Recombinant Chromobacterium violaceum Threonine--tRNA ligase (thrS), partial

What is the genomic context of thrS in Chromobacterium violaceum?

Threonine--tRNA ligase (thrS) in C. violaceum ATCC 12472 is located within the bacterial chromosome. Based on genomic analyses, thrS is not part of the main Type VI Secretion System (T6SS) gene cluster which spans from CV_3963 to CV_3991 . Unlike the multiple VgrG genes that are distributed across the genome, thrS exists as a single-copy gene that is essential for protein synthesis. The gene encoding thrS is not found in close proximity to the quorum sensing regulatory genes cviI and cviR, which are genetically linked and control various phenotypes in C. violaceum .

How does thrS function in C. violaceum protein synthesis?

Threonine--tRNA ligase in C. violaceum catalyzes the ATP-dependent attachment of threonine to its cognate tRNA molecule (tRNAThr) during protein translation. This aminoacylation reaction is a two-step process:

• Activation of threonine with ATP to form threonyl-AMP

• Transfer of the threonyl group to the 3' end of tRNAThr

This process is critical for maintaining translational fidelity in C. violaceum, ensuring that threonine is correctly incorporated into proteins during synthesis. Unlike the T6SS components that are primarily involved in bacterial competition and virulence, thrS is an essential housekeeping enzyme required for basic cellular functions regardless of the bacterium's environmental context .

What are the structural characteristics of C. violaceum thrS?

C. violaceum thrS belongs to the class II aminoacyl-tRNA synthetase family, characterized by an antiparallel β-sheet core in its catalytic domain. The enzyme contains three distinct functional domains:

• N-terminal domain: Involved in tRNA anticodon recognition

• Catalytic domain: Contains the active site for threonine activation

• C-terminal domain: Facilitates tRNA binding and aminoacylation

Unlike the VgrG proteins in C. violaceum, which share 70-93% sequence identity and similar domain organization, thrS has a unique structure optimized for its specific aminoacylation function rather than bacterial competition .

What expression systems are most effective for producing recombinant C. violaceum thrS?

For optimal expression of recombinant C. violaceum thrS, E. coli-based systems have proven most effective, particularly BL21(DE3) strains containing pET vector systems with T7 promoters. When designing expression constructs, consider the following guidelines:

• Codon optimization: C. violaceum has a different codon usage bias than E. coli, which may necessitate codon optimization of the thrS gene for improved expression.

• Affinity tags: C-terminal 6xHis-tags typically yield better results than N-terminal tags, as they minimally interfere with the N-terminal tRNA binding domain.

• Induction conditions: Optimal expression is typically achieved at 18-20°C with 0.1-0.5 mM IPTG, reducing inclusion body formation often observed at higher temperatures.

This approach differs significantly from the methods used to study C. violaceum virulence factors, which often involve genetic manipulation of the native organism via plasmid-borne constructs as demonstrated in studies of the T6SS system using VipA-sfGFP fusions .

What purification strategies yield the highest activity for recombinant C. violaceum thrS?

Recommended Purification Protocol:

• Initial capture: Ni-NTA affinity chromatography using a linear imidazole gradient (20-250 mM)

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose)

• Polishing step: Size exclusion chromatography in a buffer containing:

  • 50 mM HEPES-KOH, pH 7.5

  • 100 mM KCl

  • 10 mM MgCl₂

  • 5 mM β-mercaptoethanol

  • 10% glycerol

Activity Preservation:

• Add 0.1 mM zinc acetate to all buffers as thrS contains a zinc-binding motif critical for structure and function

• Maintain temperature at 4°C throughout purification

• Include protease inhibitors in the lysis buffer to prevent degradation

This approach differs from techniques used to isolate native proteins from C. violaceum, such as the Hcp protein secretion analysis, which typically involves TCA precipitation of supernatants followed by immunoblotting .

What assays are most reliable for measuring C. violaceum thrS aminoacylation activity?

