Recombinant Chromobacterium violaceum Putative arginyl-tRNA--protein transferase (ate)

What is Chromobacterium violaceum and why is it of interest to researchers?

Chromobacterium violaceum is a Gram-negative, β-proteobacterium that dominates diverse ecosystems in tropical and subtropical regions. Its complete genome sequence was determined by a consortium of Brazilian laboratories, providing an excellent model for studying environmental adaptation strategies . C. violaceum is notable for several reasons:

• It produces a characteristic purple pigment called violacein that protects the bacterium from oxidative damage

• It possesses approximately 500 open reading frames (ORFs) coding for transport-related membrane proteins, representing about 11% of all genes found

• It exhibits complex transport systems that likely contribute to its dominance in various ecosystems

• From a biotechnological perspective, it contains transporters of heavy metals, suggesting potential applications in bioremediation

The organism has clinical significance as well, as it can cause serious infections including pneumonia, visceral abscesses, meningitis, endocarditis, and gastrointestinal infections, with a mortality rate of up to 80% due to its tendency for hematogenous dissemination resulting in sepsis .

What is the general function of arginyl-tRNA--protein transferase in bacterial systems?

Arginyl-tRNA--protein transferase (ATE) is a tRNA-dependent enzyme that catalyzes the covalent attachment of an arginine molecule to protein substrates. This post-translational modification, known as arginylation, plays roles in:

• Protein quality control mechanisms

• Targeting of proteins for degradation via the N-end rule pathway

• Regulation of protein function and stability

• Mediating protein-protein interactions

In the enzymatic process, ATE transfers arginine from aminoacylated tRNA^Arg to the N-terminal aspartate/glutamate residues of substrate proteins . This process is part of a larger cellular system that links protein modification to fundamental processes of cellular physiology and environmental response.

How is the putative ATE gene organized in the C. violaceum genome?

The putative arginyl-tRNA--protein transferase gene in C. violaceum was identified during genome annotation of the ATCC 12472 strain. Based on genome analysis:

• The gene is part of the 4.75 Mb circular chromosome of C. violaceum

• It follows the typical organization pattern of bacterial ate genes

• Comparative genomic analysis suggests conservation of key functional domains

• The genomic context indicates potential co-transcription with genes involved in related biochemical pathways

Analysis of the C. violaceum genome revealed approximately 4,431 open reading frames (ORFs), including many transport-related proteins that mediate this bacterium's direct metabolic interactions with complex soil and aquatic environments .

What basic laboratory techniques are essential for working with recombinant C. violaceum ATE?

When working with recombinant C. violaceum ATE, researchers should employ the following fundamental techniques:

• Gene amplification and cloning:

  • PCR amplification of the ate gene from C. violaceum genomic DNA

  • Optimization of codon usage for expression system

  • Selection of appropriate vector systems with affinity tags

• Expression optimization:

  • Testing different E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Varying induction temperatures (typically 16-30°C)

  • Optimizing inducer concentration and expression duration

• Protein purification:

  • Immobilized metal affinity chromatography (IMAC)

  • Size exclusion chromatography for oligomeric state determination

  • Ion exchange chromatography for high purity preparation

• Activity assays:

  • Spectrophotometric assays monitoring arginine transfer

  • Radioactive assays using [14C]-labeled arginine-charged tRNA

  • Mass spectrometry to detect arginine addition to substrate proteins

Proper handling of C. violaceum cultures requires appropriate biosafety measures due to the organism's potential pathogenicity, as evidenced by case reports of serious infections .

How does the structure of C. violaceum putative ATE compare to human ATE1?

Comparative structural analysis between bacterial and human ATE proteins reveals significant differences:

Human ATE1:

• Forms a symmetric homodimer that dissociates upon substrate binding

• Contains a unique extended loop that wraps around tRNA^Arg, creating extensive contacts with the T-arm of the tRNA cofactor

• Includes specific residues in the substrate binding site critical for enzymatic activity

C. violaceum putative ATE (based on structural predictions and bacterial homologs):

• Likely functions as a monomer

• Has a more compact structure lacking the extended tRNA-binding loop seen in human ATE1

• Contains conserved catalytic residues but with alterations in the substrate binding pocket

• Exhibits species-specific substrate recognition mechanisms

These structural differences suggest distinct evolutionary adaptations and likely affect substrate specificity and catalytic efficiency between bacterial and eukaryotic ATE enzymes.

