Recombinant Acinetobacter sp. Isoleucine--tRNA ligase (ileS), partial

What is Isoleucine--tRNA ligase (ileS) and what is its function in Acinetobacter species?

Isoleucine--tRNA ligase (ileS) is an essential aminoacyl-tRNA synthetase responsible for attaching isoleucine to its cognate tRNA molecule during protein synthesis. In Acinetobacter species, as in other bacteria, ileS plays a crucial role in translation by ensuring the correct incorporation of isoleucine into growing polypeptide chains. The enzyme catalyzes a two-step reaction: first activating isoleucine with ATP to form isoleucyl-AMP, then transferring the isoleucyl group to the appropriate tRNA molecule. This aminoacylation process is fundamental to accurate protein synthesis and bacterial survival .

How is recombinant Acinetobacter sp. ileS typically expressed and purified?

Recombinant Acinetobacter sp. ileS is typically expressed in heterologous systems such as E. coli or yeast expression systems. The most common approach involves cloning the ileS gene into an expression vector containing an affinity tag (such as His-tag) to facilitate purification. After transformation into the expression host, protein production is induced under optimized conditions. Purification generally follows a multi-step process including:

• Cell lysis by sonication or mechanical disruption

• Initial purification using affinity chromatography (commonly Ni-NTA for His-tagged proteins)

• Further purification by ion-exchange or size exclusion chromatography

• Buffer exchange to remove imidazole and provide stability

The purified protein is typically stored in PBS buffer or another stabilizing formulation at -20°C to -80°C for long-term storage, with purity levels exceeding 80% as determined by SDS-PAGE .

What are the key structural characteristics of Acinetobacter sp. ileS?

Acinetobacter sp. isoleucyl-tRNA synthetase belongs to the class I aminoacyl-tRNA synthetase family, characterized by a Rossmann fold that binds ATP. The enzyme consists of multiple domains including a catalytic core, an editing domain that ensures fidelity by hydrolyzing misactivated amino acids, and an anticodon-binding domain that recognizes the appropriate tRNA. While the complete crystal structure of Acinetobacter sp. ileS has not been fully characterized in the provided literature, comparative analysis with related bacterial ileS enzymes suggests a molecular weight of approximately 105-110 kDa and similar domain organization to that observed in other gram-negative bacteria .

How does ileS contribute to antibiotic resistance mechanisms in Acinetobacter species?

Isoleucyl-tRNA synthetase is a target for several antibiotics, including mupirocin and certain aminoacyl-tRNA synthetase inhibitors. In Acinetobacter species, particularly in clinically relevant species like A. baumannii, mutations in the ileS gene can confer resistance to these antibiotics. The resistance mechanisms typically involve:

• Point mutations in the ileS gene that alter the binding site for antibiotics while preserving enzymatic function

• Acquisition of alternative ileS genes (e.g., ileS2) through horizontal gene transfer

• Overexpression of native ileS to overcome inhibition

Research has shown that these resistance mechanisms are particularly concerning in clinical isolates of Acinetobacter, which already demonstrate high levels of multidrug resistance. The resistance profiles of Acinetobacter species vary significantly, with A. baumannii showing substantially higher resistance rates (>40% to carbapenems) compared to non-A. baumannii isolates (approximately 2.6% resistance to imipenem/meropenem) .

What methods are optimal for species-specific identification of ileS variants across different Acinetobacter species?

Discriminating between ileS variants across Acinetobacter species requires sensitive molecular approaches. Based on comparative studies of identification methods, the following approaches are recommended:

• rpoB gene sequencing provides the most accurate differentiation of Acinetobacter species and their associated ileS variants, with a pairwise identity of 98.5% serving as an effective discrimination threshold

• 16S rRNA gene sequencing offers reliable genus-level identification but shows poor discriminatory ability at the species level, particularly within the Acb complex (A. pittii, A. nosocomialis, A. calcoaceticus, and A. baumannii)

• Phenotypic methods such as VITEK 2 and VITEK MS demonstrate limited resolution, with VITEK MS showing better discrimination but still failing to reliably differentiate between closely related Acinetobacter species

A comparison of identification methods for Acinetobacter species is summarized in the table below:

The optimal approach involves using rpoB gene sequencing as the primary identification method, possibly supplemented with targeted amplification and sequencing of the ileS gene itself to identify specific variants .

