Recombinant Chromobacterium violaceum Proline--tRNA ligase (proS), partial

Basic Research Questions

• What is the genomic context and fundamental characteristics of Proline--tRNA ligase in Chromobacterium violaceum?

Proline--tRNA ligase (ProS) in Chromobacterium violaceum ATCC 12472 is encoded within the complete genome that was sequenced by The Brazilian National Genome Project Consortium . This enzyme belongs to the class II aminoacyl-tRNA synthetase family and catalyzes the attachment of proline to its cognate tRNA in a two-step reaction:

Step 1: Proline + ATP → Prolyl-AMP + PPi
Step 2: Prolyl-AMP + tRNAPro → Prolyl-tRNAPro + AMP

The enzyme exists as a typical class II aminoacyl-tRNA synthetase with characteristic domains for ATP binding, proline recognition, and tRNA interaction. As a critical component of the protein synthesis machinery, ProS ensures translational fidelity by specifically charging tRNA molecules with proline. The gene is located within the context of other protein synthesis genes in the C. violaceum genome, which has been thoroughly characterized through genomic analysis .

• What expression systems are most effective for producing recombinant C. violaceum Proline--tRNA ligase?

The optimization of expression systems for recombinant C. violaceum ProS requires careful consideration of several factors:

Methodological approach for optimal expression:

• Clone the proS gene from C. violaceum ATCC 12472 genomic DNA using high-fidelity PCR

• Design primers with appropriate restriction sites for directional cloning

• Insert into an expression vector (pET28a or similar) with an N-terminal His6-tag

• Transform into E. coli BL21(DE3)

• Culture at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.5 mM IPTG

• Shift temperature to 18°C for 16-18 hours to maximize soluble protein production

This approach leverages established expression systems while addressing the specific characteristics of C. violaceum proteins, which may have different folding requirements compared to E. coli proteins.

• What purification strategies yield high-purity recombinant C. violaceum Proline--tRNA ligase for structural and functional studies?

A multi-step purification approach is necessary to obtain high-purity C. violaceum ProS suitable for structural and functional characterization:

Detailed methodological protocol:

• Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM DTT, and protease inhibitors

• Apply clarified lysate to Ni-NTA column pre-equilibrated with lysis buffer

• Wash with increasing imidazole concentrations (20 mM, 40 mM) to remove non-specifically bound proteins

• Elute with 250-300 mM imidazole

• If tag removal is desired, incubate with TEV protease (1:50 ratio) during overnight dialysis to reduce salt concentration

• Apply to ion exchange column (Q-Sepharose) and elute with salt gradient

• Concentrate and apply to Superdex 200 size exclusion column

• For nucleic acid-binding studies, include a heparin affinity step to remove any bound nucleic acids

This purification scheme typically yields protein with >95% purity as assessed by SDS-PAGE, suitable for crystallography, enzyme kinetics, and structural biology applications.

• What methods are recommended for assessing the aminoacylation activity of purified recombinant C. violaceum Proline--tRNA ligase?

Several complementary methods can be employed to evaluate the aminoacylation activity of purified C. violaceum ProS:

Standard aminoacylation assay protocol:

• Prepare reaction mixture containing 100 mM HEPES-KOH pH 7.5, 10 mM MgCl₂, 50 mM KCl, 5 mM DTT, 4 mM ATP, 50 μM [³H]-proline, 10 μM tRNAPro, and purified enzyme (10-100 nM)

• Incubate at 37°C and remove aliquots at different time points (0-10 minutes)

• Spot aliquots on Whatman filter paper pre-soaked with 5% trichloroacetic acid

• Wash filters extensively with 5% TCA to remove unincorporated [³H]-proline

• Quantify incorporated radioactivity by liquid scintillation counting

• Calculate initial velocities and specific activity (nmol/min/mg protein)

These methods provide complementary approaches to characterize the enzymatic activity of C. violaceum ProS, enabling researchers to select the most appropriate technique based on available equipment and specific research questions.

• How does C. violaceum Proline--tRNA ligase compare with homologous enzymes from other bacterial species?

Comparative analysis of C. violaceum ProS with homologs from other bacterial species reveals important evolutionary and functional insights:

*Based on comparative analysis with related species, as C. violaceum is a member of the Neisseriaceae family of Betaproteobacteria

Methodological approaches for comprehensive comparison:

• Multiple sequence alignment using MUSCLE or CLUSTALW algorithms

• Phylogenetic analysis using maximum likelihood methods

• Homology modeling based on available crystal structures

• Structural superposition to identify conserved catalytic residues

• Comparative biochemical analysis of substrate specificities and kinetic parameters

• Analysis of species-specific insertions or deletions that may affect function

C. violaceum ProS likely shares core catalytic mechanisms with other bacterial ProRS enzymes while possessing unique features related to its adaptation to the organism's environmental niche in tropical and subtropical regions . Understanding these differences can provide insights into evolutionary adaptation of aminoacyl-tRNA synthetases across bacterial species.

Advanced Research Questions

• What role might C. violaceum Proline--tRNA ligase play in bacterial pathogenesis and virulence?

While direct evidence for ProS involvement in C. violaceum pathogenesis is limited, several compelling connections can be explored:

C. violaceum is an opportunistic pathogen capable of causing fatal infections in humans and animals, with cases of septicemia reported in medical literature . The organism possesses several virulence factors, most notably two Type III Secretion Systems (T3SSs) encoded by Chromobacterium pathogenicity islands Cpi-1/1a and Cpi-2 .

