Recombinant Bdellovibrio bacteriovorus tRNA dimethylallyltransferase (miaA)

What is the basic function of tRNA dimethylallyltransferase (miaA) in Bdellovibrio bacteriovorus?

MiaA in Bdellovibrio bacteriovorus 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 (i(6)A) . This post-transcriptional modification is essential for proper tRNA functionality and represents a critical step in the modification pathway that ultimately produces ms2i6A-37 when followed by methylthiolation via MiaB . The modification enhances tRNA interactions with UNN target codons, which promotes reading frame maintenance and translational fidelity .

How can researchers effectively express and purify recombinant B. bacteriovorus miaA?

Methodological Approach:

• Vector Selection: For expression of recombinant B. bacteriovorus miaA, researchers should consider using expression vectors with inducible promoters like pRR48 or IPTG-inducible pTac systems, as these have been successfully used for similar tRNA modifying enzymes .

• Tagging Strategy: C-terminal tagging with epitopes such as Flag has been shown to not interfere with MiaA function in complementation assays . Consider the following plasmid construction approach:

  • Amplify the miaA gene from B. bacteriovorus genomic DNA

  • Include a ribosome binding site in the forward primer

  • Create fusion constructs with C-terminal tags

  • Clone into appropriate restriction sites (e.g., PstI and KpnI for pRR48-based vectors)

• Expression Conditions: Optimal expression is typically achieved in E. coli hosts like BL21(DE3) with induction using 100 μM IPTG, though conditions should be optimized for each specific construct .

• Purification Protocol: Affinity chromatography using the epitope tag, followed by size exclusion chromatography to ensure homogeneity.

What PCR protocols are recommended for amplifying B. bacteriovorus miaA?

For PCR amplification of B. bacteriovorus miaA, researchers should consider adopting protocols similar to those used for related genes in Bdellovibrio species:

Recommended PCR Protocol:

• Reaction mixture (50 μL): 1 μL genomic DNA, 25 μL 2× rTaq premix enzyme, 1 μL of each primer (10 μM), and sterile distilled water to complete volume

• Thermal cycling parameters: 95°C for 3 min (initial denaturation), followed by 33 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 1 min, with final extension at 72°C for 5 min

• Primer design: Target conserved regions of the miaA gene; consider using primers that have successfully amplified other IPP transferase family genes

What experimental approaches can be used to study the effects of miaA modification on translational fidelity in B. bacteriovorus?

Methodological Framework:

• Dual-Luciferase Reporter Assays: Researchers can adapt the dual-luciferase reporter system used in E. coli studies for B. bacteriovorus:

  • Create constructs containing renilla and firefly luciferases with frameshifting-prone sequences in between

  • Incorporate a Shine-Dalgarno ribosome binding site in forward primers

  • Transform these constructs into wild-type and miaA-deficient B. bacteriovorus

  • Measure relative luciferase activities to quantify frameshifting rates

• Motility Assays: Assess the impact of miaA modification on protein synthesis fidelity through motility phenotypes:

  • Use swim motility plates containing 0.2% agar in appropriate media

  • Compare motility rates between wild-type, ΔmiaA, and complemented strains

  • Calculate swim rates during logarithmic growth phase

• Comparative Proteomics:

  • Employ mass spectrometry-based proteomics to identify proteins differentially expressed between wild-type and miaA-modified strains

  • Focus analysis on proteins encoded by genes with high UNN codon content, which would be most affected by miaA modification

How do environmental conditions affect miaA expression and activity in B. bacteriovorus?

Based on extrapolation from studies of MiaA in other bacterial species, particularly E. coli, researchers should consider these key environmental factors when studying B. bacteriovorus miaA:

• Stress Response Mechanism: MiaA levels are known to shift in response to stress via post-transcriptional mechanisms . For B. bacteriovorus, researchers should examine miaA expression under:

  • Nutrient limitation conditions that mimic the transition between host-dependent and host-independent growth

  • Oxidative stress conditions that might be encountered during predation

  • pH variations that reflect different environmental niches

• Metabolic Precursor Availability: Effects of miaA overexpression or deletion can be attributed to changes in catalytic activity or availability of metabolic precursors . Researchers should investigate:

  • How dimethylallyl pyrophosphate (DMAPP) availability affects miaA activity in B. bacteriovorus

  • Whether metabolic shifts during predation cycles influence miaA function

• Experimental Design Considerations:

  • Monitor miaA expression using reporter fusions under various environmental conditions

  • Employ quantitative PCR to measure miaA transcript levels during different growth phases

  • Use western blotting with epitope-tagged miaA to track protein levels in response to environmental shifts

What is the relationship between B. bacteriovorus miaA and the bacterial predation mechanism against antibiotic-resistant pathogens?