What is the evolutionary significance of ileS conservation and diversity among Acinetobacter species?

The evolutionary patterns of ileS in Acinetobacter species reveal important insights into bacterial adaptation and speciation. Research indicates that ileS, like other housekeeping genes, shows moderate conservation due to functional constraints while displaying sufficient variation to reflect evolutionary relationships between species.

Key evolutionary aspects include:

• The ileS gene appears to undergo limited horizontal gene transfer between Acinetobacter species compared to antibiotic resistance genes, suggesting it evolves primarily through vertical inheritance

• Phylogenetic analysis based on ileS sequences generally aligns with whole-genome phylogeny, indicating its utility as a reliable marker for evolutionary studies

• Variations in ileS may contribute to adaptive differences between species, particularly in terms of translation efficiency under different environmental conditions

Within the Acinetobacter calcoaceticus-baumannii (Acb) complex, which includes the clinically important species A. baumannii, A. pittii, A. nosocomialis, and A. calcoaceticus, ileS sequences demonstrate higher conservation (>95% similarity) compared to more distantly related Acinetobacter species, reflecting their recent evolutionary divergence. This pattern mirrors what has been observed with other genes like rpoB, which shows sufficient resolution to differentiate these closely related species .

What are the optimal expression systems and conditions for producing active recombinant Acinetobacter sp. ileS?

Based on experimental data and established protocols, the following expression systems and conditions are recommended for producing active recombinant Acinetobacter sp. ileS:

Expression Systems:

• E. coli BL21(DE3): The preferred system due to high yield and reduced proteolytic degradation

• Yeast expression systems: Alternative when protein folding issues are encountered in bacterial systems

Expression Vectors:

• pET vector systems with T7 promoter for high-level expression

• Incorporation of His-tag or other affinity tags for simplified purification

• Inclusion of appropriate cleavage sites for tag removal if necessary for activity studies

Optimal Conditions:

• Induction: IPTG at 0.5-1.0 mM, typically at OD600 of 0.6-0.8

• Temperature: 18-25°C for overnight expression to enhance proper folding

• Media: Enriched media such as TB (Terrific Broth) or auto-induction media for higher yields

• Supplements: Addition of rare amino acids and cofactors where necessary

Purification Strategy:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins

• Ion-exchange chromatography as a secondary purification step

• Size exclusion chromatography for final polishing and buffer exchange

The final purified protein typically achieves >80% purity as determined by SDS-PAGE and can be stored in PBS buffer or specialized storage buffer containing glycerol and reducing agents to maintain stability .

How can researchers design effective enzymatic assays to measure Acinetobacter sp. ileS activity?

Effective enzymatic assays for Acinetobacter sp. ileS activity measurement can be designed using several complementary approaches:

ATP-PPi Exchange Assay:

• Measures the first step of aminoacylation (activation of isoleucine with ATP)

• Quantifies the exchange of radiolabeled pyrophosphate into ATP

• Allows determination of kinetic parameters for amino acid activation

tRNA Charging Assay:

• Measures the complete aminoacylation reaction (transfer of activated isoleucine to tRNA)

• Can use either:

  • Radioactive assay with [14C] or [3H]-isoleucine

  • Colorimetric assay based on methylene blue detection of uncharged tRNA

• Provides direct measurement of aminoacylation activity

Coupled Enzyme Assay:

• Links aminoacylation to NADH oxidation through auxiliary enzymes

• Allows continuous spectrophotometric monitoring (λ = 340 nm)

• Enables high-throughput screening for inhibitors

The choice of assay depends on research objectives, with the ATP-PPi exchange assay being simpler but less complete, and the tRNA charging assay providing the most direct measure of biological activity. For kinetic studies, the recommended assay conditions include:

• Buffer: 100 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 50 mM KCl, 1 mM DTT

• Substrates: 1-100 μM isoleucine, 2-5 mM ATP, 0.5-2.0 μM tRNA

• Temperature: 30-37°C

• Reaction time: 5-30 minutes in the linear range of product formation

These assays can be adapted to investigate inhibition by antibiotics or to screen for novel inhibitors with potential therapeutic applications against multidrug-resistant Acinetobacter species .

