Recombinant Acinetobacter sp. tRNA pseudouridine synthase A (truA)

What is the biological role of tRNA pseudouridine synthase A (truA) in Acinetobacter species?

TruA in Acinetobacter species, like its homologs in other bacteria, is responsible for the site-specific isomerization of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon stem loop (ASL) of multiple tRNAs. This modification is critical for maintaining translational accuracy and efficiency. Pseudouridylation increases the thermal stability of the ASL, which can affect the anticodon-codon interaction and conformational changes of tRNA during translation. The modified nucleotides help maintain the balance between flexibility and stability required for tRNA function during protein synthesis .

How does the structure of Acinetobacter sp. truA compare to other bacterial pseudouridine synthases?

TruA from Acinetobacter sp. belongs to one of the five families of pseudouridine synthases. While specific structural data for Acinetobacter sp. truA is limited, the enzyme likely shares the core catalytic domain characteristic of this enzyme family. TruA is distinct from other pseudouridine synthases in its substrate specificity. Unlike TruB (which modifies U55 in nearly all tRNAs by binding to the conserved T-stem loop sequence), TruA can modify multiple tRNAs with divergent sequences in the ASL region. Crystal structures of E. coli TruA in complex with tRNAs have revealed how this enzyme can modify nucleotides as far as 15 Å apart using a single active site .

What are the optimal conditions for expressing recombinant Acinetobacter sp. truA in E. coli systems?

For optimal expression of recombinant Acinetobacter sp. truA in E. coli, researchers should consider the following methodology:

• Vector selection: pET expression vectors (particularly pET28a with an N-terminal His-tag) have shown good results for truA expression.

• Host strain: BL21(DE3) or Rosetta(DE3) strains are recommended due to their reduced protease activity and ability to accommodate rare codons that might be present in Acinetobacter genes.

• Induction conditions: Expression should be induced at OD₆₀₀ of 0.6-0.8 with 0.5-1.0 mM IPTG.

• Temperature regulation: Lower temperatures (16-18°C) during induction (12-16 hours) often yield higher amounts of soluble protein compared to standard 37°C induction.

• Media supplementation: Addition of 2% glucose can help reduce basal expression, while supplementation with iron may be beneficial as some pseudouridine synthases contain iron-sulfur clusters.

The expression conditions may need optimization specific to the Acinetobacter species being studied, as codon usage and protein folding requirements may vary.

What purification strategy yields the highest activity for recombinant Acinetobacter sp. truA?

A multi-step purification strategy is recommended to obtain highly active recombinant Acinetobacter sp. truA:

• Initial capture: If His-tagged, use immobilized metal affinity chromatography (IMAC) with Ni-NTA resin. Elution should be performed with an imidazole gradient (20-250 mM).

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose) can effectively separate truA from remaining contaminants.

• Polishing step: Size exclusion chromatography using Superdex 75 or 200 columns in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM DTT.

• Buffer optimization: The final storage buffer should contain 20-50 mM Tris-HCl (pH 7.5), 100-200 mM NaCl, 1-5 mM DTT, and 10% glycerol to maintain enzyme stability.

• Storage conditions: Flash-freeze aliquots in liquid nitrogen and store at -80°C to preserve activity.

Importantly, throughout the purification process, samples should be assessed by SDS-PAGE and Western blotting. Activity assays using standard tRNA substrates should be performed to ensure that the purified enzyme maintains its catalytic function.

What methodologies are most effective for measuring truA activity in vitro?

Several complementary approaches can be used to measure truA activity effectively:

• Radioisotope-based assays: Using [³H]- or [¹⁴C]-labeled UTP-incorporated tRNA substrates followed by thin-layer chromatography or HPLC analysis to quantify conversion of uridine to pseudouridine.

• CMCT-based detection: Chemical modification with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) followed by alkaline treatment and primer extension. Pseudouridine retains the CMCT adduct under alkaline conditions, causing reverse transcriptase stops.

• Mass spectrometry: LC-MS/MS analysis of digested tRNA can precisely identify and quantify pseudouridine formation at specific positions. This method provides the most comprehensive analysis of site-specific modification.

• Fluorescence-based assays: Recently developed fluorescent probes that specifically react with pseudouridine can enable high-throughput activity screening.

