Recombinant Chromobacterium violaceum tRNA pseudouridine synthase A (truA)

What is Chromobacterium violaceum and why is it significant as a source for truA?

Chromobacterium violaceum is a Gram-negative bacillus commonly found in soil and water in tropical and subtropical regions worldwide. It's primarily known for producing a distinctive purple pigment called violacein, which confers antibiotic-inhibiting properties and is thought to contribute to the organism's virulence . While C. violaceum rarely causes disease in humans, it has a high fatality rate when infection occurs, making it an interesting organism for pathogenicity studies .

The significance of studying C. violaceum truA lies in understanding RNA modification mechanisms in an organism with unique biochemical properties. C. violaceum belongs to a genus with nine recognized species, including C. subtsugae, C. aquaticum, C. haemolyticum, and others . Its biochemical characteristics are similar to those of Pseudomonas and Aeromonas species, contributing to its primary multi-drug resistance characteristics that present treatment challenges .

What expression systems are most effective for recombinant C. violaceum truA production?

Escherichia coli expression systems are most commonly used for recombinant protein production of bacterial enzymes like truA. Research analyzing 11,430 recombinant protein expression experiments in E. coli reveals that success rates can be optimized by considering mRNA accessibility around translation initiation sites .

For expressing C. violaceum proteins specifically, methodological considerations include:

• Vector selection: pET expression systems with T7 promoters typically yield high expression levels for bacterial enzymes like truA.

• Codon optimization: Given that approximately 50% of recombinant proteins fail to express in host cells, codon optimization is crucial . The TIsigner web application (https://tisigner.com/tisigner) can be used to optimize translation initiation sites through synonymous codon changes within the first nine codons .

• Host strain selection: BL21(DE3) and its derivatives are generally suitable, though C. violaceum proteins may benefit from strains designed for toxic protein expression.

• Expression conditions: Lower temperatures (16-25°C) often improve solubility of enzymes like truA, with induction at mid-log phase using reduced IPTG concentrations (0.1-0.5 mM).

What antibiotic resistance properties of C. violaceum might affect cloning strategies?

Chromobacterium violaceum exhibits a distinctive antibiotic resistance profile that must be considered when designing cloning strategies. According to research, C. violaceum is resistant to:

• Ampicillin

• Penicillin

• Rifampicin

• Erythromycin

• Vancomycin

• Ethidium bromide

• Gentamicin

• Tetracycline

• Chloramphenicol

• Cotrimoxazole

• Kanamycin

• Streptomycin

• Nalidixic acid

A comprehensive antibiotic susceptibility profile is presented in Table 1:

Table 1: Antibiotic Susceptibility Profile of Chromobacterium violaceum

When designing cloning strategies, selection markers for plasmids should align with C. violaceum's susceptibility profile. Kanamycin or gentamicin resistance genes are recommended for selection markers, while ampicillin-based selection should be avoided .

How can accessibility of translation initiation sites predict successful expression of recombinant C. violaceum truA?

Translation initiation site accessibility is a critical determinant of recombinant protein expression success. Analysis of 11,430 recombinant protein expression experiments revealed that the accessibility of translation initiation sites, modeled using mRNA base-unpairing across Boltzmann's ensemble, significantly outperforms alternative features in predicting expression success .

Methodological approach for optimizing C. violaceum truA expression:

• Calculate opening energies: Analyze the region from -24 to +24 nucleotides relative to the initiation codon. An opening energy of 10 kcal/mol or below in this region is approximately twice as likely to result in successful expression .

• Apply TIsigner optimization: Use the TIsigner tool to optimize the coding sequence through synonymous substitutions in the first nine codons, which can dramatically improve accessibility without changing the protein sequence .

• Consider full ensemble average energy: Unlike minimum free energy (MFE) approaches, accessibility calculations capture the full ensemble average energy of sequences. This approach uses a 210-nucleotide region surrounding the translation initiation site (-24:24) to capture key propensities beyond the immediate region .