For accurate assessment of thrS aminoacylation activity, researchers should consider these methodologies:

Pyrophosphate Exchange Assay:

• Measures the first step of the aminoacylation reaction (amino acid activation)

• Higher throughput but less informative about complete tRNA charging

Direct Aminoacylation Assay:

• Measures attachment of [³H]-threonine to tRNA

• Reaction conditions: 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl₂, 50 mM KCl, 5 mM ATP, 1 mM DTT, 0.1 mg/ml BSA, 10 μM [³H]-threonine, 5-10 μM tRNA, and 10-50 nM thrS

• Monitor reaction by TCA precipitation and scintillation counting

ATP-PPi Exchange Assay:

• Reaction mixture contains 100 mM HEPES-KOH (pH 7.5), 10 mM MgCl₂, 2 mM ATP, 2 mM [³²P]-PPi, 2 mM threonine, and 50-100 nM thrS

• Quantify [³²P]-ATP formation by thin-layer chromatography

Unlike assays for measuring C. violaceum T6SS activity, which involve bacterial competition experiments or VipA-sfGFP fluorescence microscopy , these biochemical assays provide direct quantitative measurements of enzymatic function.

How do temperature and pH affect C. violaceum thrS activity?

C. violaceum thrS demonstrates distinctive temperature and pH dependencies that reflect its adaptation to C. violaceum's environmental niche:

Temperature Profile:

• Optimal activity: 30-37°C (coinciding with C. violaceum's growth range)

• Significant activity retention (>60%): 25-42°C

• Rapid inactivation: >45°C

• Activity at lower temperatures: Maintains ~30% activity at 20°C

pH Profile:

• Optimal pH range: 7.2-7.8

• Broader activity range: pH 6.5-8.5 (>50% activity)

• Sharper activity drop in acidic conditions than in alkaline conditions

These parameters align with C. violaceum's natural habitats in tropical and subtropical soil and water environments , but differ from optimal conditions for violacein production, which is regulated by quorum sensing and VioS repressor protein .

How can CRISPR-Cas9 be optimized for generating thrS mutants in C. violaceum?

Creating precise thrS mutants in C. violaceum requires careful CRISPR-Cas9 strategy optimization:

Protocol Recommendations:

• sgRNA Design:

  • Target sequences with minimal off-target potential using C. violaceum ATCC 12472 genome sequence

  • Avoid regions with secondary structure formation

  • Optimal PAM sites (NGG) should be selected using C. violaceum-specific tools

• Delivery Method:

  • Electroporation of ribonucleoprotein complexes (pre-assembled Cas9 protein and sgRNA)

  • Parameters: 2.5 kV, 200 Ω, 25 μF with 0.2 cm cuvettes

• Homology-Directed Repair Template:

  • Homology arms: 500-1000 bp flanking the target site

  • For complete deletion: Include start and stop codons to avoid polar effects

  • For point mutations: Incorporate silent mutations in the PAM site to prevent re-cutting

• Selection Strategy:

  • Since thrS is likely essential, use an inducible complementation system

  • Express wild-type thrS from an arabinose-inducible promoter during mutant generation

This approach differs from methods used to generate the VgrG mutants in C. violaceum, which employed traditional homologous recombination strategies but shares similarities with targeted gene disruption approaches used in pathogenicity studies .

What phenotypes are associated with thrS mutations in C. violaceum?

Mutations in thrS can produce several phenotypes in C. violaceum, categorized by their severity and cellular impact:

Complete Loss-of-Function:

• Likely lethal due to the essential nature of thrS in protein synthesis

• Requires conditional expression systems for study

Partial Loss-of-Function:

• Reduced growth rates, particularly in minimal media

• Increased sensitivity to antibiotics targeting protein synthesis

• Altered cellular morphology (elongated cells due to stress response)

Point Mutations in Catalytic Sites:

• Amino acid substitutions affecting threonine recognition can trigger stringent response

• Mutations in tRNA binding domains may lead to increased mistranslation rates

• Temperature-sensitive phenotypes at restrictive temperatures

Unlike mutations in the T6SS genes, which primarily affect bacterial competition without growth impairment , or mutations in quorum sensing genes, which affect virulence and pigment production , thrS mutations fundamentally impact cellular viability and proteome integrity.

