Recombinant Chromobacterium violaceum Isoleucine--tRNA ligase (ileS), partial

What is Chromobacterium violaceum and why is its ileS enzyme significant for research?

Chromobacterium violaceum is a Gram-negative bacterium abundant in soil and water ecosystems of tropical and subtropical regions worldwide. It is primarily known for producing violacein, a purple pigment with antimicrobial properties, and as an environmental opportunistic pathogen that can cause severe, often fatal infections in humans and animals .

The isoleucine-tRNA ligase (ileS) from C. violaceum is significant for research because:

• It catalyzes the ATP-dependent ligation of isoleucine to tRNA^Ile, a crucial step in protein synthesis that ensures translation fidelity

• It belongs to the aminoacyl-tRNA synthetase (ARS) family, which plays essential roles in all living organisms

• Understanding ileS function contributes to knowledge about C. violaceum's adaptability and potential pathogenicity

• Recombinant forms of the enzyme enable detailed structural and functional studies without requiring large cultures of potentially pathogenic bacteria

The enzyme is particularly valuable for comparative studies with other bacterial ARS enzymes and for investigating potential antimicrobial targets, as proper tRNA charging is essential for bacterial survival .

How does C. violaceum ileS compare structurally and functionally to other bacterial isoleucyl-tRNA synthetases?

C. violaceum ileS shares the fundamental catalytic functions of other bacterial isoleucyl-tRNA synthetases while exhibiting species-specific characteristics:

Structural Comparison:

• Like other bacterial ileS enzymes, C. violaceum ileS contains conserved domains for ATP binding, isoleucine recognition, and tRNA interaction

• The C. violaceum enzyme likely possesses the typical modular architecture with N-terminal catalytic domain, editing domain, and C-terminal anticodon-binding domain

• Recombinant partial forms typically retain the catalytic domains necessary for substrate binding and aminoacylation while potentially lacking non-essential regions

Functional Comparison:

• The core catalytic function of attaching isoleucine to its cognate tRNA is preserved across species

• C. violaceum ileS contains the editing function critical for distinguishing between isoleucine and the structurally similar valine

• The enzyme is expected to participate in the multi-enzyme synthetase complexes common in bacteria, similar to the IARS component recognized by anti-OJ antibodies

Researchers should note that C. violaceum has 19 aminoacyl-tRNA synthetases in total, with additional aminoacyl-tRNA synthetase-related proteins, suggesting unique adaptations in its translation machinery .

What expression systems are most suitable for producing recombinant C. violaceum ileS?

Based on experimental approaches with similar enzymes and other C. violaceum proteins, the following expression systems are recommended for recombinant C. violaceum ileS production:

E. coli Expression System (Preferred):

• BL21(DE3) strain with pET vector systems (particularly pET303/CT-His) has proven effective for other C. violaceum recombinant proteins

• IPTG induction typically yields high levels of expression

• The inclusion of a C-terminal His-tag facilitates purification via affinity chromatography

• Optimal expression temperature is typically 30°C to balance yield and solubility

Alternative Expression Systems:

• In vitro transcription/translation systems can be used for quick production of biotinylated recombinant proteins, particularly useful for ELISA development and other immunological assays

• Cold-adapted expression hosts may improve solubility, as demonstrated with other tRNA synthetases from psychrophilic organisms

For secreted expression, include the native signal peptide, which has been shown to function effectively in E. coli, as demonstrated with other C. violaceum enzymes . After expression, purification via affinity chromatography (His-tag or substrate-based matrices) is typically effective.

How can researchers optimize purification protocols for recombinant C. violaceum ileS to maintain enzymatic activity?