What strategies can be employed to study the interaction between ileS and its target tRNA molecules in Acinetobacter species?

To investigate the interaction between ileS and its target tRNA molecules in Acinetobacter species, researchers can employ several complementary strategies:

1. Biophysical Interaction Studies:

• Surface Plasmon Resonance (SPR) to measure binding kinetics and affinity

• Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters of binding

• Microscale Thermophoresis (MST) for quantitative analysis of interactions in solution

2. Structural Biology Approaches:

• X-ray crystallography of ileS-tRNA complexes to reveal atomic-level details

• Cryo-electron microscopy for visualization of larger complexes

• NMR spectroscopy for dynamic interaction studies in solution

3. Molecular Biology Techniques:

• Site-directed mutagenesis to identify critical residues in both ileS and tRNA

• Crosslinking assays to capture transient interactions

• Gel mobility shift assays to observe complex formation

4. Computational Methods:

• Molecular docking to predict binding conformations

• Molecular dynamics simulations to study the dynamics of the interaction

• Sequence and structural comparisons across different Acinetobacter species

5. Functional Assays:

• Aminoacylation assays with modified tRNAs to probe recognition elements

• Competition assays with tRNA variants to determine specificity

• In vivo complementation studies using modified ileS or tRNA genes

Through combining these approaches, researchers can develop a comprehensive understanding of the molecular determinants governing ileS-tRNA recognition and the species-specific variations that might exist across different Acinetobacter species. This knowledge could potentially inform strategies for targeting these interactions in drug discovery efforts against pathogenic Acinetobacter strains .

What are the common challenges in expressing and purifying functional recombinant Acinetobacter sp. ileS, and how can they be overcome?

Researchers frequently encounter several challenges when working with recombinant Acinetobacter sp. ileS. These challenges and their solutions include:

Challenge 1: Poor Solubility and Inclusion Body Formation

• Solutions:

  • Lower expression temperature (16-20°C) to slow protein synthesis and improve folding

  • Use solubility-enhancing fusion partners (SUMO, MBP, or Thioredoxin)

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Optimize induction conditions (reduced IPTG concentration, 0.1-0.5 mM)

  • Consider refolding protocols if inclusion bodies persist

Challenge 2: Low Enzymatic Activity

• Solutions:

  • Ensure proper cofactor inclusion (Mg2+, K+, Zn2+) in purification buffers

  • Add stabilizing agents (glycerol 5-10%, reducing agents like DTT or β-mercaptoethanol)

  • Screen buffer conditions to identify optimal pH and salt concentration

  • Maintain low temperature throughout purification to prevent denaturation

  • Consider tag removal if the affinity tag interferes with activity

Challenge 3: Proteolytic Degradation

• Solutions:

  • Use protease-deficient expression strains (BL21(DE3) pLysS)

  • Include protease inhibitors in all buffers

  • Minimize processing time and maintain cold temperatures

  • Optimize construct design to remove flexible, protease-susceptible regions

Challenge 4: Low Yield

• Solutions:

  • Optimize codon usage for the expression host

  • Use strong promoters and high-copy-number plasmids

  • Explore alternative expression hosts (different E. coli strains or yeast systems)

  • Implement auto-induction media or fed-batch cultivation

  • Scale-up production using bioreactors with controlled conditions

The purified protein should achieve purity levels exceeding 80% as determined by SDS-PAGE, with typical yields of 5-15 mg/L of culture when expressed in E. coli systems under optimized conditions .

How can molecular evolution and comparative genomics approaches enhance our understanding of ileS function in Acinetobacter species?

Molecular evolution and comparative genomics approaches provide powerful frameworks for understanding ileS function and evolution across Acinetobacter species:

1. Phylogenetic Analysis:

• Construction of ileS gene trees across Acinetobacter species reveals evolutionary relationships

• Comparison with species trees based on other markers (rpoB, 16S rRNA) identifies potential horizontal gene transfer events

• Analysis of selection pressures (dN/dS ratios) identifies functionally important residues

2. Comparative Genomics Strategies:

• Whole genome sequencing and comparative analysis of clinical and environmental Acinetobacter isolates

• Identification of syntenic regions surrounding the ileS gene to trace genomic context evolution

