Recombinant Arcobacter butzleri tRNA dimethylallyltransferase (miaA)

What is tRNA dimethylallyltransferase (MiaA) and what is its function in bacteria?

tRNA dimethylallyltransferase (MiaA) is an enzyme that catalyzes the transfer of a dimethylallyl group from dimethylallyl pyrophosphate (DMAPP) to the adenosine at position 37 (A37) adjacent to the anticodon in certain tRNAs. This modification results in N6-(Δ2-isopentenyl)adenosine (i6A), which is critical for proper codon-anticodon interactions during translation. The enzyme belongs to the P-loop NTPase family, as evidenced by the characteristic GPTAVGKS motif seen in MiaA from various species .

In bacteria like Arcobacter butzleri, MiaA plays an essential role in maintaining translational accuracy and efficiency. The i6A modification enhances base stacking interactions between the anticodon and codon, preventing frameshifting and improving decoding of codons beginning with uridine. This modification affects multiple cellular processes, including protein synthesis rates, stress responses, and potentially virulence factor expression.

MiaA works as part of a broader network of tRNA modification enzymes that collectively ensure proper tRNA function. In some bacterial species, the i6A modification can be further modified by MiaB to form 2-methylthio-N6-(Δ2-isopentenyl)adenosine (ms2i6A), creating a sequential modification pathway that fine-tunes tRNA function.

How is the miaA gene typically identified in Arcobacter butzleri genome sequences?

The miaA gene in Arcobacter butzleri is typically identified through whole genome sequencing followed by bioinformatic analysis. As seen in research studies, whole genome sequencing of A. butzleri isolates can involve a combination of technologies such as single-molecule real-time (SMRT) and Illumina sequencing . The assembled genome size for A. butzleri strains typically ranges from 2,068,991 to 2,299,734 bp, with an average of 2,163,707 bp assembled in about 39 contigs per genome .

Once the genome is sequenced, several approaches can be used to identify the miaA gene:

• Homology-based annotation: The miaA gene can be identified by comparing the genome sequence with known miaA genes from related bacterial species using tools like BLASTN or BLASTP.

• Gene prediction software: Programs like Prokka, RAST, or the NCBI Prokaryotic Genome Annotation Pipeline can predict genes in the A. butzleri genome, including the miaA gene.

• Domain-based annotation: The MiaA protein contains specific functional domains, such as the P-loop NTPase domain seen in the protein sequence provided for other bacterial MiaA proteins , which can be identified using tools like InterProScan or HMMER.

• MLST-based approaches: While multilocus sequence typing (MLST) as described for A. butzleri in research typically uses seven housekeeping genes (not including miaA), similar approaches can be used to identify and characterize the miaA gene across different isolates .

Confirmation of gene identity often requires functional validation through expression and activity assays of the recombinant protein.

How does MiaA enzyme activity affect bacterial translation and protein synthesis?

MiaA enzyme activity directly impacts bacterial translation and protein synthesis through several mechanisms:

Research has shown that mutations in the miaA gene in various bacteria can lead to pleiotropic effects, including altered growth rates, reduced virulence, and increased sensitivity to stresses. For A. butzleri, which contains numerous virulence genes like flgG, flhA, flhB, fliI, fliP, motA, cadF, cjl349, ciaB, mviN, pldA and tlyA , MiaA activity could potentially influence the expression of these factors and consequently affect pathogenicity.

Given that A. butzleri shows variable antibiotic resistance patterns , the influence of MiaA on translation could also potentially affect the expression of resistance genes and stress response factors that contribute to antibiotic tolerance.

What are the common methods for expressing recombinant bacterial MiaA proteins?

Based on published research and practices in recombinant protein expression, several methods are commonly employed for expressing recombinant bacterial MiaA proteins:

• Expression hosts:

  • Yeast has been successfully used as a host for expressing recombinant tRNA Dimethylallyltransferase (MiaA) from various bacterial species with high purity (>90%) .

  • Escherichia coli expression systems, particularly strains like BL21(DE3), are also frequently used for bacterial protein expression due to their rapid growth and high yield potential.