How can C. violaceum thrS be leveraged for studying bacterial mistranslation mechanisms?

C. violaceum thrS presents a valuable model for investigating bacterial mistranslation mechanisms and their physiological consequences:

Experimental Approach:

• Engineering editing-deficient thrS variants:

  • Create point mutations in the editing domain (typically H73A, H186A based on homology to E. coli thrS)

  • Confirm reduced editing capacity through in vitro mischarging assays with serine

• Proteomic analysis of mistranslation:

  • Use mass spectrometry to quantify threonine-to-serine substitution rates

  • Employ SILAC labeling to compare proteomes of wild-type and editing-deficient strains

• Stress response characterization:

  • RNA-seq analysis to identify upregulated stress response pathways

  • Comparative phenotypic analysis under various stressors (oxidative, heat, antibiotics)

• Evolution experiments:

  • Long-term culturing of editing-deficient strains to identify compensatory mutations

  • Whole genome sequencing to track evolutionary adaptations to chronic mistranslation

This research direction differs fundamentally from studies of C. violaceum's T6SS and T3SS systems, which focus on bacterial competition and virulence , by instead exploring fundamental aspects of protein synthesis fidelity and cellular adaptation.

What is the relationship between thrS activity and quorum sensing in C. violaceum?

While direct evidence for thrS regulation by quorum sensing is not established, several potential interactions warrant investigation:

Theoretical Connections:

• Translational regulation during quorum transitions:

  • C. violaceum's CviI/R quorum sensing system regulates numerous phenotypes

  • High cell density could necessitate changes in translation efficiency

• Stress response coordination:

  • Quorum sensing and the stringent response (potentially triggered by uncharged tRNAs) may exhibit cross-regulation

  • Both systems influence virulence factor expression

Experimental Approaches:

• Measure thrS expression levels in wild-type vs. ΔcviI and ΔcviR mutants using RT-qPCR

• Analyze thrS promoter sequences for potential CviR binding sites

• Use ChIP-seq to identify if CviR interacts with the thrS promoter region

• Create reporter fusions to monitor thrS expression in response to exogenous C10-HSL

This research direction explores potential regulatory connections between basic cellular processes (translation) and population-level behaviors (quorum sensing) that have not been directly addressed in current literature on C. violaceum .

How does C. violaceum thrS contribute to bacterial adaptation in changing environments?

C. violaceum thrS may play critical roles in environmental adaptation beyond its canonical function in protein synthesis:

Adaptation Mechanisms:

• Temperature fluctuation response:

  • thrS activity optimization at different temperatures may facilitate adaptation to environmental temperature changes

  • Potential temperature-dependent alternative folding or activity patterns

• Nutrient limitation strategies:

  • Under amino acid limitation, thrS regulation may contribute to the stringent response

  • Fine-tuning of thrS activity could help prioritize essential protein synthesis

• Biofilm formation influence:

  • Potential role in regulating translation efficiency during biofilm development

  • Connection to quorum sensing-regulated biofilm processes

Research Methodologies:

• Transcriptomic and proteomic profiling of C. violaceum under various environmental stresses

• Comparison of thrS expression and activity across environmental isolates from different niches

• Analysis of translation rates using ribosome profiling under different environmental conditions

This differs from studies focused on specific virulence mechanisms like T3SS and T6SS by examining broader adaptive responses that enable C. violaceum's survival in diverse tropical and subtropical habitats.

What structural features distinguish C. violaceum thrS from human threonyl-tRNA synthetase?

C. violaceum thrS exhibits several structural differences from human threonyl-tRNA synthetase that can be exploited for selective inhibitor development:

Key Structural Distinctions:

These structural differences, particularly in the active site architecture, provide potential targets for developing selective inhibitors. Unlike the structural studies of C. violaceum's T6SS components like VgrG proteins , which focus on protein-protein interactions for bacterial competition, structural studies of thrS emphasize enzyme-substrate interactions crucial for protein synthesis.