The relationship between miaA function and B. bacteriovorus predatory capability against antibiotic-resistant pathogens presents an important research direction:

• Predation Efficiency Connection: While direct evidence linking miaA to predation efficiency is not explicitly provided in the search results, the role of MiaA in optimizing cellular responses through proteome regulation suggests it may influence predatory functions. Studies have demonstrated that B. bacteriovorus HD100 effectively predates various clinical pathogens and their biofilms, particularly Gram-negative isolates .

• Potential Mechanistic Pathways:

  • MiaA-mediated tRNA modification likely affects translation of proteins involved in motility, attachment, and invasion during predation

  • Translational frameshifting altered by miaA modifications could affect the production of specialized predation-related proteins

• Research Approach for Investigation:

  • Generate miaA knockouts or overexpression strains in B. bacteriovorus

  • Assess predation efficiency against antibiotic-resistant clinical isolates using co-culture methods

  • Quantify biofilm degradation capabilities using crystal violet staining and scanning electron microscopy

  • Compare predation rates on different Gram-negative species to identify correlations between miaA expression and host range

What are common challenges in expressing functional recombinant B. bacteriovorus miaA and how can they be addressed?

Challenge 1: Protein Solubility Issues

• Problem: Recombinant expression often leads to inclusion body formation

• Solution:

  • Reduce induction temperature to 16-18°C

  • Use solubility-enhancing fusion tags (MBP, SUMO)

  • Optimize induction conditions (IPTG concentration, time)

  • Consider using specialized E. coli strains designed for problematic protein expression

Challenge 2: Confirming Enzymatic Activity

• Problem: Difficulty in verifying that recombinant miaA retains native prenylation activity

• Solution:

  • Develop in vitro prenylation assays using synthesized tRNA substrates

  • Implement complementation assays in miaA-deficient strains

  • Use mass spectrometry to detect formation of N6-(dimethylallyl)adenosine in substrate tRNAs

Challenge 3: Protein Degradation During Purification

• Problem: Proteolytic degradation during expression or purification

• Solution:

  • Include protease inhibitors throughout purification process

  • Use E. coli strains deficient in key proteases

  • Optimize buffer conditions to enhance stability

How can researchers verify the functionality of purified recombinant B. bacteriovorus miaA?

To confirm that purified recombinant B. bacteriovorus miaA is functionally active, researchers should implement a multi-faceted verification approach:

• Biochemical Activity Assay:

  • Set up an in vitro prenylation reaction using purified miaA, appropriate tRNA substrates, and dimethylallyl pyrophosphate

  • Detect the formation of N6-(dimethylallyl)adenosine (i6A) using mass spectrometry or HPLC analysis

• Complementation Testing:

  • Transform miaA-deficient bacterial strains with expression vectors containing the recombinant B. bacteriovorus miaA

  • Assess restoration of phenotypes known to be affected by miaA deficiency, such as translational fidelity or motility

• Structural Integrity Assessment:

  • Perform circular dichroism spectroscopy to confirm proper protein folding

  • Assess thermal stability using differential scanning fluorimetry

• Binding Assays:

  • Develop fluorescence-based or surface plasmon resonance assays to verify binding to tRNA substrates and cofactors

What potential applications exist for engineered B. bacteriovorus miaA variants in synthetic biology?

Engineered B. bacteriovorus miaA variants offer several promising applications in synthetic biology:

• Controlled Translational Frameshifting: Since MiaA affects translational frameshifting , engineered variants could be used to create controlled gene expression systems where frameshift-dependent protein production is regulated by miaA activity.

• Tunable Regulatory Systems: Given MiaA's role as a regulatory nexus , engineered variants could function as molecular switches in synthetic circuits, responding to specific environmental signals by altering translation of specific protein sets.