• Expression vectors:

  • Plasmids containing inducible promoters (such as T7, tac, or arabinose-inducible promoters) allow for controlled expression of recombinant proteins.

  • Vectors carrying appropriate selection markers and origins of replication compatible with the chosen host are essential.

• Fusion tags:

  • His-tags are commonly used for purification, as seen in commercially available recombinant MiaA proteins which utilize His tags for purification and detection .

  • Other common fusion tags include GST, MBP, and SUMO, which can enhance solubility and facilitate purification.

• Expression conditions:

  • Optimization of temperature, induction time, and inducer concentration is crucial for maximizing the yield of soluble and active recombinant MiaA.

  • Lower temperatures (15-25°C) often favor proper folding of recombinant proteins.

• Solubility enhancement:

  • Co-expression with chaperones or use of solubility-enhancing fusion partners may be necessary if MiaA tends to form inclusion bodies.

For specific applications like ELISA (as mentioned for available recombinant MiaA proteins) , high purity (>90%) is typically required, necessitating effective purification strategies following expression.

How do the structural characteristics of Arcobacter butzleri MiaA compare to those from other bacterial species?

Comparative analysis of A. butzleri MiaA with those from other bacterial species provides insights into conserved functional domains and species-specific variations. While specific structural information for A. butzleri MiaA is limited, comparisons can be made based on sequence analysis and structural predictions:

MiaA proteins from various bacterial species, including those from Borrelia, Helicobacter, Streptococcus, and Agrobacterium , share common structural features despite variations in length (ranging from 274 to 305 amino acids). The MiaA protein sequence typically begins with a characteristic P-loop NTPase domain (GPTAVGKS motif) as seen in the sequence from Borrelia duttonii (MKTNKIVFIF GPTAVGKSDI LFHFPKGVAE...) . This domain is essential for binding and hydrolyzing DMAPP, the substrate that provides the dimethylallyl group.

The structural architecture of MiaA proteins generally includes:

• N-terminal domain: Contains the P-loop NTPase fold responsible for DMAPP binding and catalysis.

• C-terminal domain: Involved in tRNA recognition and binding.

• Interdomain linker: Provides flexibility necessary for conformational changes during catalysis.

Comparing A. butzleri MiaA with those from other bacterial species would involve:

• Sequence alignment to identify conserved catalytic residues and variable regions that might confer species-specific properties.

• Structural modeling based on crystal structures from model organisms to predict the three-dimensional architecture.

• Phylogenetic analysis to understand evolutionary relationships between MiaA proteins from different bacterial taxa.

Given A. butzleri's position as an emerging pathogen with distinctive antibiotic resistance patterns , its MiaA might possess unique structural features that could potentially be targeted for species-specific inhibition, though this requires experimental validation.

What are the optimal conditions for assessing the enzymatic activity of recombinant A. butzleri MiaA?

The optimal conditions for assessing enzymatic activity of recombinant A. butzleri MiaA would typically include carefully optimized buffer components, substrate preparations, and detection methods:

• Buffer composition:

  • pH: Most bacterial enzymes, including tRNA modification enzymes, function optimally at pH 7.0-8.0

  • Salt concentration: 50-100 mM NaCl or KCl to maintain ionic strength

  • Divalent cations: Mg²⁺ (1-5 mM) is usually required as a cofactor for MiaA activity

  • Reducing agents: DTT or β-mercaptoethanol (1-5 mM) to maintain cysteine residues in reduced state

• Substrate preparation:

  • tRNA substrate: Either total tRNA extracted from miaA-deficient bacteria or in vitro transcribed tRNA containing an A37 target site

  • DMAPP: Freshly prepared dimethylallyl pyrophosphate at concentrations typically between 10-100 μM

  • Ensuring substrate quality is critical for reliable activity measurements

• Reaction conditions:

  • Temperature: For A. butzleri proteins, temperatures around 30-37°C are typically suitable, considering the organism's growth temperature

  • Incubation time: 15-60 minutes depending on enzyme concentration and activity

  • Enzyme concentration: Typically in the range of 0.1-1 μM purified recombinant protein