Optimizing purification protocols for recombinant C. violaceum ileS requires careful consideration of multiple factors to preserve the enzyme's native conformation and catalytic activity:

Recommended Purification Strategy:

• Cell Lysis Optimization:

  • Use gentle lysis buffers (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol)

  • Add protease inhibitors to prevent degradation

  • Include 1-5 mM DTT or 2-mercaptoethanol to maintain thiol groups

  • Perform lysis at 4°C to minimize denaturation

• Affinity Chromatography:

  • For His-tagged constructs, use Ni-NTA or TALON resins with imidazole gradient elution

  • Alternatively, substrate affinity purification using tRNA^Ile-coupled resins provides higher specificity

  • Consider chitin matrix affinity chromatography, which has proven effective for other C. violaceum enzymes

• Additional Purification Steps:

  • Ion exchange chromatography (typically Q-Sepharose) at pH 8.0

  • Size exclusion chromatography in 25 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl₂, 10% glycerol

Activity Preservation Measures:

• Include 5-10 mM MgCl₂ in all buffers as it's essential for synthetase activity

• Add 1-2 mM ATP to stabilize the enzyme's conformation

• Maintain 10-15% glycerol in storage buffers to prevent freezing damage

• Store purified enzyme at -80°C in small aliquots to avoid freeze-thaw cycles

Researchers should validate enzyme activity after purification using aminoacylation assays with radiolabeled isoleucine or more modern high-throughput spectrophotometric pyrophosphate detection methods.

What methodological approaches can be used to study the role of C. violaceum ileS in bacterial pathogenicity?

Investigating C. violaceum ileS's potential role in pathogenicity requires multidisciplinary approaches that connect translation fidelity with virulence mechanisms:

Genetic Manipulation Approaches:

• Generate conditional ileS mutants using CRISPR-Cas9 or transposon mutagenesis systems

• Create point mutations in the editing domain to induce mistranslation stress

• Develop fluorescent protein fusions to track ileS localization during infection stages

Functional Assays:

• Measure mistranslation rates under infection-relevant conditions using reporter systems

• Compare aminoacylation efficiency between virulent and avirulent strains

• Assess changes in ileS activity during biofilm formation, which is essential for C. violaceum pathogenicity

Infection Models:

• Use established mouse infection models for C. violaceum, focusing on the connection between translation fidelity and virulence

• Monitor changes in ileS expression during interaction with host cells

• Investigate potential interactions between ileS and the type III secretion systems (T3SSs), which play pivotal roles in C. violaceum virulence

Table 1: Experimental Methods for Studying ileS Role in Pathogenicity

Since C. violaceum infections can lead to fatal sepsis, researchers should investigate whether ileS activity affects toxin production or bacterial adaptation to host environments .

How can researchers effectively design experiments to study the interaction between C. violaceum ileS and quorum sensing systems?

C. violaceum is well-known for its quorum sensing (QS) system, which regulates various physiological processes including violacein production and biofilm formation . Designing experiments to investigate potential interactions between ileS and QS requires careful planning:

Experimental Design Approach:

• Expression Analysis Under QS Manipulation:

  • Cultivate C. violaceum wild-type strain ATCC 31532 and its QS-deficient mutant NCTC 13274 with and without N-hexanoyl-L-homoserine lactone (C6-HSL)

  • Quantify ileS expression using RT-qPCR and Western blotting

  • Compare ileS enzymatic activity in cell extracts from QS-positive and QS-negative conditions

• Biofilm-Translation Relationship Studies:

  • Develop dual-reporter systems to simultaneously monitor biofilm formation and translation accuracy

  • Use atomic force microscopy (AFM) to examine morphological changes in bacterial cells while tracking ileS localization

  • Assess whether biofilm matrix components affect ileS aminoacylation efficiency

• Stress Response Integration:

  • Introduce controlled mistranslation by manipulating ileS activity

  • Monitor effects on C6-HSL production and QS-dependent gene expression

  • Determine whether translation stress triggers QS-dependent adaptations

Advanced Methods for Deeper Mechanistic Insights:

• Ribosome profiling to assess translation efficiency under different QS states

• Chromatin immunoprecipitation (ChIP) to identify potential regulatory interactions between QS transcription factors and ileS gene

• Metabolomics to detect changes in amino acid pools that might affect both QS signaling and ileS function

The experimental approach should account for C. violaceum's morphological differentiation during biofilm development, which involves membrane invaginations and polymer matrix extrusions directed by QS autoinducers .

What are the critical factors to consider when developing an ELISA assay using recombinant C. violaceum ileS?

