Recombinant Burkholderia multivorans tRNA pseudouridine synthase A (truA)

Basic Research Questions

• What is the biological function of Burkholderia multivorans tRNA pseudouridine synthase A (truA)?

TruA is a highly conserved pseudouridine synthase that modifies uridines at positions 38, 39, and/or 40 in the anticodon stem loop (ASL) of multiple tRNAs. This enzyme is critical for translational accuracy and efficiency in B. multivorans. Unlike other pseudouridine synthases that modify specific positions in tRNAs with conserved sequences, TruA exhibits "site promiscuity" - it can modify nucleotides that are as far as 15 Å apart using a single active site across tRNAs with divergent sequences . This unique characteristic allows TruA to modify multiple positions in the ASL region of various tRNAs, contributing to B. multivorans' adaptability in different host environments, particularly in cystic fibrosis patients where it is the dominant Burkholderia pathogen recovered from lung infections .

• How do researchers express and purify recombinant B. multivorans truA for in vitro studies?

Methodology for expression and purification:

• Clone the B. multivorans truA gene into an expression vector (commonly pET-based systems) with a 6×His-tag for purification

• Transform into an E. coli expression strain (BL21(DE3) or similar)

• Culture in LB medium supplemented with appropriate antibiotics at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with IPTG (0.5-1 mM) at 16-18°C overnight

• Harvest cells by centrifugation and lyse using sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and 5% glycerol

• Purify using Ni-NTA affinity chromatography followed by size exclusion chromatography

• Assess protein purity using SDS-PAGE and Western blotting

• Determine protein concentration using Bradford assay or spectrophotometric methods

The purified protein should be stored in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% glycerol at -80°C for long-term storage or at -20°C for short-term use.

• What are the lineage-specific differences in truA among B. multivorans strains?

B. multivorans populations separate into two distinct evolutionary clades: lineage 1 (n=58 genomes) and lineage 2 (n=221 genomes) . While specific truA variations haven't been fully characterized between these lineages, genomic analyses reveal:

Importantly, no lineage-specific phenotypic differences have been demonstrated despite considerable inter-strain variance . Researchers studying truA should consider these lineage distinctions when selecting reference strains for recombinant expression and functional studies.

Advanced Research Questions

• How can researchers determine the substrate specificity of B. multivorans truA compared to other bacterial truA enzymes?

To comprehensively characterize substrate specificity, implement this methodological approach:

• In vitro pseudouridylation assays:

  • Synthesize or transcribe various tRNA substrates with different sequences in the anticodon stem loop

  • Incubate purified recombinant truA with tRNA substrates under optimal conditions (37°C, pH 7.5)

  • Detect pseudouridine formation using:
    a) CMC (N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide) derivatization followed by primer extension
    b) Mass spectrometry to identify modified nucleosides
    c) Thin-layer chromatography after nuclease digestion

• Structural analysis:

  • Perform crystallographic studies of B. multivorans truA in complex with different tRNA substrates

  • Map the binding interface using hydrogen-deuterium exchange mass spectrometry

  • Compare with crystal structures of E. coli TruA-tRNA complexes which capture three stages of the reaction mechanism

• Mutagenesis studies:

  • Create variants of tRNA substrates with mutations at positions 38-40

  • Generate truA mutants with alterations in key catalytic residues (based on E. coli TruA homology)

  • Analyze how changes affect enzyme activity and substrate recognition

• Comparative genomic analysis:

  • Compare truA sequences across Burkholderia species and other bacterial genera

  • Identify conserved and divergent regions that may contribute to substrate specificity

These approaches will reveal whether B. multivorans truA exhibits similar "regional specificity" as observed in E. coli truA, which can modify nucleotides that are as far as 15 Å apart using a single active site .

• What methodologies are most effective for studying the kinetics of tRNA modification by B. multivorans truA?

For comprehensive kinetic analysis, researchers should employ:

• Real-time monitoring of pseudouridylation:

  • Use fluorescently labeled tRNA substrates

  • Monitor conformational changes during modification using FRET (Förster Resonance Energy Transfer)

  • Apply stopped-flow techniques to capture rapid kinetics

• Steady-state kinetic analysis:

  • Determine Km, kcat, and kcat/Km values for different tRNA substrates

  • Use tritium release assays to quantify pseudouridine formation rates

  • Vary substrate concentrations to generate Michaelis-Menten plots

• Pre-steady-state kinetics:

  • Apply rapid-quench techniques to identify rate-limiting steps

  • Determine binding constants using surface plasmon resonance (SPR)

  • Use isothermal titration calorimetry (ITC) to determine thermodynamic parameters

• Single-molecule studies:

  • Perform single-molecule FRET to observe individual enzyme-substrate interactions

  • Track conformational changes during catalysis

  • Determine dwell times at different stages of the reaction

A complete dataset should include temperature-dependent kinetics (10-50°C) and pH profiles (pH 5.0-9.0) to identify optimal conditions and understand the catalytic mechanism. Based on studies of E. coli TruA, researchers should investigate how B. multivorans TruA utilizes the intrinsic flexibility of the ASL for site promiscuity while avoiding overstabilization of intrinsically stable tRNAs .

• How does B. multivorans truA function differ in biofilm formation versus planktonic growth?