• Detection methods:

  • HPLC analysis of nucleosides after tRNA hydrolysis to detect i6A formation

  • Mass spectrometry to identify modified nucleosides

  • Radioactive assays using [¹⁴C] or [³H]-labeled DMAPP

  • Coupled enzymatic assays measuring pyrophosphate release

• Controls:

  • Negative control: Reaction without enzyme or with heat-inactivated enzyme

  • Positive control: Reaction with well-characterized MiaA from another organism

Given that recombinant MiaA proteins are often produced with >90% purity for applications like ELISA , similar purity levels would be desirable for enzymatic assays to minimize interference from contaminants. Optimization of these conditions would be necessary for the specific recombinant A. butzleri MiaA, as optimal conditions may vary between MiaA enzymes from different bacterial species.

How can site-directed mutagenesis of A. butzleri MiaA help elucidate its catalytic mechanism?

Site-directed mutagenesis of A. butzleri MiaA can be a powerful approach to elucidate its catalytic mechanism by systematically altering specific amino acids and observing the effects on enzyme activity. This approach provides insights into structure-function relationships that are difficult to obtain through other methods.

Key applications of site-directed mutagenesis for studying A. butzleri MiaA include:

• Identifying catalytic residues: By mutating conserved amino acids in the predicted active site (such as the P-loop motif GPTAVGKS seen in related MiaA proteins ), researchers can determine which residues are essential for catalysis. Mutations would typically target:

  • Residues involved in DMAPP binding

  • Residues involved in tRNA recognition and binding

  • Residues potentially involved in acid/base catalysis

• Understanding substrate specificity: Mutating residues in regions predicted to interact with the tRNA substrate can reveal the molecular basis for tRNA recognition and selectivity.

• Investigating conformational changes: Mutations that potentially affect enzyme dynamics or domain movements can provide insights into conformational changes during catalysis.

• Exploring evolutionary conservation: Mutating highly conserved residues versus variable regions can reveal which structural elements are fundamental to all MiaA enzymes versus those that confer species-specific properties.

A systematic mutagenesis approach would typically involve:

• Alanine scanning mutagenesis of conserved residues

• Conservative substitutions to maintain chemical properties while altering specific features

• Drastic substitutions to completely change the properties of key residues

• Kinetic analysis of mutants to determine effects on Km and kcat

• Structural analysis of selected mutants to observe any conformational changes

For A. butzleri MiaA specifically, targeting residues that are uniquely conserved in this species compared to other bacteria could help identify features that might be relevant to pathogenicity or adaptation to specific niches, potentially connecting MiaA function to the organism's biology as an emerging pathogen .

How do post-translational modifications affect the activity of recombinant A. butzleri MiaA?

Post-translational modifications (PTMs) can significantly influence the activity, stability, localization, and interactions of recombinant A. butzleri MiaA. While specific information about PTMs in A. butzleri MiaA is limited, several potential impacts can be identified based on knowledge of bacterial enzyme regulation:

• Phosphorylation:

  • Serine, threonine, or tyrosine phosphorylation can regulate enzyme activity by inducing conformational changes

  • In bacteria, phosphorylation often serves as a regulatory mechanism responding to environmental signals

  • For MiaA, phosphorylation could potentially modulate its activity based on cellular energy status or stress conditions

• Acetylation:

  • N-terminal or lysine acetylation can affect protein stability and activity

  • In bacteria, acetylation often responds to metabolic states and carbon source availability

  • Acetylation of MiaA could connect tRNA modification to the metabolic state of A. butzleri

• Methylation:

  • Protein methylation can alter enzyme activity and protein-protein interactions

  • For MiaA, methylation could potentially fine-tune substrate recognition or catalytic efficiency

• Proteolytic processing:

  • Limited proteolysis can activate or inactivate enzymes or release them from membrane associations

  • For recombinant MiaA, unexpected proteolytic events during expression or purification could lead to heterogeneous preparations with variable activity

• Expression system considerations:

  • When expressing recombinant A. butzleri MiaA in heterologous systems (e.g., E. coli or yeast as mentioned for other MiaA proteins ), the PTM patterns may differ from the native bacterium

  • This could lead to functional differences between recombinant and native MiaA

To properly characterize the impact of PTMs on recombinant A. butzleri MiaA, techniques such as mass spectrometry could be employed to identify and map PTMs on the recombinant protein and correlate specific PTMs with enzymatic activity. Understanding these modifications is crucial for ensuring that recombinant MiaA accurately represents the native enzyme's activity.

