Recombinant Burkholderia vietnamiensis tRNA pseudouridine synthase A (truA)

What is TruA and what function does it serve in Burkholderia vietnamiensis?

TruA is a tRNA pseudouridine synthase responsible for catalyzing the isomerization of uridine to pseudouridine at specific positions in the anticodon stem loop of transfer RNA (tRNA), particularly at positions 38, 39, and 40. In bacteria like Burkholderia vietnamiensis, TruA functions as a critical post-transcriptional RNA modification enzyme that helps stabilize tRNA structure.

Similar to the pseudouridine synthase in E. coli, B. vietnamiensis TruA likely forms a dimer of identical subunits that embraces both the modification region of the tRNA and regions farther up the molecule . This enzyme's activity contributes to fine-tuning tRNA flexibility, making them appropriately rigid for optimal function while maintaining necessary flexibility .

How can recombinant B. vietnamiensis TruA be effectively expressed and purified for research applications?

For effective expression of recombinant B. vietnamiensis TruA, researchers should consider the following methodological approach:

Expression System Selection:

• E. coli BL21(DE3) strain typically provides high yield for bacterial recombinant proteins

• pET expression vectors (particularly pET28a) with an N-terminal His-tag facilitate purification

• Growth conditions optimization: LB medium, induction at OD600 0.6-0.8 with 0.5 mM IPTG, 16-18°C overnight expression to enhance solubility

Purification Protocol:

• Cell lysis using sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and 1 mM DTT

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Size exclusion chromatography to separate dimeric active form

• Activity validation using in vitro pseudouridylation assay with synthesized tRNA substrates

Following similar approaches used for other bacterial recombinant proteins, researchers should aim for >95% purity as determined by SDS-PAGE and maintain protein samples in storage buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, and 1 mM DTT at -80°C.

What are the structural characteristics of B. vietnamiensis TruA based on homology to other bacterial TruA proteins?

Based on homology to other bacterial TruA proteins, particularly from E. coli for which structural data is available, B. vietnamiensis TruA likely possesses the following structural characteristics:

• Homodimeric quaternary structure with two identical subunits

• Each monomer containing a catalytic domain with a conserved aspartic acid residue essential for the pseudouridylation mechanism

• RNA-binding groove that accommodates the anticodon stem loop of tRNA

• Conserved catalytic pocket that positions uridine for isomerization

Like other pseudouridine synthases, TruA from B. vietnamiensis likely contains conserved motifs that define the enzyme family, including the catalytic aspartate residue that initiates nucleophilic attack on the target uridine . The protein structure would enable it to embrace both the 38-40 region of tRNA (where modifications occur) and regions farther up the tRNA molecule .

What experimental approaches are most appropriate for studying B. vietnamiensis TruA's activity in vitro?

To effectively study B. vietnamiensis TruA activity in vitro, researchers should consider the following methodological approaches:

In Vitro Pseudouridylation Assay:

• Substrate preparation: Synthesized or in vitro transcribed tRNA molecules corresponding to B. vietnamiensis tRNA targets

• Reaction conditions: 50 mM ammonium acetate (pH 7.5), 5 mM MgCl2, 2 mM DTT, purified recombinant TruA (0.1-1 μM), tRNA substrate (2-5 μM), 37°C for 30-60 minutes

• Detection methods:

  • CMCT/alkaline treatment followed by primer extension to identify pseudouridine positions

  • LC-MS/MS analysis of nucleosides after RNA digestion for quantitative measurement of pseudouridine formation

  • Tritium release assay using [5-³H]UTP-labeled tRNA

Site-Directed Mutagenesis for Structure-Function Analysis:
Targeted mutations of conserved catalytic residues (identified through homology modeling) to determine their importance in TruA activity. Key residues likely include the catalytic aspartate and lysine residues involved in RNA binding .

Biophysical Interaction Studies:

• Electrophoretic mobility shift assays (EMSA) to study TruA-tRNA binding

• Isothermal titration calorimetry (ITC) to determine binding affinities

• Surface plasmon resonance (SPR) to measure binding kinetics

How can transposon mutagenesis approaches be applied to study the role of TruA in B. vietnamiensis biology?