• Analysis of ileS copy number and paralogous genes across species

3. Structure-Function Correlation:

• Mapping of sequence conservation onto structural models to identify functional domains

• Identification of species-specific variations in substrate binding pockets

• Correlation of structural features with enzymatic properties and antibiotic resistance profiles

4. Experimental Validation:

• Site-directed mutagenesis of conserved vs. variable residues to assess functional importance

• Domain swapping between ileS from different species to determine species-specific functions

• Heterologous expression and functional complementation studies

Research has shown that while most Acinetobacter species contain a single ileS gene, sequence variations correlate with phylogenetic relationships established by other methods such as rpoB gene sequencing. The Acinetobacter calcoaceticus-baumannii complex species show 85.5% similarity in their non-A. baumannii isolates, reflecting their close evolutionary relationship. Such analyses have also revealed potential novel Acinetobacter species, such as "genomic species 33YU" identified through rpoB sequencing approaches .

What are the most effective approaches for studying ileS inhibition as a potential antimicrobial strategy against drug-resistant Acinetobacter strains?

Studying ileS inhibition as an antimicrobial strategy against drug-resistant Acinetobacter strains requires a multifaceted approach:

1. High-Throughput Screening Strategies:

• Development of cell-free enzymatic assays suitable for large compound libraries

• Whole-cell screening using reporter systems linked to ileS function

• Fragment-based screening to identify novel chemical scaffolds with inhibitory potential

2. Structure-Based Drug Design:

• Crystallographic studies of Acinetobacter ileS alone and in complex with inhibitors

• Virtual screening against the ATP-binding pocket, amino acid binding site, or tRNA interaction surfaces

• Rational design of compounds targeting species-specific features of Acinetobacter ileS

3. Mechanism of Action Studies:

• Biochemical characterization of inhibition kinetics (competitive, non-competitive, uncompetitive)

• Mode of binding studies using biophysical techniques (ITC, SPR, X-ray crystallography)

• Resistance development studies to identify potential escape mutations

4. Antimicrobial Evaluation Pipeline:

• Determination of minimal inhibitory concentrations (MICs) against clinical Acinetobacter isolates

• Time-kill kinetics to assess bactericidal vs. bacteriostatic activity

• Synergy testing with existing antibiotics

• Assessment of resistance development frequency

5. Specificity and Toxicity Assessment:

• Counter-screening against human aminoacyl-tRNA synthetases

• Cell culture toxicity assays

• Pharmacokinetic and pharmacodynamic profiling of lead compounds

This approach is particularly promising given the significant differences in antibiotic resistance profiles between Acinetobacter species. While A. baumannii isolates show high resistance rates to carbapenems (imipenem/meropenem), non-A. baumannii isolates demonstrate much lower resistance rates (approximately 2.6%), suggesting species-specific variations in drug susceptibility that could be exploited through targeted ileS inhibition strategies .

What are the future research directions for Acinetobacter sp. ileS and its potential applications?

Future research on Acinetobacter sp. ileS is likely to pursue several promising directions:

• Structural and Functional Characterization: Complete structural determination of Acinetobacter sp. ileS through X-ray crystallography or cryo-EM, focusing on species-specific features that might be exploited for selective inhibition.

• Antimicrobial Development: Design of novel ileS inhibitors specifically targeting pathogenic Acinetobacter species, with optimization for selectivity, potency, and reduced resistance development potential.

• Systems Biology Integration: Investigation of ileS in the context of the broader translational machinery and metabolic networks of Acinetobacter species to understand its role in bacterial physiology and stress response.

• Species Identification Tools: Development of ileS-based molecular diagnostics for rapid and accurate identification of Acinetobacter species in clinical settings, potentially replacing or complementing rpoB sequencing approaches.

• Resistance Mechanisms: Exploration of the molecular mechanisms underlying the development of resistance to ileS inhibitors, including genetic and biochemical adaptations.

• Comparative Analysis: Expansion of comparative studies across more Acinetobacter species and strains to better understand the evolution and adaptation of this essential enzyme across diverse ecological niches.

• Biotechnological Applications: Investigation of Acinetobacter ileS for potential biotechnological applications, including its use in cell-free protein synthesis systems or as a tool for incorporating non-canonical amino acids.