B. multivorans strains show considerable capacity for biofilm formation . The role of truA may differ significantly between these growth states:

Methodological approach to study these differences:

• Compare truA expression using RT-qPCR and Western blotting between planktonic and biofilm cells

• Analyze tRNA modification profiles using mass spectrometry

• Perform ribosome profiling to identify differentially translated mRNAs

• Create truA knockout or knockdown strains and assess impact on biofilm formation capacity

• Use confocal microscopy with fluorescently tagged truA to visualize localization within biofilm structures

This research is particularly relevant since B. multivorans clinical isolates show considerable inter-strain variance but most are capable of biofilm formation , which contributes to persistence in cystic fibrosis lungs.

• What role might truA play in B. multivorans pathogenicity, particularly in cystic fibrosis lung infections?

In pathogenic settings, B. multivorans truA likely plays several critical roles:

• Adaptation to stress conditions:

  • truA-mediated tRNA modifications may enhance translational fidelity under the oxidative stress conditions present in CF lungs

  • This allows efficient translation of stress response proteins during infection

• Intracellular survival mechanisms:

  • B. multivorans can replicate and survive within macrophages

  • truA may contribute to this capability by maintaining translational efficiency during phagosomal stress

  • Unlike B. cenocepacia (which delays phagosomal maturation), B. multivorans uses different intramacrophage survival strategies

• Antibiotic resistance contributions:

  • B. multivorans exhibits antibiotic collateral sensitivity patterns

  • truA-mediated tRNA modifications may influence expression of resistance genes

  • Consistent pseudouridylation may stabilize translation of antibiotic resistance determinants like AmpC β-lactamase

• Biofilm formation influence:

  • B. multivorans clinical isolates show biofilm formation capacity

  • Proper tRNA modification by truA could support the protein synthesis required for extracellular matrix production

  • Modification of specific tRNAs may regulate the translation of biofilm-related genes

Research methodology should include:

• Creation of truA mutants with altered enzyme activity (similar to D48A, D90A mutations in E. coli TruB )

• Evaluation of mutant strain virulence in cystic fibrosis cell models and animal models

• Comparison of clinical isolates with varying virulence for truA sequence and expression differences

• How can site-directed mutagenesis be used to investigate key catalytic residues in B. multivorans truA?

Based on conserved structures of pseudouridine synthases, researchers should implement this comprehensive mutagenesis strategy:

• Target residue selection:

  • Based on homology with E. coli TruA, target:
    a) Catalytic aspartate residues (equivalent to D48, D90 in E. coli TruB )
    b) RNA-binding residues (equivalent to K64 in E. coli TruB )
    c) Residues in the active site pocket that may confer substrate specificity

• Mutagenesis protocol:

  • Use QuikChange or Q5 site-directed mutagenesis on truA cloned in expression vector

  • Create alanine substitutions (to abolish function) and conservative substitutions (to alter function)

  • Generate double and triple mutants to investigate synergistic effects

• Functional characterization:

  • Express and purify mutant proteins using the same protocol as wild-type

  • Assess structural integrity using circular dichroism spectroscopy

  • Perform in vitro pseudouridylation assays with various tRNA substrates

  • Measure binding affinity using electrophoretic mobility shift assays (EMSA)

  • Determine enzyme kinetics for each mutant

• Structural analysis:

  • Obtain crystal structures of key mutants

  • Compare to wild-type enzyme structure

  • Use molecular dynamics simulations to understand structural perturbations

• In vivo complementation:

  • Introduce mutant truA genes into truA-deficient B. multivorans

  • Assess ability to restore wild-type phenotypes (growth rate, stress resistance)

  • Evaluate impacts on virulence in infection models

This approach will identify which residues are essential for catalysis versus substrate binding, and potentially reveal B. multivorans-specific functional features of truA.

• What are the challenges in developing specific inhibitors of B. multivorans truA for research purposes?

Several technical challenges complicate the development of specific truA inhibitors:

Research strategies to overcome these challenges:

• Structure-based design targeting B. multivorans-specific surface features

• Fragment-based screening approach combining multiple weak binders

• Development of competitive tRNA mimics as chemical probes

• Application of click chemistry for target-guided synthesis

These inhibitors would serve as valuable research tools for investigating truA's role in B. multivorans physiology and pathogenesis.

• How does genomic variation in truA across B. multivorans lineages correlate with phenotypic differences?

Comprehensive analysis of B. multivorans genomic data reveals patterns in truA variation:

• Phylogenomic distribution:

  • B. multivorans separates into two distinct evolutionary lineages

  • truA sequence conservation is generally high (>95%) across both lineages

  • Single nucleotide polymorphisms (SNPs) in truA are rare but may be lineage-associated

• Correlation with phenotypic traits:

  • Despite genomic differences between lineages, no lineage-specific phenotypic differences have been demonstrated in B. multivorans

  • Clinical isolates show considerable inter-strain variance but most are motile and capable of biofilm formation

  • truA sequence variations do not strongly correlate with obvious phenotypic differences

• Methodological approach for correlation studies:

  • Collect diverse B. multivorans isolates (environmental and clinical sources)

  • Sequence full genomes and extract truA sequences

  • Assess phenotypes: growth rates, biofilm formation, antibiotic resistance

  • Perform comparative genomics and phylogenetic analyses

  • Use statistical methods to identify correlations between truA variants and phenotypes

• Experimental validation:

  • Introduce specific truA variants into reference strains using allelic exchange

  • Assess impact on tRNA modification patterns

  • Compare phenotypes of isogenic strains differing only in truA sequence