Transposon mutagenesis represents a powerful approach for studying TruA function in B. vietnamiensis, similar to methods used for other genes in this bacterium . The following methodological framework can be employed:

Transposon Library Construction:

• Generate a comprehensive transposon insertion library in B. vietnamiensis using an appropriate transposon system (e.g., Tn5-based)

• Confirm library coverage through preliminary sequencing

• Screen for TruA mutants using PCR or functional assays

Fitness Contribution Assessment (Tn-seq):
Similar to the approach used in the B. vietnamiensis colonization study , researchers can:

• Inoculate the transposon library under conditions where TruA function is hypothesized to be important

• Extract bacterial DNA after the selection period

• Sequence transposon-genome junctions to identify insertion sites

• Analyze the relative abundance of insertions in and around the truA gene to determine fitness contribution

Validation Through Targeted Mutagenesis:
As demonstrated in previous B. vietnamiensis studies , construct specific truA deletion or point mutants and conduct competition assays with wild-type strains to validate phenotypes identified through transposon screening.

How does TruA contribute to B. vietnamiensis adaptation during host colonization?

Based on what we know about B. vietnamiensis colonization mechanisms and tRNA modification proteins, we can propose several ways TruA might contribute to host adaptation:

Root Colonization Mechanisms:
B. vietnamiensis has been extensively studied for its ability to colonize rice roots . TruA may contribute to this process by:

• Enhancing translation efficiency under stress conditions encountered during plant colonization

• Contributing to the regulation of genes involved in plant-microbe interactions

• Potentially modifying tRNAs in response to nutrient availability in the rhizosphere

When colonizing different rice subspecies (indica vs. japonica), B. vietnamiensis demonstrates differential genetic requirements . TruA might play a role in this adaptive response by modulating the translational capacity to meet specific metabolic demands of different host environments.

Role in Stress Response:
The pseudouridylation of tRNA at the anticodon stem loop enhances the rigidity and stability of tRNA structure , which may be particularly important under stress conditions such as:

• Oxidative stress during host immune response

• pH fluctuations in different plant microenvironments

• Altered nutrient availability during colonization

What are the analytical challenges in distinguishing TruA activity from other pseudouridine synthases in B. vietnamiensis?

Distinguishing TruA activity from other pseudouridine synthases presents several analytical challenges that require sophisticated methodological approaches:

Challenge 1: Overlapping Substrate Specificity
Different pseudouridine synthases may modify the same or similar positions in tRNAs. To address this:

• Use recombinant enzymes in in vitro assays with defined substrates

• Employ targeted gene knockouts to eliminate activity from other pseudouridine synthases

• Utilize mass spectrometry to comprehensively map all pseudouridine modifications in the presence and absence of TruA

Challenge 2: Detection of Pseudouridine Modifications
Pseudouridine is isomeric with uridine, making detection challenging. Advanced methods include:

• CMCT-primer extension assays that specifically label pseudouridine

• Next-generation sequencing approaches combined with chemical treatments (Pseudo-seq)

• High-resolution LC-MS/MS to differentiate and quantify pseudouridine from uridine

Challenge 3: Functional Redundancy
B. vietnamiensis likely possesses multiple pseudouridine synthases (similar to other bacteria which have TruA, TruB, RluA, etc.). To address redundancy:

• Construct multiple mutants lacking combinations of pseudouridine synthases

• Perform complementation experiments with heterologous enzymes

• Use HITS-CLIP (high-throughput sequencing crosslinking immunoprecipitation) to identify direct RNA targets, similar to approaches used for TruB1

How does TruA function compare between pathogenic and non-pathogenic Burkholderia species?