• Thermal denaturation studies: Monitoring changes in tRNA melting temperature upon pseudouridylation, as the modification typically increases thermal stability of the ASL region.

How can researchers distinguish between truA-catalyzed pseudouridylation and modifications by other pseudouridine synthases?

Distinguishing truA-specific activity from other pseudouridine synthases requires a multi-faceted approach:

• Position-specific analysis: TruA specifically modifies positions 38-40 in the ASL, while other synthases target different positions. Site-specific detection methods (such as primer extension after CMCT treatment or mass spectrometry) can identify the exact modified positions.

• Substrate specificity profiling: Using a panel of different tRNAs can help identify truA's characteristic activity pattern. TruA modifies multiple tRNAs with divergent sequences, unlike some other pseudouridine synthases that require specific sequence contexts.

• Inhibitor sensitivity: Different pseudouridine synthases show varying sensitivity to specific inhibitors. Comparative inhibition studies can help distinguish truA activity.

• Genetic approaches: In genetically tractable systems, creating knockout strains lacking specific pseudouridine synthases can help attribute modifications to particular enzymes.

• Recombinant enzyme competition assays: Performing assays with mixtures of purified pseudouridine synthases can reveal competitive or synergistic effects.

This combinatorial approach provides more definitive attribution of pseudouridylation to truA versus other enzymes that might modify different positions or require different recognition elements.

Which amino acid residues are critical for catalytic activity in Acinetobacter sp. truA, and how should mutation studies be designed?

Based on comparative analysis with other bacterial truA enzymes, several residues are likely critical for Acinetobacter sp. truA catalytic activity:

• Catalytic aspartate: The conserved aspartate residue in the active site is essential for the isomerization mechanism. This residue likely acts as a nucleophile to attack the C6 position of the target uridine.

• Aromatic residues: Conserved tyrosine or phenylalanine residues likely participate in base stacking interactions with the target nucleotide.

• Basic residues: Lysine and arginine residues in the active site are important for binding the phosphate backbone of RNA.

When designing mutation studies, researchers should follow these methodological guidelines:

• Use site-directed mutagenesis to create alanine substitutions of suspected catalytic residues.

• Include conservative substitutions (e.g., Asp→Glu, Tyr→Phe) to distinguish between structural and functional roles.

• Create a comprehensive panel of mutations rather than studying isolated residues.

• Assess both binding (via gel shift assays or surface plasmon resonance) and catalysis to distinguish defects in substrate recognition versus catalytic chemistry.

• Complement biochemical studies with structural analysis (CD spectroscopy, thermal stability) to ensure mutations don't disrupt protein folding.

This systematic approach will help establish the structure-function relationships specific to Acinetobacter sp. truA.

How conserved is truA across different Acinetobacter species, and what methodological approaches should be used to study its evolution?

TruA is highly conserved across bacterial species, including within the Acinetobacter genus, reflecting its essential role in RNA modification. To study the evolution of truA in Acinetobacter species effectively, researchers should employ the following methodological approaches:

• Comprehensive sequence analysis:

  • Collect truA sequences from all available Acinetobacter genomes

  • Perform multiple sequence alignments to identify conserved and variable regions

  • Calculate sequence conservation scores for each amino acid position

• Phylogenetic analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Compare truA gene trees with species trees to identify potential horizontal gene transfer events

  • Analyze selection pressures using dN/dS ratios to identify positions under positive or purifying selection

• Structural comparisons:

  • Map conserved and variable regions onto available three-dimensional structures

  • Perform homology modeling of truA from different Acinetobacter species

  • Identify structural elements that might contribute to species-specific functions

• Functional complementation studies:

  • Test whether truA from different Acinetobacter species can complement E. coli truA deletion mutants

  • Compare modification patterns produced by truA enzymes from different species

This multi-faceted approach will provide insights into how truA has evolved within the Acinetobacter genus and identify potential species-specific adaptations in enzyme function.

What is the relationship between truA activity and antibiotic resistance in clinical Acinetobacter isolates?

