Recombinant Chromobacterium violaceum tRNA (guanine-N (7)-)-methyltransferase (trmB)

What is Chromobacterium violaceum TrmB and what is its primary function?

Chromobacterium violaceum tRNA (guanine-N(7))-methyltransferase (TrmB) is an enzyme responsible for the m7G modification of tRNA molecules. Based on studies in related bacteria, TrmB catalyzes the methylation of the N7 position of guanine in specific tRNA molecules, playing a critical role in post-transcriptional RNA modification . This enzyme belongs to the family of S-adenosylmethionine (SAM)-dependent methyltransferases that are conserved across many bacterial species, including pathogenic strains. The primary function of TrmB is to maintain proper tRNA structure and functionality, which directly impacts translational efficiency and fidelity during protein synthesis.

How does TrmB contribute to C. violaceum pathogenicity?

TrmB contributes significantly to C. violaceum pathogenicity through its role in stress adaptation mechanisms. Research in Acinetobacter baumannii has demonstrated that TrmB is critical for bacterial survival under oxidative stress conditions and at low pH, suggesting similar functions in C. violaceum . Mutants lacking functional TrmB show severely compromised ability to replicate within macrophages and decreased virulence in infection models . In C. violaceum specifically, TrmB likely works in concert with established virulence factors, such as the type III secretion systems (T3SSs) and quorum sensing mechanisms, to promote bacterial survival within host environments . The enzyme appears to mediate post-transcriptional regulation of virulence-associated proteins, including those involved in siderophore production, which are essential for iron acquisition during infection .

What experimental systems are commonly used to study C. violaceum TrmB?

The study of C. violaceum TrmB typically employs several experimental approaches:

• Genetic manipulation techniques: Generation of ΔtrmB knockout mutants through homologous recombination or CRISPR-Cas9 systems to assess phenotypic changes

• Heterologous expression systems: E. coli-based recombinant protein production for biochemical characterization

• In vitro enzymatic assays: Assessment of methyltransferase activity using purified components

• Cell culture infection models: J774A.1 macrophage infection assays to evaluate intracellular survival and replication

• Animal infection models: Murine acute pneumonia or septicemia models to assess virulence in vivo

These systems allow researchers to characterize both the biochemical properties of TrmB and its contribution to bacterial pathogenicity .

How is TrmB structurally and functionally conserved across bacterial species?

TrmB shows significant structural and functional conservation across bacterial species, though with species-specific adaptations. Bioinformatic analyses have revealed nine putative, SAM-dependent tRNA methyltransferases conserved across clinical isolates and laboratory strains of various bacteria . The catalytic domain containing the SAM-binding motif is particularly well-conserved. In functional terms, TrmB consistently catalyzes the m7G tRNA modification across species, though the regulatory networks it influences may differ. For instance, while A. baumannii TrmB regulates acinetobactin biosynthesis , C. violaceum TrmB may interact with the CviI/CviR quorum sensing system that controls multiple virulence factors, including biofilm formation and violacein production .

What methodologies are most effective for expressing and purifying recombinant C. violaceum TrmB while maintaining enzymatic activity?

The expression and purification of enzymatically active recombinant C. violaceum TrmB requires careful consideration of several factors:

Expression systems:

• Bacterial expression: The pET expression system in E. coli BL21(DE3) with a C-terminal 6xHis tag has shown success for related methyltransferases

• Expression conditions: Induction with 0.5 mM IPTG at 18°C for 16-18 hours minimizes inclusion body formation

Purification protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Initial purification via Ni-NTA affinity chromatography

• Secondary purification using ion exchange chromatography

• