Recombinant Acinetobacter sp. (Dimethylallyl)adenosine tRNA methylthiotransferase MiaB (miaB)

What is Acinetobacter baumannii and why is it significant for MiaB research?

Acinetobacter baumannii is a gram-negative, non-motile, strictly aerobic bacterial species widely distributed in soil and water environments. Its significance lies in its remarkable genomic plasticity and ability to develop multidrug resistance through various mechanisms. A. baumannii has emerged as a critical nosocomial pathogen in intensive care units, capable of causing life-threatening infections including bacteremia, pneumonia, and meningitis .

The organism's genomic flexibility makes it particularly valuable for recombinant protein expression studies, including MiaB research. A. baumannii can acquire genetic material through horizontal gene transfer and homologous recombination, allowing for genetic exchange between different clonal lineages . This property makes it both a challenging pathogen to control and an interesting model for studying recombinant protein expression and function.

What is (Dimethylallyl)adenosine tRNA Methylthiotransferase MiaB and what cellular functions does it perform?

(Dimethylallyl)adenosine tRNA Methylthiotransferase MiaB is a bifunctional radical S-adenosylmethionine (SAM) enzyme involved in the thiolation and methylation of transfer RNA (tRNA) . Specifically, MiaB catalyzes the methylthiolation of N6-(dimethylallyl)adenosine (i6A), leading to the formation of 2-methylthio-N6-(dimethylallyl)adenosine (ms2i6A) at position 37 in tRNAs that read codons beginning with uridine .

The enzyme contains two essential 4Fe-4S clusters with distinct functions:

• The first cluster is coordinated with three cysteines and an exchangeable S-adenosyl-L-methionine molecule

• The second cluster is thought to act as the sulfur donor, with a polysulfide group bound to it

The enzymatic reaction proceeds in multiple steps:

• The polysulfide group on the second iron-sulfur cluster is methylated

• The SAM-[4Fe-4S] cluster forms the 5'-dAdo radical

• This radical abstracts a hydrogen atom from the substrate

• The substrate is then methylthiolated by the methylated polysulfide group

This post-transcriptional modification of tRNA is crucial for ensuring correct codon recognition and translational fidelity.

How can one differentiate between native and recombinant MiaB expression in Acinetobacter species?

Differentiating between native and recombinant MiaB expression requires a multi-faceted approach:

Molecular Tagging Method:

• Incorporate histidine or FLAG tags to recombinant MiaB constructs

• Perform Western blot analysis using anti-His or anti-FLAG antibodies

• Native MiaB will not produce signals with these antibodies

Expression Level Analysis:

• Quantitative PCR (qPCR) to measure miaB transcript levels

• Compare expression levels between wild-type and recombinant strains

• Significant overexpression indicates successful recombinant production

Enzymatic Activity Comparison:

• Isolate tRNA from both native and recombinant strains

• Analyze the proportion of ms2i6A-modified tRNAs using liquid chromatography-mass spectrometry (LC-MS)

• Enhanced modification rates in recombinant strains confirm functional expression

Protein Purification Profile:

• Use size-exclusion chromatography to isolate the MiaB protein

• Compare elution profiles - recombinant protein with tags will show altered retention times

• Analyze Fe-S cluster incorporation using UV-visible spectroscopy (characteristic absorbance at ~410 nm)

When working with Acinetobacter species, it's important to account for their genomic plasticity, as horizontal gene transfer events may complicate expression analysis .

What vectors and expression systems are most effective for recombinant MiaB production in Acinetobacter species?

Successful recombinant MiaB expression in Acinetobacter species requires careful selection of vectors and expression systems tailored to this challenging organism:

Recommended Vector Systems:

Methodological Considerations:

• Iron-Sulfur Cluster Assembly: Since MiaB requires two 4Fe-4S clusters for proper function, expression systems should be supplemented with iron sources (50-100 μM ferric ammonium citrate) and operated under microaerobic conditions to facilitate cluster assembly .

• Codon Optimization: Acinetobacter species have distinct codon usage patterns. Synthesize MiaB coding sequences optimized for Acinetobacter codon bias to improve expression yields.

• Induction Parameters: For inducible systems, IPTG concentrations between 0.1-0.5 mM with induction at mid-log phase (OD600 ~0.6) typically yield optimal results.

• Temperature Modulation: Lowering culture temperatures to 18-22°C after induction can improve proper folding of MiaB and prevent inclusion body formation.

When using Acinetobacter as an expression host, consider that its intrinsic antibiotic resistance mechanisms may interfere with selection systems. The presence of aminoglycoside-modifying enzymes in many Acinetobacter strains may necessitate alternative selection strategies beyond kanamycin or gentamicin resistance .

What purification strategies yield the highest activity for recombinant MiaB from Acinetobacter?

Purifying active recombinant MiaB from Acinetobacter requires specialized techniques to preserve the oxygen-sensitive iron-sulfur clusters essential for enzymatic activity:

Multi-step Purification Protocol:

• Anaerobic Cell Lysis

  • Harvest cells in a sealed centrifuge bottle

  • Transfer to anaerobic chamber with <1 ppm O2

  • Resuspend in buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, 1 mM DTT

  • Add lysozyme (1 mg/ml) and DNase I (5 μg/ml)

  • Sonicate in sealed tubes on ice

• Affinity Chromatography

  • For His-tagged constructs, use Ni-NTA resin pre-equilibrated with anaerobic buffer

  • Include 5 mM β-mercaptoethanol in all buffers

  • Elute with 250 mM imidazole under strict anaerobic conditions

• Iron-Sulfur Cluster Reconstitution

  • Incubate purified protein with 8-molar excess Fe(NH4)2(SO4)2 and Na2S

  • Perform in anaerobic chamber for 4 hours at 15°C

  • Remove excess iron and sulfide by desalting

• Size Exclusion Chromatography

  • Final polishing step using Superdex 200 column

  • Elution buffer: 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM DTT

Activity Preservation Strategies:

The activity of purified MiaB is highly dependent on maintaining intact iron-sulfur clusters. Thermal stability analysis has shown that MiaB from Thermotoga maritima maintains activity at higher temperatures, suggesting that homologous protein from thermophilic Acinetobacter strains may offer advantages for structural and functional studies .

Storage recommendations include flash-freezing in liquid nitrogen with 30% glycerol and storing under argon at -80°C, which preserves >80% activity for up to 6 months.

How does antibiotic resistance in Acinetobacter strains influence MiaB expression studies?

Antibiotic resistance in Acinetobacter strains presents significant challenges for recombinant MiaB expression studies that must be strategically addressed:

Common Resistance Mechanisms Affecting Research:

Acinetobacter baumannii exhibits resistance to multiple antibiotic classes through various mechanisms:

• Aminoglycoside-modifying enzymes: The presence of aminoglycoside-3'-phosphotransferase VI (found in 28% of clinical isolates), adenylytransferases, and acetyltransferases undermines kanamycin, gentamicin, and other aminoglycoside selection markers .

• β-lactamases: Cephalosporinase activity is found in 98% of strains, with TEM-type lactamases present in approximately 16%, rendering ampicillin selection unreliable .

• Membrane protein alterations: Changes in membrane proteins can prevent antibiotics from attaching to bacterial cells, influencing permeability to induction agents like IPTG .

Methodological Adaptations:

Strain Selection Guidance:

When selecting Acinetobacter strains for MiaB expression studies, prioritize strains with defined susceptibility profiles. Based on susceptibility data, imipenem-susceptible strains offer advantages as hosts since this antibiotic remains effective against many clinical isolates . For work requiring genetically tractable strains, consider the naturally competent Acinetobacter baylyi ADP1, which offers ease of genetic manipulation while maintaining relevant characteristics for MiaB studies.

How do recombination events in Acinetobacter species affect the functional properties of MiaB?

Recombination events in Acinetobacter species can significantly alter the functional properties of MiaB through several mechanisms, creating both research challenges and opportunities:

Homologous Recombination Effects:

Acinetobacter species demonstrate remarkable genomic plasticity through homologous recombination, which enables genetic exchange between different clonal lineages . This phenomenon has been well-documented for the pmrCAB operon in A. baumannii, where recombination regions can vary in length from 5 kb to over 21 kb . Similar recombination events affecting the miaB gene would result in chimeric enzymes with potentially altered properties.

Such recombinations can lead to:

• Altered substrate specificity

• Modified enzyme kinetics

• Changed thermal stability profiles

• Differential iron-sulfur cluster binding capabilities

• Altered interactions with other cellular components

Experimental Evidence and Methodological Approaches:

To characterize recombination effects on MiaB function, researchers can employ:

• Comparative Genomic Analysis:

  • Sequence miaB genes from multiple Acinetobacter isolates

  • Use tools like SplitsTree with phi test for recombination (P < 0.05 indicates significant recombination)

  • Construct phylogenetic networks to visualize recombination events

• Protein Variant Analysis:

  • Express and purify MiaB variants identified through genomic analysis

  • Compare enzymatic parameters (kcat/KM) using in vitro methylthiolation assays

  • Perform thermal shift assays to determine stability differences

• Domain Swapping Experiments:

  • Create chimeric MiaB proteins by swapping domains between variants

  • Map functional differences to specific protein regions

  • Correlate with natural recombination breakpoints

Research has shown that recombination in Acinetobacter is not always driven by antibiotic pressure, as recombination events have been observed in both susceptible and resistant isolates . This suggests that MiaB variants resulting from recombination may reflect adaptations to different cellular environments rather than direct selective pressures on the enzyme itself.

What is the relationship between MiaB activity and antibiotic resistance mechanisms in Acinetobacter?

The relationship between MiaB activity and antibiotic resistance mechanisms in Acinetobacter represents an underexplored frontier with significant implications for both basic science and therapeutic development:

Translational Fidelity Connection:

As a tRNA modification enzyme, MiaB catalyzes the formation of 2-methylthio-N6-(dimethylallyl)adenosine (ms2i6A) at position 37 in tRNAs that read codons beginning with uridine . This modification enhances translational accuracy and efficiency, which could impact the production of:

• Membrane proteins involved in antibiotic efflux

• Enzymes that modify or degrade antibiotics

• Regulatory proteins that control resistance gene expression

Research Methodological Approach:

To investigate this relationship, researchers should consider:

• Comparative Transcriptomics:

  • Generate miaB knockout or overexpression strains in Acinetobacter

  • Perform RNA-seq under various antibiotic stress conditions

  • Analyze differential expression of resistance determinants

• Proteome-wide Translation Efficiency Analysis:

  • Implement ribosome profiling to measure translation efficiency

  • Focus on genes encoding aminoglycoside-modifying enzymes

  • Correlate with MiaB activity levels

• Minimum Inhibitory Concentration (MIC) Profiling:

• Metabolomic Analysis:

  • Measure S-adenosylmethionine (SAM) pools under antibiotic stress

  • Determine if competition for SAM between MiaB and other methyltransferases affects resistance

Theoretical Model:

Current evidence suggests that the aminoglycoside-modifying enzyme APH(3')-VI is present in approximately 28% of clinical Acinetobacter isolates . This enzyme, which phosphorylates and inactivates aminoglycosides, requires precise translation for proper function. MiaB-catalyzed tRNA modifications could potentially enhance the translation efficiency of this and other resistance determinants, creating an indirect link between MiaB activity and antibiotic resistance.

What methodologies are most effective for analyzing the 4Fe-4S clusters in recombinant MiaB from Acinetobacter?

The 4Fe-4S clusters in MiaB are crucial for its enzymatic function, requiring specialized methodologies for accurate characterization in recombinant proteins from Acinetobacter:

Spectroscopic Characterization Techniques:

• UV-Visible Absorption Spectroscopy:

  • Primary screening method for Fe-S cluster presence

  • Characteristic absorption bands at ~325 nm and ~410 nm

  • Quantify iron loading using extinction coefficient ε410 ≈ 15,000 M−1cm−1 per cluster

  • Sample preparation requires strict anaerobic conditions using sealed quartz cuvettes

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Differentiate between the two distinct 4Fe-4S clusters

  • The radical SAM cluster gives characteristic g-values of 2.03, 1.93, and 1.89 upon reduction

  • The auxiliary cluster shows distinct spectral features

  • Sample preparation protocol:
    a. Concentrate protein to 200-300 μM in anaerobic chamber
    b. Add sodium dithionite (10-fold excess) for reduction
    c. Transfer to EPR tubes and freeze immediately in liquid nitrogen

• Mössbauer Spectroscopy:

  • Provides detailed information on iron oxidation states

  • Requires 57Fe enrichment during protein expression

  • Growth medium supplementation protocol:
    a. Deplete medium of iron using Chelex-100 resin
    b. Supplement with 40 μM 57Fe(III)-citrate
    c. Add 100 μM cysteine to facilitate iron-sulfur cluster assembly

Functional Assay Methodologies:

• SAM Cleavage Assay:

  • Measures the reductive cleavage of SAM to 5'-deoxyadenosine

  • Indicates functional radical SAM cluster

  • Analysis by HPLC with the following conditions:

    • Column: C18 reverse phase

    • Mobile phase: 0.1% TFA in water/acetonitrile gradient

    • Detection: UV absorbance at 260 nm

• Methylthiolation Activity Assay:

  • Direct measurement of MiaB's native function

  • Requires in vitro transcribed tRNA substrate containing i6A modification

  • Product detection by LC-MS/MS

  • Reaction components:

    • 10 μM MiaB

    • 100 μM tRNA substrate

    • 1 mM SAM

    • 5 mM dithionite

    • 50 mM HEPES pH 7.5

    • 150 mM NaCl

Cluster Stability Analysis:

Research has shown that the two 4Fe-4S clusters in MiaB have different stability profiles, with the auxiliary cluster being more oxygen-sensitive . To measure this differential sensitivity in recombinant Acinetobacter MiaB:

• Expose protein samples to increasing oxygen concentrations (0-1000 ppm)

• Monitor cluster degradation by UV-visible spectroscopy

• Plot first-order decay constants against oxygen concentration

• Calculate half-life values for each cluster

This methodological approach reveals important structure-function relationships in MiaB and provides insights into optimizing expression and purification conditions for maximum enzymatic activity.

How can recombinant Acinetobacter MiaB be utilized to study bacterial adaptation to environmental stresses?

Recombinant Acinetobacter MiaB serves as a powerful model system for investigating bacterial adaptation to environmental stresses through tRNA modification mechanisms:

Experimental Framework:

• Stress Response Study Design:

  MiaB-catalyzed tRNA modifications affect translational efficiency and accuracy, particularly for codons beginning with uridine . This allows researchers to examine how environmental stresses reshape the translatome through modulation of MiaB activity.

  Implementation protocol:

  • Generate reporter constructs with synonymous codons (UNN vs. non-UNN)

  • Transform into wildtype and MiaB-deficient Acinetobacter

  • Subject to various stressors (temperature, pH, oxidative stress)

  • Measure reporter expression via fluorescence or luminescence

• Comparative Genomics Analysis:

  Acinetobacter species isolated from different environments show variations in their miaB genes due to homologous recombination, similar to observed recombination in the pmrCAB operon . These variations may represent adaptations to specific ecological niches.

  Analytical methodology:

  • Sequence miaB from Acinetobacter isolates from diverse environments

  • Perform phylogenetic analysis and recombination detection

  • Correlate sequence variations with environmental parameters

  • Express variant proteins to determine biochemical differences

Research Applications Table:

Methodological Considerations:

When utilizing recombinant MiaB for stress response studies, researchers must account for Acinetobacter's genomic plasticity. The organism's ability to undergo homologous recombination without requiring mobile genetic elements necessitates careful genetic stability monitoring during experiments. Regular sequencing of the recombinant miaB construct throughout stress experiments is recommended to detect potential genetic changes.

Additionally, researchers should consider the complex interplay between MiaB activity and other tRNA modification systems. Comprehensive analysis requires quantitative measurement of multiple tRNA modifications simultaneously using techniques like liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).

What are the most challenging technical issues when studying recombinant MiaB expression in multidrug-resistant Acinetobacter strains?

Working with recombinant MiaB in multidrug-resistant Acinetobacter strains presents unique technical challenges that require sophisticated methodological solutions:

Challenge 1: Selection Marker Limitations

Multidrug-resistant Acinetobacter strains often harbor resistance mechanisms against common antibiotics used as selection markers. Clinical isolates show variable resistance patterns:

• 67% resistant to gentamicin

• 50% resistant to tobramycin

• 28% resistant to amikacin

• 34% resistant to netilmicin

Solution Approaches:

• Counter-selection systems:

  • Implement sacB-based negative selection (sensitivity to sucrose)

  • Use tellurite resistance (telB) as an alternative marker

  • Develop CRISPR-Cas9 based marker-free systems

• Resistance testing protocol:

  • Perform comprehensive susceptibility testing prior to transformation

  • Identify susceptibility gaps for selection strategy

  • Develop custom selection markers based on strain-specific susceptibility profiles

Challenge 2: Iron-Sulfur Cluster Assembly

MiaB requires two intact 4Fe-4S clusters for activity . Antibiotic stress and resistance mechanisms can interfere with iron homeostasis and Fe-S cluster assembly pathways.

Solution Approaches:

• Co-expression strategies:

  • Co-express key components of iron-sulfur cluster (ISC) assembly machinery

  • Implement vector designs carrying both miaB and isc operon genes

  • Optimize expression ratios through dual-promoter systems

• Medium supplementation protocol:

  • Enrich medium with 100 μM ferric ammonium citrate

  • Add 5 mM cysteine as sulfur source

  • Include 50 μM pyridoxal phosphate to enhance cysteine desulfurase activity

  • Maintain microaerobic conditions (2-5% O2)

Challenge 3: Genetic Instability Due to Recombination

Acinetobacter demonstrates extensive genomic plasticity through homologous recombination , potentially affecting stability of recombinant constructs.

Solution Approaches:

• Recombination monitoring system:

  • Flank transgene with distinct fluorescent markers

  • Loss of fluorescence indicates recombination events

  • Implement regular PCR screening to detect genetic changes

• Recombination-resistant design:

  • Optimize codon usage to reduce sequence similarity with endogenous genes

  • Incorporate genetic insulator elements

  • Use integration sites distant from recombination hotspots

Challenge 4: Methodology for Activity Verification

Confirming functional expression of MiaB in multidrug-resistant backgrounds requires specialized approaches.

Technical Solution: Integrated LC-MS/MS Protocol

• Extract total tRNA from recombinant strains

• Digest with nuclease P1 and bacterial alkaline phosphatase

• Separate nucleosides using C18 reverse-phase chromatography

• Detect ms2i6A by mass spectrometry (molecular ion m/z 382.1)

• Quantify relative to unmodified adenosine

• Compare with profiles from control strains

This comprehensive approach addresses the multiple layers of technical challenges inherent to working with recombinant MiaB in multidrug-resistant Acinetobacter strains, enabling successful experimental outcomes despite the complex resistance background.

What emerging technologies could enhance our understanding of MiaB function in Acinetobacter species?

Emerging technologies offer unprecedented opportunities to deepen our understanding of MiaB function in Acinetobacter species, potentially revealing new aspects of tRNA modification biology and bacterial adaptation:

Next-Generation Structural Biology Approaches:

• Cryo-Electron Microscopy:

  • Near-atomic resolution structures of MiaB bound to tRNA substrates

  • Visualization of conformational changes during catalysis

  • Methodological advances needed: Grid preparation techniques to maintain anaerobic conditions for oxygen-sensitive Fe-S clusters

• Time-Resolved X-ray Crystallography:

  • Capturing intermediate states during methylthiolation reaction

  • Observing radical formation and transfer processes

  • Technical requirements: Microcrystal growth optimization and rapid freezing methods

Advanced 'Omics Integration:

• Epitranscriptomics:

  • Nanopore direct RNA sequencing to map tRNA modifications in vivo

  • NAIL-MS (Nucleic Acid Isotope Labeling coupled with Mass Spectrometry) to track modification dynamics

  • Methodological workflow:
    a. Pulse-chase isotope labeling of Acinetobacter cultures
    b. tRNA isolation at defined timepoints
    c. LC-MS/MS analysis to quantify modification rates

• Translatome Profiling:

  • Ribosome profiling comparing wild-type and MiaB-deficient strains

  • Measurement of codon-specific translation rates

  • Identification of genes most affected by ms2i6A modification

  • Implementation requires adaptation of protocols for Acinetobacter's high GC content

Synthetic Biology Tools:

• Engineered MiaB Variants:

  • CRISPR-based saturation mutagenesis of miaB

  • Selection systems to identify variants with enhanced activity or altered specificity

  • Deep mutational scanning to create comprehensive fitness landscapes

• Orthogonal Translation Systems:

  • Introduction of synthetic tRNAs with alternative modification sites

  • Engineering MiaB variants to recognize these substrates

  • Creation of "orthogonal" translation subsystems in Acinetobacter

Integration of Computational Methods:

• Quantum Mechanics/Molecular Mechanics (QM/MM) Simulations:

  • Modeling radical formation and transfer during catalysis

  • Calculating energy barriers for methylthiolation reactions

  • Predicting effects of amino acid substitutions on catalytic efficiency

• AlphaFold2-Enhanced Structural Predictions:

  • Modeling MiaB structural variants from different Acinetobacter strains

  • Predicting protein-protein interaction interfaces

  • Identifying potential regulatory binding partners

These emerging technologies promise to reveal how MiaB contributes to Acinetobacter's remarkable adaptability across environments and its ability to develop resistance to multiple antibiotics through fine-tuning of translational processes. The integration of these approaches will likely uncover previously unknown connections between tRNA modification and bacterial stress responses.

How might recombinant MiaB research contribute to addressing multidrug resistance in Acinetobacter?

Recombinant MiaB research holds promising potential for addressing the critical challenge of multidrug resistance in Acinetobacter species through several innovative approaches:

Translational Regulation of Resistance Mechanisms:

MiaB-catalyzed tRNA modifications influence translational efficiency, particularly for codons beginning with uridine . Research into how these modifications affect the expression of resistance determinants could reveal new intervention strategies.

Research Strategy Framework:

• Codon Usage Analysis of Resistance Genes:

  • Analyze codon bias in aminoglycoside-modifying enzymes and other resistance determinants

  • Map UNN codon frequency in key resistance genes

  • Identify resistance genes likely affected by MiaB activity

• Translation Efficiency Measurement:

  • Implement ribosome profiling in wild-type vs. MiaB-deficient strains

  • Focus on translation efficiency of identified resistance genes

  • Correlate with antibiotic susceptibility profiles

Potential Applications in Resistance Management:

Mechanistic Insights from Current Research:

Studies of pmrCAB recombination in Acinetobacter have demonstrated how homologous recombination contributes to colistin resistance . Similar investigations into miaB recombination could reveal how tRNA modification systems evolve in response to antibiotic pressure.

The remarkable finding that mobile genetic elements were not required for pmrCAB transfer suggests that natural competence and homologous recombination are sufficient for resistance gene spread in Acinetobacter . This highlights the importance of understanding the basic biology of these processes, which MiaB research can contribute to.

Theoretical Model: MiaB-Dependent Translation Regulation Hypothesis

• Antibiotic stress induces changes in MiaB expression/activity

• Altered tRNA modification patterns shift translation efficiency of specific mRNAs

• Resistance gene expression is enhanced or suppressed

• Population-level antibiotic susceptibility is modulated

Validating this model requires integrating recombinant MiaB studies with systems biology approaches to map the complete network of interactions between tRNA modification, translation regulation, and resistance mechanisms in Acinetobacter.