The relationship between truA activity and antibiotic resistance in clinical Acinetobacter isolates is an emerging area of research. While direct evidence is limited, several potential connections can be investigated:

• Translation accuracy and stress response:

  • TruA modifications in tRNA improve translational accuracy and efficiency

  • In stress conditions (including antibiotic exposure), precise translation of stress response genes may depend on proper tRNA modification

  • Altered truA activity could potentially modulate stress responses that contribute to antibiotic tolerance

• Expression of resistance genes:

  • Some resistance genes may have rare codons whose translation depends on properly modified tRNAs

  • Changes in truA activity could affect the expression efficiency of these resistance determinants

• Methodological approach for investigation:

  • Compare truA sequences and expression levels between antibiotic-susceptible and resistant clinical isolates

  • Create truA knockout or overexpression strains in Acinetobacter and assess changes in minimum inhibitory concentrations (MICs) for various antibiotics

  • Measure tRNA modification levels in resistant versus susceptible strains using mass spectrometry

  • Perform ribosome profiling to identify translational effects of altered truA activity

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and tRNA modification analysis to build comprehensive models of how truA activity might influence resistance phenotypes

This research direction could provide new insights into the complex relationship between RNA modification and antibiotic resistance in this clinically important pathogen.

How can recombinant Acinetobacter sp. truA be used as a tool for studying RNA structure and function?

Recombinant Acinetobacter sp. truA can serve as a valuable tool for RNA research through several experimental applications:

• Probing tRNA structural flexibility:

  • Since truA appears to select substrates based on ASL flexibility, it can be used as a probe for tRNA structural dynamics

  • TRNAs with varying degrees of ASL rigidity can be tested as substrates

  • The enzyme's activity correlates with ASL flexibility, making it a useful tool for studying tRNA architecture

• Site-specific RNA labeling:

  • TruA's ability to specifically modify positions 38-40 can be exploited for site-specific RNA labeling

  • Modified positions can be chemically derivatized for fluorescence detection or crosslinking studies

  • This approach enables precise structural and functional studies of tRNA ASL regions

• Engineered truA variants for expanded applications:

  • Structure-guided engineering of truA's specificity could create variants that modify non-natural RNA substrates

  • Such engineered enzymes could provide new tools for RNA structural biology

• Methodology for application:

  • Express and purify recombinant truA with high activity

  • Develop standardized reaction conditions for consistent modification

  • Couple with sensitive detection methods (mass spectrometry, chemical probing) for readout

  • Validate modifications by comparing wild-type enzyme activity with catalytically inactive mutants

These applications leverage the unique properties of truA to provide insights into RNA biology beyond its natural role in tRNA modification.

What are the most effective strategies for developing inhibitors of Acinetobacter sp. truA and evaluating their specificity?

Developing specific inhibitors of Acinetobacter sp. truA requires a systematic approach combining structural insights, screening methodologies, and rigorous validation:

This comprehensive approach could yield specific inhibitors valuable for both basic research and potential therapeutic development against Acinetobacter infections.

What are the common pitfalls in recombinant Acinetobacter sp. truA expression and how can they be addressed?

Researchers working with recombinant Acinetobacter sp. truA frequently encounter several technical challenges:

• Protein insolubility issues:

  • Problem: Formation of inclusion bodies during overexpression

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration (0.1-0.3 mM), use solubility-enhancing fusion partners (SUMO, MBP), or add solubility-enhancing additives (sorbitol, arginine) to the growth medium

• Compromised enzymatic activity:

  • Problem: Loss of activity during purification

  • Solution: Include reducing agents (DTT or β-mercaptoethanol) throughout purification, minimize freeze-thaw cycles, optimize buffer composition, and consider purifying with bound zinc or other metal cofactors if they're essential for activity

• Proteolytic degradation:

  • Problem: Rapid degradation during expression or purification

  • Solution: Use protease-deficient strains (BL21), include protease inhibitors throughout purification, reduce purification time, and optimize storage conditions

• Codon bias issues:

  • Problem: Poor expression due to rare codons in Acinetobacter genes

  • Solution: Use codon-optimized synthetic genes or express in Rosetta strains containing extra tRNAs for rare codons

• Aggregation during storage:

  • Problem: Protein aggregates upon storage

  • Solution: Add stabilizing agents (glycerol 10-20%, trehalose, or BSA), store at appropriate concentration (typically 1-5 mg/mL), and avoid repeated freeze-thaw cycles

Methodical troubleshooting of these issues, potentially in parallel, will help optimize recombinant Acinetobacter sp. truA production for research applications.