• Enhanced Predatory Capabilities: Modified miaA could potentially optimize the predatory capabilities of B. bacteriovorus against specific pathogens, particularly important for applications against antibiotic-resistant bacteria .

• Biosensors Development: MiaA's sensitivity to stress conditions could be exploited to develop biosensors that detect specific environmental stressors through reporter gene expression.

How might miaA manipulation be used to enhance B. bacteriovorus as a therapeutic agent against antibiotic-resistant infections?

Manipulating miaA expression or activity presents several strategies for enhancing B. bacteriovorus as a therapeutic against antibiotic-resistant infections:

• Optimized Predation Efficiency:

  • Fine-tuning miaA expression levels could enhance predatory capabilities against specific pathogens

  • Research shows B. bacteriovorus effectively predates various clinical pathogens, particularly Gram-negative isolates like Pseudomonas aeruginosa and Acinetobacter baumannii, though with variable efficiency

• Enhanced Host Range Engineering:

  • Modifying miaA to alter the proteome composition might expand predatory host range

  • Interestingly, studies have shown that B. bacteriovorus can inhibit some Gram-positive bacteria like Staphylococci species despite conventional understanding that it predates primarily on Gram-negative bacteria

• Biofilm Disruption Capabilities:

  • Optimization of miaA could enhance expression of proteins involved in biofilm penetration and degradation

  • B. bacteriovorus has demonstrated effectiveness against bacterial biofilms as shown by co-culture methods and crystal violet staining

• Stress Tolerance Improvement:

  • Given MiaA's role in stress response , engineering miaA variants could enhance B. bacteriovorus survival under therapeutic administration conditions

• Research Design Considerations:

  • Create miaA variants with altered regulatory properties

  • Test predation efficiency against antibiotic-resistant clinical isolates in both planktonic and biofilm growth conditions

  • Evaluate therapeutic potential using appropriate infection models

How does B. bacteriovorus miaA compare structurally and functionally to miaA from other bacterial species?

While detailed comparative structural analysis is not provided in the search results, functional comparison can be extrapolated from available data:

• Structural Conservation:

  • B. bacteriovorus miaA belongs to the IPP transferase family , suggesting structural similarity to other bacterial miaA proteins

  • The 308 amino acid length and 34.7 kDa mass are comparable to miaA from other bacterial species

  • Key functional domains are likely conserved across bacterial species, as indicated by the similar catalytic function

• Functional Comparison with E. coli MiaA:

  • Both catalyze the prenylation of adenosine-37 in tRNAs that read UNN codons

  • In E. coli, MiaA has been shown to be crucial for fitness and virulence, particularly in ExPEC strains

  • Both are involved in optimizing translational fidelity and preventing frameshifting

• Evolutionary Conservation:

  • The ms2i6A-37 modification pathway is highly conserved in both prokaryotes and eukaryotes, though specific enzymes have diverged in evolutionarily distant organisms

  • In prokaryotes, MiaA and MiaB homologues are relatively well conserved and appear to function similarly across bacterial species

What insights can be gained by comparing the regulation and activity of miaA across different Bdellovibrio strains?

Comparative analysis of miaA across Bdellovibrio strains could provide valuable insights:

• Host Range Correlation:

  • Different Bdellovibrio strains show variable predation efficiency against different bacterial hosts

  • Comparing miaA expression and activity between strains with different host specificities could reveal correlations between miaA function and predatory range

• Environmental Adaptation:

  • Bdellovibrio strains isolated from different environments (freshwater, soil, clinical samples) may show adaptations in miaA regulation

  • Comparative analysis could identify regulatory mechanisms that enable adaptation to specific niches

• Methodological Approach for Strain Comparison:

  • Sequence comparison of miaA genes and promoter regions across strains

  • Expression analysis under standardized conditions

  • Functional complementation experiments between strains

  • Predation efficiency testing against standardized host panels

• Research Design Considerations:

  • Include diverse Bdellovibrio strains, such as the well-characterized HD100 strain and environmental isolates

  • Compare host-dependent and host-independent variants of the same strain

  • Standardize experimental conditions to enable valid cross-strain comparisons