The relationship between opening energy and expression success follows a sigmoidal pattern with defined upper and lower bounds on translation initiation rates. Figure 1 demonstrates how accessibility calculations differ from traditional MFE predictions:

Figure 1: Accessibility vs. MFE for Translation Initiation Prediction

• Region analyzed: -24 to +24 nt relative to start codon

• Opening energy threshold: ≤10 kcal/mol indicates higher expression probability

• Area under ROC curve: 0.70-0.78 for accessibility vs. 0.55-0.60 for MFE

Experimental validation confirms that optimizing accessibility leads to higher protein production, though this may occur at the expense of slower cell growth due to the protein cost where cell growth is constrained during overexpression .

What strategies can resolve contradictory findings in C. violaceum truA research?

Contradictions in research findings are common in biomedical literature and require systematic approaches for resolution. A study analyzing contradictions in biomedical research identified 58 apparent contradictions from 2,236 candidate contradictory pairs . To resolve contradictions in C. violaceum truA research, consider the following methodological approach:

• Systematic literature review: Extract semantic predications (subject-relation-object triples) from the literature to identify potentially contradictory claims .

• Categorize contextual characteristics: Classify contradictions into five main categories:

  • Internal to the experimental system (e.g., strain differences)

  • External to the experimental system (e.g., environmental conditions)

  • Endogenous/exogenous factors

  • Known controversies in the field

  • True contradictions in literature

• Experimental design to address contradictions: When contradictory findings emerge, design experiments that explicitly test both hypotheses simultaneously with appropriate controls.

Table 2: Contradictions Resolution Framework for C. violaceum truA Research

When applying this framework to C. violaceum truA research, particular attention should be paid to the impact of the violacein pigment, which may interfere with spectrophotometric assays commonly used for enzyme activity measurements .

What are the optimal methods to assay C. violaceum truA activity in vitro?

To effectively assay the activity of recombinant C. violaceum truA in vitro, researchers should employ a combination of approaches that account for the unique properties of the enzyme and potential interference from violacein pigment.

Primary Activity Assay Methods:

• Radioactive assay using [³H]UTP-labeled tRNA substrates:

  • Incubate purified truA with [³H]UTP-labeled tRNA

  • Digest the tRNA to nucleosides using nuclease P1 and alkaline phosphatase

  • Separate nucleosides by HPLC

  • Quantify pseudouridine formation by scintillation counting

• CMCT-primer extension assay:

  • Treat tRNA with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT)

  • CMCT specifically modifies pseudouridine

  • Perform reverse transcription with labeled primers

  • Analyze resulting cDNA by gel electrophoresis

  • Stops at modified positions indicate pseudouridine sites

• LC-MS/MS analysis:

  • Digest tRNA substrate to nucleosides

  • Analyze by liquid chromatography-tandem mass spectrometry

  • Monitor specific transitions for uridine and pseudouridine

  • Calculate pseudouridine/uridine ratios to quantify activity

Special Considerations for C. violaceum truA:

• Control for potential interference from violacein pigment, which has a deep purple color and may interfere with spectrophotometric assays

• Include purification steps using ion-exchange chromatography to ensure removal of violacein from enzyme preparations

• Consider using non-pigmented variants of C. violaceum or heterologous expression systems to avoid pigment interference

• Validate activity in multiple buffer systems, as C. violaceum proteins may have different pH optima compared to model organisms

How does the multi-drug resistance of C. violaceum impact purification strategies for recombinant truA?

The inherent multi-drug resistance of Chromobacterium violaceum presents unique challenges for recombinant protein purification strategies. Research indicates that C. violaceum with dark violet color (due to violacein) exhibits resistance to various antibiotics including vancomycin, ampicillin, and linezolid, while remaining susceptible to colistin, oxacillin, gentamicin, norfloxacin, chloramphenicol, and amikacin .