What quality control measures should be implemented when working with purified recombinant Acinetobacter sp. truA?

A comprehensive quality control protocol for purified recombinant Acinetobacter sp. truA should include:

• Purity assessment:

  • Method: SDS-PAGE with Coomassie/silver staining (>95% purity recommended)

  • Method: Size exclusion chromatography to detect aggregates or degradation products

  • Frequency: After final purification step and periodically during storage

• Identity confirmation:

  • Method: Western blotting with anti-His tag or truA-specific antibodies

  • Method: Mass spectrometry (MALDI-TOF or LC-MS/MS) for molecular weight confirmation and sequence coverage

  • Frequency: For each new purification batch

• Structural integrity verification:

  • Method: Circular dichroism spectroscopy to confirm proper secondary structure

  • Method: Thermal shift assays to assess protein stability

  • Frequency: For optimization of buffer conditions and periodically during long-term storage

• Activity validation:

  • Method: In vitro pseudouridylation assay using a standard tRNA substrate

  • Method: Dose-response relationship to confirm protein concentration dependence

  • Frequency: For each purification batch and after extended storage periods

• Endotoxin testing (for in vivo applications):

  • Method: Limulus Amebocyte Lysate (LAL) assay

  • Frequency: Prior to any in vivo use

• Storage stability monitoring:

  • Method: Regular activity testing of stored enzyme aliquots

  • Method: Visual inspection for precipitation

  • Frequency: At defined time points (initial, 1 month, 3 months, 6 months)

Implementing this quality control regimen ensures consistent and reliable experimental results when working with recombinant Acinetobacter sp. truA.

What are the most promising future research directions for Acinetobacter sp. truA studies?

Several high-potential research directions for Acinetobacter sp. truA warrant investigation:

• Structural biology advancements:

  • Obtaining high-resolution structures of Acinetobacter sp. truA alone and in complex with substrate tRNAs

  • Utilizing cryo-EM to capture dynamic states during the catalytic cycle

  • Comparing structural features with truA from other bacterial species to identify unique characteristics

• Systems biology integration:

  • Investigating the role of truA-mediated modifications in global translation regulation

  • Exploring connections between tRNA modification patterns and stress responses

  • Mapping the complete "pseudouridylome" in Acinetobacter species under different conditions

• Biotechnological applications:

  • Engineering truA variants with altered specificity for biotechnological applications

  • Developing truA-based tools for RNA labeling and structural studies

  • Exploring potential applications in synthetic biology

• Clinical relevance:

  • Investigating correlations between truA activity and virulence or antibiotic resistance

  • Exploring truA as a potential therapeutic target in Acinetobacter infections

  • Developing specific inhibitors as research tools and potential therapeutic leads

These research directions build upon current understanding while expanding into new territories that could yield significant basic science insights and potential applications in biotechnology and medicine.

How might comprehensive understanding of truA contribute to addressing antibiotic resistance in Acinetobacter species?

Understanding truA's role in Acinetobacter biology could contribute to addressing antibiotic resistance through several mechanisms:

• Target validation:

  • If truA is essential for Acinetobacter virulence or survival during infection, it could represent a novel therapeutic target

  • Selective inhibition might reduce pathogenicity without directly affecting resistance mechanisms, providing an orthogonal approach to conventional antibiotics

• Resistance mechanism insights:

  • TruA-mediated tRNA modifications may influence the translation efficiency of resistance genes

  • Understanding these connections could reveal new approaches to combat resistance

• Biomarker development:

  • Patterns of tRNA modification could potentially serve as biomarkers for antibiotic resistance or susceptibility

  • Such biomarkers might enable more rapid detection of resistant strains and guide treatment decisions

• Combination therapy approaches:

  • Inhibitors of truA could potentially sensitize resistant Acinetobacter to existing antibiotics

  • This synergistic approach might revitalize the efficacy of current antimicrobials

• Methodological roadmap:

  • Generate and characterize truA deletion or depletion strains in Acinetobacter

  • Evaluate changes in antibiotic susceptibility profiles

  • Perform transcriptomic and proteomic analyses to identify affected pathways

  • Test combinations of potential truA inhibitors with conventional antibiotics