What methodological approaches are optimal for characterizing the tRNA specificity of C. violaceum ATE?

To characterize tRNA specificity of C. violaceum ATE, researchers should consider the following methodological approaches:

• In vitro transcription and aminoacylation of tRNA^Arg variants:

  • Preparation of tRNA^Arg transcripts with modifications in key recognition elements

  • Aminoacylation using recombinant or purified ArgRS (arginyl-tRNA synthetase)

  • Testing modified tRNAs in ATE transfer assays

• RNA-protein interaction analyses:

  • Electrophoretic mobility shift assays (EMSA) with labeled tRNA^Arg

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • RNA footprinting to identify protected regions upon ATE binding

• Structural biology approaches:

  • X-ray crystallography of ATE-tRNA^Arg complexes

  • Cryo-electron microscopy for larger complexes

  • NMR studies of labeled tRNA-ATE interactions

• Computational modeling:

  • Molecular dynamics simulations of ATE-tRNA interactions

  • Sequence-based prediction of recognition elements

  • Docking studies comparing bacterial and eukaryotic tRNA binding

These approaches would help elucidate the molecular basis for tRNA selection, which appears to involve an extended loop structure in human ATE1 that creates contacts with the T-arm of tRNA^Arg . Determining whether similar mechanisms exist in the bacterial enzyme would provide valuable evolutionary insights.

How might the C. violaceum ATE function within the context of bacterial transport systems?

C. violaceum possesses a complex transport apparatus that likely contributes to its dominance in various ecosystems . The putative ATE might function within this context in several ways:

• Regulation of membrane transporter activity:

  • Post-translational arginylation may modify the activity or stability of key transporters

  • Potential modification of approximately 212 primary active transporters, 154 electrochemical potential-driven transporters, or 62 channels/pores identified in C. violaceum

• Integration with stress response pathways:

  • Modification of proteins involved in heavy metal transport and detoxification

  • Regulation of iron acquisition systems, including those involving FhuA, TonB, ExbB, ExbD, and FhuC complexes identified in the C. violaceum genome

• Environmental adaptation mechanisms:

  • Potential roles in modifying proteins critical for sensing environmental conditions

  • Possible contribution to biofilm formation and virulence factor expression

The electrochemical potential-driven transporters, which account for 31.5% of all annotated ORFs related to transport in C. violaceum , might be particularly affected by ATE-mediated regulation, thereby influencing nutrient uptake and multidrug resistance capabilities.

What experimental approaches can differentiate between putative and confirmed ATE activity in C. violaceum?

To move beyond putative annotation and confirm actual ATE activity in C. violaceum, researchers should implement the following experimental approaches:

• Gene knockout and complementation studies:

  • CRISPR-Cas9 or homologous recombination-based gene deletion

  • Phenotypic analysis of knockout strains

  • Complementation with wild-type and mutant versions to confirm function

• In vitro biochemical characterization:

  • Arginylation assay using purified recombinant protein

  • Substrate specificity profiling with peptide libraries

  • Kinetic analysis with various substrates and tRNA species

• In vivo target identification:

  • Proteomics approaches to identify arginylated proteins in C. violaceum

  • Comparison of proteome profiles between wild-type and ate-knockout strains

  • Stable isotope labeling to track arginine incorporation into proteins

• Functional validation:

  • Site-directed mutagenesis of predicted catalytic residues

  • Rescue experiments in heterologous systems

  • Cross-species complementation with known ATE enzymes

The structural characterization approach used for human ATE1, revealing its homodimeric structure and unique tRNA-binding loop , could be adapted for the C. violaceum enzyme to provide definitive evidence of its function and mechanism.

What is the relationship between C. violaceum ATE activity and bacterial pathogenicity?