What expression systems are most effective for producing functional recombinant A. butzleri MiaA?

Based on published research and practical considerations for recombinant protein expression, several expression systems can be considered for producing functional recombinant A. butzleri MiaA:

• Yeast expression systems:

  • Yeast has been successfully used as a host for expressing recombinant tRNA Dimethylallyltransferase (MiaA) from various bacterial species including Borrelia duttonii, Helicobacter pylori, and others .

  • Yeast systems like Saccharomyces cerevisiae or Pichia pastoris can provide a eukaryotic environment with protein folding machinery that might be beneficial for complex proteins.

  • Advantages include post-translational modification capabilities and secretion of the recombinant protein into the medium.

• E. coli expression systems:

  • E. coli remains the most commonly used host for bacterial protein expression due to its:

    • Rapid growth and high yield

    • Well-established genetic tools

    • Simple media requirements

  • For A. butzleri MiaA, E. coli strains designed for improved expression of potentially toxic proteins (like BL21(DE3)pLysS, C41(DE3), or C43(DE3)) might be appropriate.

• Cell-free expression systems:

  • For proteins that might be toxic to living cells, cell-free systems based on E. coli or wheat germ extracts can be considered.

  • These systems allow for rapid protein production and can be scaled for high-throughput screening.

Optimization considerations for expressing functional A. butzleri MiaA would include:

• Codon optimization:

  • Adapting the A. butzleri miaA gene codons to the preferred codons of the expression host

• Fusion tags:

  • His-tags are commonly used as shown in commercial recombinant MiaA proteins

  • Solubility-enhancing tags like MBP, SUMO, or Trx may improve yield of soluble protein

• Expression conditions:

  • Temperature (typically lower temperatures of 16-25°C favor proper folding)

  • Induction parameters (inducer concentration and timing)

  • Media composition

The optimal expression system would ultimately need to be determined empirically by comparing protein yield, solubility, and enzymatic activity across different systems.

What purification strategies optimize yield and activity of recombinant A. butzleri MiaA?

Effective purification of recombinant A. butzleri MiaA requires strategies that maximize both yield and enzymatic activity. Based on standard protein purification principles and the information that commercial recombinant MiaA proteins are typically produced with >90% purity , the following strategies are recommended:

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins is suitable for His-tagged MiaA proteins as described in commercial preparations .

  • Optimization of imidazole concentrations in washing and elution buffers can improve purity while maintaining high yield.

  • For purity levels >90% as mentioned for commercial MiaA proteins , a polishing step after initial affinity purification is typically necessary.

• Buffer optimization:

  • Stabilizing additives: Glycerol (10-20%), reducing agents (DTT or β-mercaptoethanol), and specific ions can enhance stability.

  • pH optimization: Typically, MiaA enzymes are most stable at neutral to slightly alkaline pH (7.0-8.0).

  • Salt concentration: Moderate salt concentrations (100-300 mM NaCl) often prevent aggregation while maintaining activity.

• Chromatographic polishing steps:

  • Size exclusion chromatography (SEC) to separate monomeric protein from aggregates and remove impurities of different sizes.

  • Ion exchange chromatography (IEX) as a second dimension of purification based on charge properties.

  • Hydrophobic interaction chromatography (HIC) if appropriate for the specific properties of A. butzleri MiaA.

• Solubility enhancement strategies:

  • Screening different detergents or lipids if membrane association is a concern.

  • Addition of stabilizing ligands or cofactors during purification.

  • Use of arginine or proline as aggregation suppressors in buffers.

• Activity preservation approaches:

  • Rapid processing to minimize time between cell lysis and final purification.