Methodological approaches to address these challenges:

• Selection of appropriate expression hosts: Using E. coli BL21(DE3) or similar strains for heterologous expression circumvents the antibiotic resistance issues of native C. violaceum.

• Affinity tag design considerations:

  • His-tag purification may be complicated by violacein binding to nickel columns

  • Alternative tags such as GST or MBP can provide better specificity

  • Consider dual tagging strategies (His-tag plus a second affinity tag)

• Specialized purification protocol:

  Table 3: Optimized Purification Protocol for C. violaceum truA

  Step	Method	Conditions	Special Considerations
  Cell lysis	Sonication or French press	In 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol	Include additional protease inhibitors due to C. violaceum proteases
  Clarification	Centrifugation	20,000 × g, 30 min, 4°C	Multiple clarification steps to remove violacein
  Capture	Affinity chromatography	Based on chosen tag (His, GST, etc.)	Include 1% Triton X-100 in initial washes to remove violacein
  Intermediate	Ion exchange	MonoQ column, pH 8.0, salt gradient	Separates different forms of truA and removes pigment
  Polishing	Size exclusion	Superdex 200, 20 mM HEPES pH 7.5, 150 mM NaCl	Ensures homogeneity of final preparation
  Quality control	Activity assay + SDS-PAGE	Standard conditions	Verify absence of violacein by absorbance at 575 nm

• Violacein removal strategies:

  • Ethanol precipitation steps can selectively precipitate proteins while keeping violacein in solution

  • Hydrophobic interaction chromatography can separate violacein-bound protein fractions

  • Activated charcoal treatment can remove residual violacein but may reduce protein yield

• Storage considerations:

  • Include reducing agents (5 mM DTT or 2 mM β-mercaptoethanol) to prevent oxidation

  • Add glycerol (20-30%) to prevent freeze-thaw damage

  • Aliquot and flash-freeze in liquid nitrogen for long-term storage

The presence of violacein can affect downstream applications, particularly spectrophotometric assays. Therefore, additional quality control steps to verify the absence of violacein contamination are essential for accurate enzyme characterization .

What structural and functional aspects of C. violaceum truA contribute to RNA modification specificity?

tRNA pseudouridine synthase A (truA) catalyzes the isomerization of uridine to pseudouridine at positions 38, 39, and 40 in the anticodon stem and loop of transfer RNAs. While specific structural data for C. violaceum truA is limited, comparative analysis with truA from other bacterial species provides insights into its likely structural and functional properties.

Key structural elements expected in C. violaceum truA:

• Catalytic domain: Contains the active site with conserved aspartic acid residue essential for catalysis

• RNA-binding domain: Includes positively charged residues that interact with the negatively charged RNA backbone

• Loop structures: Flexible loops that recognize specific features of the anticodon stem-loop

• Dimer interface: truA typically functions as a homodimer, with the interface contributing to substrate specificity

Factors affecting substrate specificity:

• tRNA recognition elements: Specific nucleotides in tRNA substrates that are recognized by truA

• Conformational changes: Induced-fit mechanisms that occur upon tRNA binding

• Sequence conservation: Comparison with other bacterial truA enzymes suggests conservation of key residues

Methodological approaches to study C. violaceum truA structure-function relationships:

• Homology modeling: Generate structural models based on crystal structures of truA from other organisms

• Site-directed mutagenesis: Systematically mutate conserved residues to assess their role in:

  • Catalysis

  • Substrate binding

  • Specificity for positions 38-40

  • Dimer formation

• Chimeric enzyme construction: Create chimeric enzymes with domains from different pseudouridine synthases to identify specificity determinants

• Cross-linking studies: Identify RNA-protein contact points using UV cross-linking followed by mass spectrometry

• NMR analysis: Investigate dynamic aspects of enzyme-substrate interactions in solution

Table 4: Predicted Conserved Motifs in C. violaceum truA Based on Comparative Analysis

Understanding these structural and functional aspects is essential for engineering C. violaceum truA with altered specificity or enhanced activity for biotechnological applications.