The relationship between ATE activity and C. violaceum pathogenicity remains to be fully elucidated, but several connections can be hypothesized based on known virulence mechanisms:

• Regulation of virulence factors:

  • ATE may modify proteins involved in toxin production or secretion

  • Arginylation might influence the stability or activity of invasion-related proteins

• Stress response during host infection:

  • ATE activity could be upregulated during oxidative stress encountered in host environments

  • Modification of protective proteins may enhance bacterial survival in hostile conditions

• Immune evasion mechanisms:

  • Arginylation might alter surface proteins to evade host recognition

  • Modified proteins could interfere with complement activation or phagocytosis

C. violaceum infections, though uncommon, have a significant mortality rate (up to 80%) due to their tendency for hematogenous dissemination resulting in sepsis . The organism has been reported to cause pneumonia, visceral abscesses, meningitis, endocarditis, and gastrointestinal infections . Understanding the role of ATE in these pathogenic processes could provide insights into novel therapeutic approaches.

What purification strategy yields the highest activity for recombinant C. violaceum ATE?

Based on experiences with related enzymes, the following purification strategy is recommended for obtaining high-activity recombinant C. violaceum ATE:

• Expression optimization:

  • Use pET-based vectors with C-terminal His6 tag

  • Express in E. coli Rosetta(DE3) strain to address codon bias

  • Induce with 0.2 mM IPTG at 18°C for 16-18 hours

• Cell lysis and initial purification:

  • Use buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol

  • Include protease inhibitors and DNase I

  • Perform initial IMAC using Ni-NTA resin with gradient elution (20-300 mM imidazole)

• Secondary purification:

  • Perform ion exchange chromatography using Q-Sepharose

  • Use size exclusion chromatography (Superdex 200) to obtain homogeneous preparation

  • Final buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol

• Activity preservation:

  • Store enzyme with addition of 0.5 mM EDTA and 0.5 mM TCEP

  • Flash-freeze in small aliquots and store at -80°C

  • Avoid repeated freeze-thaw cycles

This strategy typically yields >95% pure protein with specific activity comparable to or higher than that of other characterized bacterial aminoacyl-transferases.

How can researchers develop a high-throughput screening assay for C. violaceum ATE inhibitors?

A robust high-throughput screening assay for C. violaceum ATE inhibitors can be developed using the following approach:

• Fluorescence-based activity assay:

  • Synthetic peptide substrates with N-terminal Asp/Glu labeled with fluorescence quencher

  • Fluorescently labeled arginine-charged tRNA^Arg

  • Transfer of arginine releases fluorophore from quencher proximity, increasing signal

• Assay optimization:

  • 384-well plate format, 50 μL reaction volume

  • Buffer conditions: 50 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl2, 1 mM DTT

  • Enzyme concentration: 10-50 nM (determined in pilot studies)

  • Substrate concentrations at approximately K_m values

• Screening parameters:

  • Z' factor >0.7 for statistical robustness

  • Signal-to-background ratio >5

  • DMSO tolerance up to 2%

  • Read points at 0, 15, and 30 minutes

• Counter-screening and validation:

  • Secondary assay using radioactive [14C]-Arg-tRNA

  • Selectivity panel against human ATE1 and other related enzymes

  • Dose-response determination for promising hits

This approach enables the efficient screening of compound libraries while providing data suitable for structure-activity relationship studies of identified inhibitors.

What are the optimal conditions for studying oligomerization states of recombinant C. violaceum ATE?

Unlike human ATE1, which forms a symmetric homodimer that dissociates upon substrate binding , the oligomerization state of C. violaceum ATE remains to be definitively characterized. To study this, researchers should employ the following methods:

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • Buffer conditions: 25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT

  • Protein concentration range: 0.1-5 mg/mL

  • Temperature: 4°C and 25°C to assess temperature dependence

  • Addition of tRNA^Arg and/or substrate peptides to assess dissociation

• Analytical ultracentrifugation (AUC):

  • Sedimentation velocity experiments at 40,000-60,000 rpm

  • Sample concentration range: 0.2-2 mg/mL

  • Analysis using SEDFIT or UltraScan software

  • Calculation of sedimentation coefficient (s) and molecular weight

• Native mass spectrometry:

  • Gentle ionization conditions to preserve native complexes

  • Buffer exchange to ammonium acetate before analysis

  • Comparison of spectra with and without substrates/cofactors

• Crosslinking studies:

  • Chemical crosslinking with BS3 or glutaraldehyde

  • Analysis by SDS-PAGE and mass spectrometry

  • Identification of inter-subunit crosslinked peptides

These complementary approaches will provide a comprehensive understanding of the oligomerization behavior of C. violaceum ATE and how it compares to its human counterpart.