Developing a reliable ELISA assay using recombinant C. violaceum ileS requires careful consideration of several critical factors to ensure specificity, sensitivity, and reproducibility:

Protein Production and Quality Control:

• Express recombinant C. violaceum ileS using in vitro transcription/translation systems to produce biotinylated protein, following methodologies proven successful with other aminoacyl-tRNA synthetases

• Verify protein integrity via SDS-PAGE (aim for >85% purity) and Western blotting

• Confirm enzymatic activity through aminoacylation assays to ensure proper folding

Assay Development Considerations:

• Optimize coating conditions: 0.1-1.0 μg/well of recombinant ileS in carbonate buffer (pH 9.6) overnight at 4°C

• Block with 2-3% BSA in PBS to minimize background

• Use streptavidin-coated plates for biotinylated recombinant ileS to ensure proper orientation

• Develop a reference standard using immunoprecipitation-confirmed positive and negative samples

Validation Parameters:

• Establish analytical sensitivity and specificity using known positive and negative controls

• Determine reproducibility through intra-assay and inter-assay variation studies

• Perform cross-reactivity testing against other aminoacyl-tRNA synthetases, particularly KARS

Table 2: ELISA Optimization Parameters for Recombinant C. violaceum ileS Detection

When establishing cutoff values, researchers should aim for high specificity (>90%) while maintaining acceptable sensitivity, as demonstrated in successful ELISA development for other aminoacyl-tRNA synthetases such as KARS and IARS .

How can recombinant C. violaceum ileS be applied in structural biology studies?

Recombinant C. violaceum ileS presents valuable opportunities for structural biology investigations that can reveal fundamental insights into aminoacyl-tRNA synthetase mechanisms:

Crystallization Approaches:

• Screen crystallization conditions using vapor diffusion methods with PEG-based precipitants

• Consider co-crystallization with substrates (ATP, isoleucine, tRNA) to capture different functional states

• Employ surface entropy reduction mutations to enhance crystallizability while preserving catalytic function

• Use truncated constructs that retain core catalytic domains for initial crystallization attempts

Cryo-EM Studies:

• Generate ileS complexes with tRNA and other synthetases for single-particle cryo-EM analysis

• Apply GraFix method to stabilize multi-protein complexes for structural determination

• Investigate potential interactions with ribosomes or other translation machinery components

Structural Dynamics Investigations:

• Implement hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes during substrate binding

• Apply small-angle X-ray scattering (SAXS) to characterize solution behavior of full-length and truncated constructs

• Develop FRET-based assays using strategically placed fluorophores to monitor conformational changes during catalysis

Researchers should leverage the unusual morphological differentiation observed in C. violaceum to investigate potential structural adaptations of ileS that might contribute to the bacterium's environmental adaptability and pathogenicity.

What research methodologies can address the contradictory data on aminoacyl-tRNA synthetase inhibition as an antimicrobial strategy?

The field of aminoacyl-tRNA synthetase inhibition presents some contradictory findings that require nuanced methodological approaches to resolve:

Reconciling Contradictory Data:

• Implement parallel testing of ileS inhibitors against multiple bacterial species to identify species-specific effects

• Compare results from different assay systems (in vitro enzymatic, cell-based, infection models) to identify discrepancies

• Develop resistant mutants and characterize compensatory mechanisms that might explain clinical failures of synthetase inhibitors

Advanced Methodological Approaches:

• Apply chemical genetics using activity-based protein profiling to identify off-target effects of ileS inhibitors

• Implement systems biology approaches to map metabolic consequences of ileS inhibition

• Develop multi-omics pipelines to compare transcriptomic, proteomic, and metabolomic responses to ileS inhibition across different bacterial species

Novel Research Directions:

• Investigate the interplay between ileS inhibition and C. violaceum's violacein production, which has antibiotic-inhibiting properties

• Examine whether ileS inhibition affects the type III secretion systems crucial for C. violaceum virulence

• Develop combination strategies targeting both ileS and quorum sensing to simultaneously disrupt translation and virulence factor production

The violacein pigment produced by C. violaceum confers resistance to various antibiotics , suggesting that successful antimicrobial strategies might need to address both translation fidelity and pigment production pathways.