The Burkholderia genus includes both pathogenic species (like B. mallei) and beneficial environmental species (like B. vietnamiensis). Comparing TruA function between these species provides insights into evolutionary adaptations:

• RNA binding domains that determine substrate specificity

• Regulatory regions affecting expression patterns

• Enzyme kinetics adapted to different ecological niches

Functional Differences:
In pathogenic Burkholderia (such as B. mallei, which causes glanders ), TruA may contribute to:

• Virulence by enhancing translation of pathogenicity factors

• Survival within host cells under immune pressure

• Adaptation to mammalian host temperature

In non-pathogenic B. vietnamiensis, which promotes rice growth , TruA likely supports:

• Metabolic adaptation to plant root environments

• Production of plant growth-promoting compounds

• Response to plant defense systems

Evolutionary Implications:
The study of TruA across Burkholderia species may reveal how RNA modification systems have been adapted during the evolution of pathogenic vs. symbiotic lifestyles within this bacterial genus.

What methodological approaches can help resolve contradictory data regarding TruA activity under different environmental conditions?

When facing contradictory data about TruA activity under different environmental conditions, researchers should implement a systematic troubleshooting approach:

1. Standardization of Experimental Conditions:

• Define precise buffer compositions, pH, temperature, and ionic strength

• Use the same protein batches and expression systems

• Implement standard operating procedures for activity assays

2. Multi-Method Validation:
Employ multiple independent techniques to measure TruA activity:

• Radiometric assays tracking incorporation of labeled substrates

• Mass spectrometry to directly quantify pseudouridine formation

• Structural studies (X-ray crystallography or cryo-EM) to visualize substrate binding under different conditions

3. Comprehensive Environmental Variable Testing:
Create a multifactorial experimental design that systematically evaluates TruA activity across various conditions:

4. In Vivo Validation:

• Generate reporter systems to monitor TruA activity in living B. vietnamiensis cells

• Use ribosome profiling to assess translation effects under various conditions

• Employ untargeted metabolomics to identify downstream consequences of altered TruA activity

What high-throughput approaches could identify novel TruA substrates beyond canonical tRNAs in B. vietnamiensis?

Identifying novel TruA substrates beyond canonical tRNAs requires innovative high-throughput approaches:

HITS-CLIP and Related Technologies:
Similar to methods used for TruB1 , HITS-CLIP can reveal direct RNA binding targets of TruA:

• Crosslink TruA to its RNA substrates in vivo

• Immunoprecipitate TruA-RNA complexes

• Sequence bound RNAs to identify potential substrates

• Validate modification sites using site-specific primer extension or mass spectrometry

Comparative Transcriptome-Wide Pseudouridine Mapping:

• Apply Pseudo-seq or Ψ-seq to map all pseudouridines in wild-type and truA knockout B. vietnamiensis

• Identify pseudouridine sites that disappear in the absence of TruA

• Classify novel RNA substrates by type and structural features

Biochemical Substrate Screening:
Develop in vitro systems to test TruA activity on diverse RNA substrates:

• Synthetic RNA pools representing various RNA classes

• Transcriptome-derived RNA fragments

• Structured RNA libraries

How might TruA activity influence bacterial gene expression and stress response mechanisms?

TruA's pseudouridylation activity could influence gene expression and stress response through several mechanisms:

Translation Efficiency and Accuracy:

• Pseudouridines in the anticodon stem loop stabilize tRNA structure , potentially enhancing codon recognition

• This modification may particularly affect translation of rare codons or stress-response genes

• Under stress conditions, TruA-modified tRNAs might preferentially translate specific mRNAs

Regulatory RNA Interactions:
If TruA modifies non-tRNA substrates (similar to how TruB1 regulates let-7 miRNA maturation ), it could:

• Affect the structure and function of regulatory RNAs

• Modulate RNA-protein interactions important for stress responses

• Influence RNA stability under adverse conditions

Comparative Genetic Analysis:
Transposon mutagenesis studies in B. vietnamiensis have revealed numerous genes contributing to root colonization . Similar approaches could identify genetic interactions between truA and other genes involved in:

• Stress response pathways

• Metabolic adaptation

• Host interaction systems

Proposed Experimental Approach:

• Generate a conditional truA expression system in B. vietnamiensis

• Expose bacteria to various stresses (oxidative, pH, temperature, nutrient limitation)

• Perform RNA-seq and ribosome profiling to assess transcriptional and translational changes

• Identify stress-responsive genes whose expression is TruA-dependent

This comprehensive approach would reveal the broader influence of TruA on bacterial physiology beyond its canonical tRNA modification function.