Potential roles of ProS in pathogenesis include:

• Regulation of Virulence Factor Translation:

  • ProS activity may modulate translation efficiency of virulence factors during infection

  • The T3SS components require precise temporal expression, potentially regulated at the translational level

• Stress Adaptation During Infection:

  • ProS may function differently under host-associated stress conditions

  • Altered aminoacylation efficiency could enable rapid adaptation to the host environment

• Potential Moonlighting Functions:

  • Similar to other aminoacyl-tRNA synthetases, ProS may have non-canonical functions

  • These could include signaling roles or direct interactions with host components

Experimental approaches to investigate these hypotheses:

• Generate conditional ProS mutants and evaluate virulence in mouse infection models

• Examine protein synthesis patterns during infection using ribosome profiling

• Assess ProS expression during different stages of infection

• Test for direct interactions between ProS and components of the T3SS machinery

The Cpi-1/1a T3SS has been demonstrated as a major virulence determinant for C. violaceum , and understanding ProS's potential regulatory role could provide new insights into pathogenesis mechanisms.

• How can structural studies of C. violaceum Proline--tRNA ligase inform drug development against bacterial infections?

Structural studies of C. violaceum ProS can provide valuable insights for antimicrobial development:

Experimental strategy for structure-based drug discovery:

• Solve high-resolution crystal structures of C. violaceum ProS in multiple states:

  • Apo-enzyme

  • Enzyme-ATP complex

  • Enzyme-proline complex

  • Enzyme-prolyl-AMP complex

  • Enzyme-tRNA complex

• Identify druggable pockets through computational analysis:

  • Molecular dynamics simulations to identify transient pockets

  • Fragment-based screening against crystallographic structures

  • Virtual screening of compound libraries

• Develop and validate binding assays:

  • Thermal shift assays to detect stabilizing compounds

  • Surface plasmon resonance for binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• Establish structure-activity relationships:

  • Medicinal chemistry optimization of hit compounds

  • Co-crystallization with promising inhibitors

  • Activity assays against C. violaceum and other pathogenic bacteria

Such approaches could potentially lead to new antimicrobial compounds effective against C. violaceum infections, which are often fatal and resistant to many conventional antibiotics . Additionally, targeting aminoacyl-tRNA synthetases represents an established antimicrobial strategy with precedents in clinical use.

• What experimental approaches can determine whether C. violaceum Proline--tRNA ligase possesses editing activity against misacylated tRNAs?

Determining the editing activity of C. violaceum ProS requires specialized methodological approaches:

Detailed post-transfer editing assay protocol:

• Prepare mischarged [³H]-Ala-tRNAPro using an editing-deficient ProRS variant

• Purify the mischarged tRNA by phenol extraction and ethanol precipitation

• Incubate purified mischarged tRNA (1 μM) with wild-type C. violaceum ProS (100 nM)

• At various time points (0-30 minutes), take aliquots and precipitate tRNA with TCA

• Measure the decrease in acid-precipitable radioactivity over time

• Calculate deacylation rate constants (kdeacyl) for the editing reaction

• Compare with deacylation rates for correctly charged Prolyl-tRNAPro (control)

Editing activity is critical for preventing mistranslation of proline codons with alanine or cysteine, which could be particularly important during C. violaceum infection processes. Given C. violaceum's environmental versatility and pathogenic potential , its ProS may have evolved specific editing mechanisms to maintain translational fidelity under diverse conditions.

• How might post-translational modifications regulate C. violaceum Proline--tRNA ligase activity during environmental adaptation?

Post-translational modifications (PTMs) of C. violaceum ProS could serve as regulatory mechanisms during environmental adaptation:

Comprehensive experimental approach:

C. violaceum's ability to adapt to diverse environments, from soil and water to human hosts , suggests sophisticated regulatory mechanisms may control its core physiological processes. Understanding how ProS activity is modulated through PTMs could reveal critical adaptation mechanisms relevant to both environmental persistence and pathogenicity.

• What role might C. violaceum Proline--tRNA ligase play in bacterial stress responses, and how can this be experimentally validated?

Investigating the potential role of C. violaceum ProS in stress responses requires targeted experimental approaches:

Comprehensive experimental design:

• Generate conditional ProS mutants in C. violaceum:

  • Construct strains with ProS under inducible promoter control

  • Create point mutations in key functional domains

  • Develop depletion systems to reduce ProS levels

• Expose these strains to various stresses and assess:

  • Growth kinetics under stress conditions

  • Global translation rates using puromycin incorporation

  • Specific stress response protein synthesis

  • Mistranslation frequency using reporter systems

  • Virulence factor expression and secretion

• Monitor ProS expression and modifications:

  • Quantitative RT-PCR for transcriptional regulation

  • Western blotting for protein levels and modifications

  • Fluorescent tagging for subcellular localization during stress

• Assess impact on pathogenicity:

  • Mouse infection models comparing wild-type and ProS variant strains

  • Ex vivo macrophage infection assays

  • T3SS functionality tests with ProS depletion

C. violaceum's remarkable adaptability to diverse environmental conditions and its capacity to switch from a free-living to pathogenic lifestyle suggest sophisticated stress response mechanisms. ProS may function as both an essential housekeeping enzyme and a regulatory component in stress adaptation networks, particularly in coordinating protein synthesis with environmental challenges.