

  • In vitro tRNA guanine transglycosylation assay using synthetic or natural tRNA substrates

  • Monitoring the incorporation of radiolabeled or fluorescently labeled queuine or analogs

  • Mass spectrometry to detect modified tRNAs, similar to the nanopore sequencing approach for detecting queuosine and precursors

• Substrate binding analysis:

  • Surface plasmon resonance (SPR) to measure binding kinetics to tRNA substrates

  • Isothermal titration calorimetry (ITC) to determine binding thermodynamics

  • Fluorescence anisotropy to measure tRNA binding
    A comprehensive enzymatic characterization would include determination of kinetic parameters (Km, kcat, kcat/Km) for both tRNA substrates and queuine/preQ1 substrates.

How can nanopore sequencing be used to study E. ictaluri tgt activity?

Nanopore sequencing offers a powerful approach for studying E. ictaluri tgt activity by directly detecting queuosine modifications in tRNAs . The methodology involves:

• tRNA substrate preparation:

  • Purification of specific tRNAs (tRNAAsn, tRNAAsp, tRNAHis, tRNATyr) from E. ictaluri or synthetic constructs

  • In vitro modification with recombinant tgt enzyme in the presence of queuine or precursors

• Direct RNA nanopore sequencing:

  • Adapter ligation to tRNA samples

  • Nanopore library preparation maintaining RNA integrity

  • Sequencing on platforms like Oxford Nanopore Technologies

• Data analysis:

  • Comparison of ionic current signals between unmodified and modified tRNAs

  • Detection of characteristic signal perturbations at the modified position

  • Quantification of modification percentages in mixed populations
    This approach enables high-throughput analysis of tgt activity on multiple tRNA substrates simultaneously, as well as detection of intermediate modification states with various queuosine precursors like preQ0 and preQ1 .

What mutagenesis approaches are recommended for studying E. ictaluri tgt function?

Targeted mutagenesis of E. ictaluri tgt can be performed using approaches similar to those described for E. ictaluri T3SS regulatory genes :

• For gene knockout in E. ictaluri:

  • Design constructs with flanking homologous regions

  • Cloning into suicide vectors like pGP704 or pRE107

  • Selection of recombinants using antibiotic markers and counter-selection with sucrose for sacB-based systems

  • Verification by PCR and DNA sequencing

• For site-directed mutagenesis of catalytic residues:

  • Target conserved active site residues based on structural homology

  • Employ overlap extension PCR or commercial site-directed mutagenesis kits

  • Validate mutations by sequencing

• For complementation studies:

  • Clone wild-type or mutant tgt genes with native promoters into vectors like pBBR1-MCS4

  • Introduce plasmids by conjugation or electroporation

  • Verify complementation by assessing enzyme activity or phenotype restoration
    The conjugation and selection protocols established for E. ictaluri regulatory gene studies provide a methodological framework applicable to tgt mutagenesis experiments .

How can I set up an in vitro assay to measure E. ictaluri tgt activity?

A robust in vitro assay for E. ictaluri tgt activity can be established as follows:

• Reagents and substrates:

  • Purified recombinant E. ictaluri tgt enzyme

  • Substrate tRNAs (either in vitro transcribed or purified from cells)

  • Queuine or precursor molecules (preQ0, preQ1)

  • Appropriate buffer system (typically Tris or HEPES buffer, pH 7.0-8.0, with Mg2+)

• Reaction setup:

  • Combine enzyme (10-100 nM), tRNA substrate (1-5 μM), and queuine/precursor (10-100 μM)

  • Incubate at optimal temperature (typically 30-37°C) for 30-60 minutes

  • Terminate reaction by phenol-chloroform extraction or heat inactivation

• Activity measurement:

  • Direct detection of modified tRNAs using nanopore sequencing

  • HPLC or mass spectrometry analysis of nucleoside composition after tRNA hydrolysis

  • Radiometric assay using 3H- or 14C-labeled substrates and scintillation counting

• Kinetic analysis:

  • Vary substrate concentrations and initial velocity measurements

  • Determine Km, Vmax, and catalytic efficiency (kcat/Km)

  • Evaluate inhibitors or cofactor requirements
    The assay should include appropriate controls such as heat-inactivated enzyme, no-enzyme reactions, and reactions with known tgt inhibitors to validate specificity.

How does E. ictaluri tgt activity change under conditions that mimic the phagosomal environment?