How might comparative studies between bacterial and human ATE inform drug development?

Comparative studies between C. violaceum ATE and human ATE1 could inform antimicrobial drug development through several avenues:

• Structural distinctions as targets for selectivity:

  • Human ATE1's unique extended loop that wraps around tRNA^Arg is absent in bacterial ATEs

  • Differences in substrate binding sites could be exploited for selective inhibition

  • Distinct oligomerization states (human ATE1 is dimeric while bacterial ATEs may be monomeric)

• Differential substrate recognition:

  • Mapping substrate specificity differences between bacterial and human enzymes

  • Identification of bacterial-specific recognition motifs

  • Development of substrate-competitive inhibitors selective for bacterial ATEs

• Evolutionary conservation analysis:

  • Identification of residues conserved across bacterial ATEs but distinct from eukaryotic versions

  • Targeting of bacterial-specific catalytic mechanisms

  • Development of transition-state analogs based on bacterial-specific reaction coordinates

• Phenotypic consequences of inhibition:

  • Determine effects of ATE inhibition on bacterial viability versus human cell toxicity

  • Assess impact on C. violaceum virulence and pathogenicity

  • Evaluate potential for resistance development

Given the clinical significance of C. violaceum infections, which can lead to severe outcomes including sepsis with mortality rates up to 80% , development of selective inhibitors could have therapeutic value.

What gene editing approaches are most effective for studying ATE function in C. violaceum?

For genetic manipulation of the ate gene in C. violaceum, researchers should consider these approaches:

• CRISPR-Cas9 system optimized for C. violaceum:

  • Design of sgRNAs targeting the ate gene with minimal off-target effects

  • Optimization of Cas9 expression in C. violaceum

  • Development of template-guided repair for precise gene editing

  • Protocol modifications accounting for C. violaceum's high GC content

• Homologous recombination-based approaches:

  • Construction of suicide vectors containing homology arms

  • Selection of appropriate antibiotic resistance markers

  • Two-step selection process with counter-selectable markers

  • Verification of genetic modifications by PCR and sequencing

• Inducible expression systems:

  • Development of tetracycline-inducible promoter systems

  • Creation of rhamnose or arabinose-inducible expression systems

  • Tunable control of ATE expression levels for dose-dependent studies

  • Verification of expression control by RT-qPCR and Western blotting

• Transposon mutagenesis for phenotypic screening:

  • Random insertion libraries to identify genetic interactions with ate

  • High-throughput screening for phenotypes related to ATE function

  • Identification of synthetic lethal or synthetic rescue relationships

These genetic approaches will facilitate comprehensive functional characterization of ATE in C. violaceum and its potential roles in the bacterium's environmental adaptation and pathogenicity.

What is the potential role of C. violaceum ATE in bacterial adaptation to environmental stressors?

Given C. violaceum's complex transport systems and environmental adaptability , its putative ATE may play important roles in stress response:

• Heavy metal resistance:

  • Potential modification of heavy metal transporters identified in C. violaceum

  • Regulation of detoxification proteins under metal stress conditions

  • Modification of metal-binding proteins to alter affinity or stability

• Iron acquisition and metabolism:

  • Potential regulation of the multiple components of iron transport systems identified in C. violaceum, including FhuA, TonB, ExbB, ExbD, and FhuC

  • Modification of siderophore biosynthesis enzymes

  • Regulation of iron storage proteins under varying iron availability

• Response to oxidative stress:

  • Modification of antioxidant enzymes or regulators

  • Alteration of protein stability under oxidative conditions

  • Regulation of violacein production, which protects against oxidative damage

• Adaptation to pH and temperature fluctuations:

  • Modification of membrane transporters involved in pH homeostasis

  • Regulation of temperature-sensitive proteins

  • Alteration of protein half-lives under stress conditions

Research into these aspects would enhance understanding of how C. violaceum dominates diverse ecosystems in tropical and subtropical regions and could provide insights into bacterial adaptation mechanisms more broadly.