  • Maintenance of cold temperatures throughout purification.

  • Addition of protease inhibitors to prevent degradation.

  • Storage in small aliquots at -80°C with cryoprotectants to maintain long-term activity.

• Quality control:

  • SDS-PAGE and Western blotting to confirm purity and identity.

  • Mass spectrometry to verify the intact mass and detect any modifications.

  • Activity assays at multiple stages of purification to track specific activity.

These strategies should be optimized based on the specific properties of A. butzleri MiaA and the intended downstream applications, such as enzymatic assays or structural studies.

How can isothermal titration calorimetry be used to characterize MiaA-substrate interactions?

Isothermal Titration Calorimetry (ITC) is a powerful technique for characterizing the thermodynamic parameters of biomolecular interactions, including those between enzymes like MiaA and their substrates. For studying A. butzleri MiaA-substrate interactions, ITC can be implemented as follows:

Using ITC in conjunction with other techniques such as surface plasmon resonance (SPR) or microscale thermophoresis (MST) can provide a comprehensive characterization of MiaA-substrate interactions that is crucial for understanding the enzyme's mechanism.

What computational approaches are useful for predicting MiaA substrate specificity?

Computational approaches can significantly enhance our understanding of MiaA substrate specificity, helping to predict which tRNAs might be preferentially modified and how variations in the enzyme might affect its function. Several powerful computational methods can be employed:

By combining these computational approaches with experimental validation, researchers can develop a comprehensive model of A. butzleri MiaA substrate specificity, which could inform studies on bacterial translation, stress responses, and potentially antimicrobial development.

How can CRISPR-Cas9 be utilized to study the physiological effects of miaA deletion in A. butzleri?

CRISPR-Cas9 technology offers a precise and efficient approach for studying the physiological effects of miaA deletion in A. butzleri. While not specifically documented for A. butzleri miaA in the search results, this methodology can be adapted based on general CRISPR principles and knowledge of bacterial genetics:

• CRISPR-Cas9 system design for A. butzleri:

  • Selection of appropriate Cas9 variant: SpCas9 or smaller variants like SaCas9 could be used depending on transformation efficiency.

  • Design of sgRNAs targeting the miaA gene: Multiple guides should be designed to target different regions of the gene, with minimal off-target potential.

  • Selection of a suitable delivery method: Plasmid-based systems or direct ribonucleoprotein (RNP) delivery depending on A. butzleri transformation efficiency.

• Generation of miaA knockout strains:

  • CRISPR-mediated cleavage can be used to introduce double-strand breaks in the miaA gene.

  • Repair templates can be provided to either:

    • Introduce a complete deletion of the miaA gene

    • Create point mutations in catalytic residues

    • Insert reporter genes for expression analysis

  • Screening and verification of mutants by PCR, sequencing, and activity assays.

• Physiological characterization:

  • Growth phenotype analysis under different conditions (temperature, pH, nutrient availability).

  • Antibiotic susceptibility testing: Given A. butzleri's variable resistance to nalidixic acid, ciprofloxacin, clindamycin, chloramphenicol, and florfenicol , changes in susceptibility would be particularly interesting to assess.

  • Analysis of virulence factor expression, especially the virulence genes commonly found in A. butzleri isolates .

• Molecular characterization:

  • Transcriptomic analysis (RNA-seq) to identify genes with altered expression in the miaA mutant.

  • Proteomic analysis to identify changes in protein abundance, particularly of proteins encoded by genes rich in codons read by i6A-modified tRNAs.

  • tRNA modification analysis to confirm the absence of i6A modifications and identify any compensatory changes in other modification pathways.

• Complementation studies:

  • Reintroduction of wild-type miaA to confirm phenotype rescue.

  • Introduction of miaA variants with specific mutations to study structure-function relationships.

  • Cross-species complementation with miaA from other bacteria to assess functional conservation.

CRISPR-Cas9 offers advantages over traditional homologous recombination methods in terms of efficiency and precision, potentially allowing for more rapid generation of multiple mutants to comprehensively study miaA function in A. butzleri and its role in this emerging pathogen's biology.