E. ictaluri is known to survive in the phagosomal environment, which is characterized by low pH and phosphate limitation . Investigating tgt activity under these conditions would provide insights into its potential role during infection:

• Experimental approach:

  • Culture E. ictaluri under conditions mimicking the phagosome (pH 5.5, phosphate limitation)

  • Extract tRNAs and analyze queuosine modification levels using nanopore sequencing

  • Measure tgt expression (qPCR, Western blotting) under these conditions compared to standard growth conditions

  • Compare recombinant tgt enzyme activity at different pH values (4.5-7.5) and phosphate concentrations

• Expected findings and interpretations:

  • If tgt activity is maintained or upregulated under phagosomal conditions, it may suggest a role in adaptation to the intracellular environment

  • Correlation with expression patterns of T3SS components would indicate potential coordination with virulence mechanisms

  • pH-dependent changes in enzyme kinetics could reveal regulatory mechanisms during infection
    This research would connect tgt function with the established pathogenesis mechanisms of E. ictaluri, particularly its adaptation to the intracellular environment of macrophages .

What is the relationship between the E. ictaluri tgt enzyme and the EsrAB-EsrC regulatory system?

The relationship between tgt and the EsrAB-EsrC regulatory system that controls T3SS expression represents an intriguing area for investigation:

• Hypothesized connections:

  • tgt-mediated tRNA modifications may influence translation efficiency of EsrA, EsrB, or EsrC proteins

  • Queuosine-modified tRNAs could affect codon usage bias in T3SS genes

  • The tgt gene itself might be regulated by EsrB or EsrC transcription factors

• Experimental approaches:

  • Analyze tgt expression in EsrA, EsrB, and EsrC mutants using qPCR and Western blotting

  • Examine queuosine modification levels in tRNAs extracted from these mutants

  • Create a tgt knockout in E. ictaluri and measure expression of T3SS genes

  • Assess intracellular replication and virulence of tgt mutants compared to T3SS regulatory mutants

• Data analysis and interpretation:

  • Correlate tgt expression patterns with T3SS gene expression profiles

  • Compare phenotypic effects of tgt mutation with those of EsrA, EsrB, and EsrC mutations

  • Examine intracellular replication in head-kidney-derived macrophages (HKDM) of tgt mutants versus wild-type
    This investigation would provide insights into whether tgt functions independently or as part of the regulatory network controlling E. ictaluri virulence.

How can structural biology approaches be used to design inhibitors of E. ictaluri tgt?

Structure-based design of E. ictaluri tgt inhibitors would follow several key steps:

• Structural determination:

  • X-ray crystallography of recombinant E. ictaluri tgt, alone and in complex with substrates

  • Homology modeling based on related bacterial tgt structures if experimental structures are unavailable

  • Molecular dynamics simulations to understand binding pocket flexibility

• Virtual screening approach:

  • Identification of the active site and potential allosteric sites

  • Structure-based virtual screening of compound libraries

  • Molecular docking and scoring of potential inhibitors

  • QSAR analysis to optimize lead compounds

• Experimental validation:

  • Synthesis or acquisition of candidate inhibitors

  • In vitro enzyme inhibition assays with purified recombinant E. ictaluri tgt

  • Co-crystallization of tgt with promising inhibitors

  • Cellular assays to evaluate inhibition of queuosine modification in vivo

• Specificity considerations:

  • Comparison with eukaryotic tgt to ensure selective targeting

  • Assessment of activity against other bacterial tgt enzymes

  • Structure-activity relationship studies to enhance selectivity
    Inhibitor design could target unique features of the E. ictaluri tgt compared to host enzymes, potentially leading to novel antibacterial agents specific for this fish pathogen.

How can I overcome solubility issues with recombinant E. ictaluri tgt?

Solubility challenges with recombinant E. ictaluri tgt can be addressed through multiple strategies:

What are common pitfalls in E. ictaluri tgt activity assays and how can they be avoided?

Common pitfalls in tgt activity assays include:

• tRNA substrate quality issues:

  • Pitfall: Degraded or improperly folded tRNA substrates

  • Solution: Verify tRNA integrity by gel electrophoresis; include folding step (heat denaturation followed by slow cooling) before assays

• Enzyme stability problems:

  • Pitfall: Loss of activity during storage or assay

  • Solution: Add stabilizing agents (glycerol, reducing agents); avoid freeze-thaw cycles; prepare fresh dilutions for each assay

• Interference from contaminating nucleases:

  • Pitfall: tRNA degradation during assay

  • Solution: Include RNase inhibitors; ensure high purity of enzyme preparations

• Detection sensitivity limitations:

  • Pitfall: Inability to detect low levels of modification

  • Solution: Employ sensitive methods like nanopore sequencing or mass spectrometry; optimize reaction conditions to increase modification yields

• Substrate specificity issues:

  • Pitfall: Incorrect tRNA substrates or base substrate

  • Solution: Ensure tRNAs contain the correct anticodon; verify queuine or precursor quality by analytical methods
    Appropriate controls, including reactions with known bacterial tgt enzymes like E. coli tgt, can help validate the assay system and identify specific issues with the E. ictaluri enzyme.

How does E. ictaluri tgt compare to tgt enzymes from other bacterial pathogens?

