Recombinant Dinoroseobacter shibae tRNA dimethylallyltransferase (miaA)

What is tRNA dimethylallyltransferase (miaA) and what is its function in Dinoroseobacter shibae?

tRNA dimethylallyltransferase (miaA) is an essential enzyme that catalyzes the transfer of a dimethylallyl group onto the adenine at position 37 in tRNAs that read codons beginning with uridine, leading to the formation of N6-(dimethylallyl)adenosine (i6A) . In D. shibae, this enzyme belongs to the IPP transferase family and plays a critical role in tRNA modification, which affects translational efficiency and fidelity.

The enzyme's function can be biochemically verified through:

• In vitro tRNA modification assays

• Mass spectrometry analysis of modified tRNAs

• Complementation studies in miaA-deficient strains

What are the key structural and biochemical properties of recombinant D. shibae miaA?

Recombinant D. shibae miaA protein has the following characteristics:

• Length: 296 amino acids

• Molecular weight: 32.5 kDa

• Belongs to the IPP transferase family

• Complete amino acid sequence available for expression systems

Experimental approaches to characterize the protein include:

• SDS-PAGE analysis for molecular weight confirmation

• Circular dichroism for secondary structure analysis

• Size exclusion chromatography for oligomeric state determination

What growth conditions are recommended for culturing D. shibae prior to miaA analysis?

For optimal culturing of D. shibae before miaA analysis, the following conditions have been established:

• Growth medium: Artificial seawater medium supplemented with 16.9 mM succinate

• Temperature: 30°C

• Agitation: 160 rpm

• Harvesting typically at OD₅₇₈ of 0.5 for mid-log phase cells

• Culture in triplicate for statistical validity

For stress response studies involving miaA:

• Expose cultures to oxidative stress agents such as hydrogen peroxide (10-30 mM), paraquat (10-90 μM), or diamide (0.5-1 mM)

• Harvest cells at different time points (0, 30, 60, 120, and 180 min) after stress exposure

• Cell disruption can be performed using cell homogenization methods (e.g., FastPrep-24™)

How should researchers design experiments to study the role of miaA in D. shibae's adaptation to oxidative stress?

Designing effective experiments to study miaA's role in oxidative stress adaptation requires a systematic approach:

Experimental Design Framework:

• Control and variable groups setup

  • Wild-type D. shibae strain as control

  • miaA deletion mutant or overexpression strain as experimental group

  • Multiple biological replicates (minimum n=3)

• Stress conditions

  • Apply gradient concentrations of oxidative stressors (H₂O₂, paraquat, diamide)

  • Monitor time-dependent responses at multiple timepoints (0-180 min)

  • Include recovery phase experiments

• Multi-omics integration approach

  • Transcriptomics: RNA-seq to measure differential gene expression

  • Proteomics: GeLC-MS/MS to quantify protein levels

  • Metabolomics: Monitor changes in cellular metabolites

• Validation methods

  • qRT-PCR for gene expression verification

  • Western blot for protein level confirmation

  • Enzymatic activity assays for functional validation

Analysis of Results:

• Compare stress response profiles between wild-type and miaA-modified strains

• Measure survival rates, growth kinetics, and recovery efficiency

• Identify genes co-regulated with miaA under stress conditions

What protein purification strategy yields optimal activity for recombinant D. shibae miaA?

Based on characteristics of the miaA protein and similar enzymes in the IPP transferase family, the following purification strategy is recommended:

Optimized Purification Protocol:

• Expression system selection

  • E. coli BL21(DE3) with pET expression vectors

  • Induction with 0.5 mM IPTG at mid-log phase

  • Growth at 18°C post-induction to enhance solubility

• Lysis and initial purification

  • Buffer composition: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Addition of protease inhibitor cocktail

  • Sonication or French press for cell disruption

  • Clarification by centrifugation at 20,000 × g for 30 min

• Affinity chromatography

  • Ni-NTA column for His-tagged protein

  • Imidazole gradient elution (20-250 mM)

• Secondary purification

  • Size exclusion chromatography using Superdex 200

  • Buffer exchange to remove imidazole

• Quality control assessments

  • SDS-PAGE for purity evaluation (>95%)

  • Dynamic light scattering for aggregation analysis

  • Thermal shift assay for stability assessment

Activity Preservation:

• Addition of 1 mM DTT to prevent oxidation of cysteine residues

• Storage at -80°C in small aliquots with 10% glycerol

• Avoid repeated freeze-thaw cycles

How does miaA expression change in response to different environmental stressors in D. shibae?

The expression and regulation of miaA in D. shibae shows distinct patterns under various environmental stressors:

Table 1: Differential Regulation of miaA Under Various Stress Conditions

Key Findings:

• The regulation of miaA appears to be integrated with broader stress response networks in D. shibae

• Iron-responsive regulator RirA may influence miaA expression under oxidative stress conditions

• Correlation between miaA expression and protection mechanisms for DNA and proteins suggests a role in stress adaptation

Methodological Approach:

• GeLC-MS/MS proteomics to quantify protein level changes

• RT-qPCR validation of gene expression changes

• Western blot analysis with specific antibodies

• Reporter gene fusions to monitor promoter activity in real-time

What is the relationship between miaA function and the iron-responsive regulator RirA in D. shibae?