What methodologies are most effective for crystallizing C. violaceum thrS?

Successfully crystallizing C. violaceum thrS requires optimization of multiple parameters:

Recommended Crystallization Protocol:

• Sample preparation:

  • Purify to >95% homogeneity by SEC

  • Concentrate to 8-12 mg/ml in 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT

  • Include 2 mM ATP or ATP analogs to stabilize conformation

• Crystallization screening:

  • Initial screens: Commercial sparse matrix screens (Hampton, Molecular Dimensions)

  • Optimization focus: PEG-based conditions (PEG 3350-8000, 15-25%)

  • Buffer range: pH 6.5-8.0 (HEPES, Tris, phosphate)

  • Additives: 10-200 mM divalent cations (Mg²⁺, Mn²⁺)

• Co-crystallization strategies:

  • With substrate analogs: non-hydrolyzable ATP analogs (AMPPNP)

  • With threonine or threonine analogs: 5-10 mM concentration

  • With tRNA: Purified tRNAᵀʰʳ at 1:1 molar ratio

• Crystal optimization:

  • Microseeding to improve crystal size and quality

  • Temperature gradient (4°C to 20°C)

  • Vapor diffusion (hanging or sitting drop) methods preferable

This methodological approach provides detailed guidance beyond what is typically described in C. violaceum research papers, which often focus on genetic and phenotypic analyses rather than structural biology techniques .

How does C. violaceum thrS compare to orthologous enzymes from other pathogenic bacteria?

Comparative analysis reveals important differences between C. violaceum thrS and orthologous enzymes from other pathogenic bacteria:

Comparative Features:

The comparison highlights that while all bacterial thrS enzymes share core catalytic functions, C. violaceum thrS most closely resembles the enzyme from Pseudomonas aeruginosa, a bacterial species that C. violaceum can outcompete through its T6SS . This comparative analysis provides context beyond the specific study of C. violaceum virulence mechanisms by positioning its translational machinery within the broader bacterial phylogeny.

How has C. violaceum thrS evolved in relation to the organism's ecological niche?

The evolution of C. violaceum thrS reflects adaptations to the bacterium's specific ecological niche:

Evolutionary Adaptations:

This evolutionary perspective complements studies of C. violaceum's pathogenicity by highlighting how both essential cellular machinery and virulence factors have adapted to the bacterium's primarily environmental lifestyle with occasional opportunistic infections.

How can C. violaceum thrS be utilized for developing antimicrobial compounds?

C. violaceum thrS presents opportunities for antimicrobial development through several approaches:

Antimicrobial Development Strategies:

• Structure-based inhibitor design:

  • Target unique features of the ATP binding pocket

  • Develop compounds that interfere with threonine recognition

  • Focus on differences between bacterial and human orthologous enzymes

• Fragment-based drug discovery:

  • Identify small molecule fragments binding to different thrS regions

  • Combine fragments with favorable binding properties

  • Optimize for bacterial selectivity

• High-throughput screening methodology:

  • Primary assay: ATP-PPi exchange inhibition (Z' factor >0.7)

  • Secondary assay: Aminoacylation inhibition

  • Counter-screen: Activity against human threonyl-tRNA synthetase

• In silico approaches:

  • Molecular docking against C. violaceum thrS model

  • Virtual screening of compound libraries

  • Molecular dynamics simulations to identify transient binding pockets

Targeting thrS offers an alternative approach to combating C. violaceum infections compared to strategies focused on virulence mechanisms , particularly important given C. violaceum's intrinsic resistance to many antibiotics and the high mortality of infections .

What challenges and solutions exist for expressing active C. violaceum thrS in heterologous systems?

Expressing functionally active C. violaceum thrS in heterologous systems presents specific challenges and solutions:

Expression Challenges and Solutions:

Unlike the expression systems used for studying C. violaceum virulence factors, which often involve genetic manipulation directly in C. violaceum , heterologous expression of thrS typically requires optimization in E. coli or other model organisms, presenting unique technical challenges beyond those encountered in native expression studies.