A comparative analysis of E. ictaluri tgt with other bacterial tgt enzymes reveals important similarities and differences:

How do the mechanisms of E. ictaluri tgt differ from eukaryotic tgt enzymes?

Bacterial tgt enzymes like that from E. ictaluri differ significantly from eukaryotic counterparts:

• Substrate specificity:

  • Bacterial tgt (including E. ictaluri): Inserts preQ1 into tRNA, which is further modified to queuosine

  • Eukaryotic tgt: Directly incorporates queuine (obtained from diet or microbiome) into tRNA

• Enzyme structure:

  • Bacterial tgt: Functions as a monomeric or homodimeric enzyme

  • Eukaryotic tgt: Functions as a heterodimer (QTRT1/QTRT2 complex)

• Biosynthetic pathway:

  • Bacterial systems (including E. ictaluri): Complete de novo biosynthesis pathway from GTP to preQ0 to preQ1 to queuosine

  • Eukaryotes: Cannot synthesize queuosine; must obtain queuine from bacterial sources

• Implications for inhibitor design:

  • These fundamental differences provide opportunities for selective targeting of bacterial tgt

  • Inhibitors targeting the preQ1 binding site would be specific for bacterial enzymes

  • The unique catalytic mechanism of bacterial tgt offers additional targets for selective inhibition
    These distinctions are important when considering tgt as an antibacterial target, as inhibitors can potentially be designed to specifically target the bacterial enzyme without affecting the structurally distinct eukaryotic counterpart.

What role might E. ictaluri tgt play in adaptation to the host environment?

Future research should investigate the potential role of E. ictaluri tgt in adaptation to the catfish host environment:

• Hypothesized adaptive functions:

  • Modulation of translation efficiency under stress conditions in the phagosome (low pH, nutrient limitation)

  • Fine-tuning expression of virulence factors regulated by the EsrAB-EsrC system

  • Contribution to bacterial fitness during different stages of infection

• Experimental approaches:

  • Create conditional tgt mutants to study essentiality under different conditions

  • Perform RNA-seq analysis of tgt mutants under conditions mimicking the phagosomal environment

  • Track queuosine modification levels during different stages of infection

  • Correlate tgt expression with expression of T3SS and T6SS components

• Expected insights:

  • Understanding whether tgt activity represents a specific adaptation to the catfish host

  • Determining if tgt function is coordinated with known virulence mechanisms

  • Identifying potential new therapeutic targets for controlling E. ictaluri infections
    This research direction would provide valuable insights into the broader role of tRNA modifications in bacterial pathogenesis.

What novel approaches could be developed for high-throughput screening of E. ictaluri tgt inhibitors?

Innovative approaches for high-throughput screening of E. ictaluri tgt inhibitors could include:

• FRET-based assay system:

  • Design fluorescently labeled tRNA substrates and queuine analogs

  • Measure FRET signal changes upon successful incorporation

  • Adapt to 384- or 1536-well plate format for high-throughput screening

• Nanopore-based screening:

  • Leverage nanopore technology for direct detection of queuosine modification

  • Develop miniaturized, parallel nanopore arrays for multiple compound testing

  • Employ machine learning algorithms to analyze modification patterns

• Cell-based reporter systems:

  • Design E. ictaluri strains with reporter genes dependent on tgt function

  • Screen compounds in infected cell culture models

  • Monitor reporter signal as a proxy for tgt inhibition

• Fragment-based drug discovery:

  • Use NMR or thermal shift assays to screen fragment libraries

  • Identify binding fragments for subsequent optimization

  • Employ structure-guided approaches to develop high-affinity inhibitors
    These approaches would accelerate the discovery of potential inhibitors with applications in aquaculture disease control.

How might systems biology approaches enhance our understanding of E. ictaluri tgt in the context of pathogenesis?

Systems biology approaches offer powerful tools to understand E. ictaluri tgt in the broader context of pathogenesis:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and tgt mutant strains

  • Identify regulatory networks connecting tgt function to virulence mechanisms

  • Map changes in tRNA modification profiles to alterations in the proteome

• Host-pathogen interaction modeling:

  • Develop mathematical models of E. ictaluri infection incorporating tgt function

  • Simulate effects of tgt inhibition on bacterial fitness and virulence

  • Predict optimal intervention strategies based on model outcomes

• Comparative systems analysis:

  • Compare regulatory networks across related fish pathogens

  • Identify conserved and unique roles of tgt in different host environments

  • Determine whether tgt represents a common vulnerability across multiple pathogens

• Single-cell approaches:

  • Apply single-cell RNA-seq to infected host cells

  • Correlate tgt activity with heterogeneous bacterial populations during infection

  • Identify subpopulations with distinct tgt expression or modification profiles
    These systems-level approaches would place tgt function in the broader context of E. ictaluri pathogenesis mechanisms, potentially revealing unexpected connections with established virulence systems like the T3SS regulated by EsrAB-EsrC .