The relationship between miaA and the iron-responsive regulator RirA reveals important insights into regulatory networks in D. shibae:

Key Observations:

• RirA is downregulated by various stressors (peroxide, superoxide, and thiol stress)

• A rirA deletion mutant showed improved adaptation to peroxide stress

• In the rirA deletion mutant, 139 proteins were upregulated, including proteins associated with protection and repair of DNA and proteins (e.g., ClpB, Hsp20, RecA, and thioredoxin-like proteins)

Experimental approach to study this relationship:

• Comparative proteomics:

  • Analyze protein expression profiles in wild-type vs. rirA deletion mutant

  • Focus on changes in miaA levels and related tRNA modification enzymes

• ChIP-seq analysis:

  • Determine if RirA directly binds to the miaA promoter region

  • Map the RirA regulon in D. shibae

• Physiological studies:

  • Compare growth kinetics and stress resistance between wild-type, rirA mutant, and miaA mutant strains

  • Measure tRNA modification levels in each strain under various stress conditions

• Genetic complementation:

  • Construct strains with controlled expression of RirA and miaA

  • Test for restoration of wild-type phenotypes

What experimental design considerations are critical when studying the function of recombinant D. shibae miaA in vitro?

When designing experiments to study recombinant D. shibae miaA function in vitro, researchers should consider:

Critical Experimental Design Factors:

• Enzyme activity assay optimization:

  • Substrate selection: Purified tRNAs vs. synthetic oligonucleotides

  • Buffer composition: pH optimization (typically 7.5-8.0), salt concentration, divalent cations (Mg²⁺)

  • Detection methods: Radiometric assays with ³H-labeled dimethylallyl pyrophosphate vs. LC-MS/MS

• Reaction conditions:

  • Temperature range: 25-37°C (optimal around 30°C for D. shibae proteins)

  • Time course: 15-60 minutes with multiple sampling points

  • Enzyme concentration titration

• Controls and validations:

  • Negative controls: Heat-inactivated enzyme, no substrate, no enzyme

  • Positive controls: Characterized miaA from model organisms (E. coli)

  • Substrate specificity testing with different tRNA species

• Data analysis approach:

  • Enzyme kinetics calculations (Km, Vmax, kcat)

  • Statistical analysis across replicates (minimum triplicates)

  • Comparison to published values for related enzymes

Potential Pitfalls and Solutions:

• Enzyme instability: Add stabilizing agents (glycerol, reducing agents)

• Low activity: Optimize co-factor concentrations and reaction conditions

• Non-specific activity: Include competitor nucleic acids

How can researchers investigate the role of miaA in D. shibae's symbiotic relationships with marine microalgae?

Investigating miaA's role in D. shibae's symbiotic relationships with marine microalgae requires specialized experimental approaches:

Experimental Strategy:

• Co-culture system establishment:

  • D. shibae wild-type and miaA mutant strains with model algal partners (e.g., Prorocentrum minimum)

  • Controlled environmental conditions (light cycles, temperature, nutrient availability)

  • Long-term monitoring (weeks to months)

• Multi-level analysis:

  • Growth kinetics of both partners

  • Metabolite exchange profiling via targeted metabolomics

  • Transcriptome analysis of both organisms during different interaction phases

  • Microscopy to observe physical interactions

• Genetic manipulation approaches:

  • Complementation with wild-type or mutant miaA variants

  • Controlled expression systems (inducible promoters)

  • Fluorescently tagged miaA to monitor localization

• Specific hypotheses to test:

  • Does miaA affect vitamin production/transfer to algal partners?

  • Is miaA expression altered during different phases of symbiosis?

  • Does miaA influence stress resistance of D. shibae during symbiosis?

Data Collection and Analysis:

• Time-course sampling for transcriptomics and proteomics

• Quantification of exchanged metabolites

• Statistical comparison between wild-type and mutant co-cultures

What techniques are most effective for analyzing the differential expression of miaA under anaerobic conditions in D. shibae?

D. shibae can grow anaerobically using nitrate as a terminal electron acceptor , making it important to understand miaA regulation under these conditions:

Recommended Techniques:

• Experimental setup for anaerobic growth:

  • Chemostat culture with controlled oxygen depletion

  • Nitrate supplementation as terminal electron acceptor

  • Time-resolved sampling during transition from aerobic to anaerobic conditions

• Gene expression analysis:

  • RNA-seq for genome-wide transcriptional changes

  • RT-qPCR for targeted analysis of miaA and related genes

  • Northern blotting to detect transcript size and stability

  • Reporter gene fusions (e.g., miaA promoter::GFP) for real-time monitoring

• Protein-level analysis:

  • Western blotting with specific antibodies

  • Targeted proteomics (MRM-MS) for quantification

  • Activity assays to correlate expression with function

• Data integration approach:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate miaA expression with other adaptation processes

  • Network analysis to identify co-regulated genes

Table 2: Comparison of Techniques for Analyzing miaA Expression Under Anaerobic Conditions

To effectively analyze the data:

• Apply appropriate normalization methods for each technique

• Use time-series analysis to track expression dynamics

• Compare with other stress responses to identify common regulatory mechanisms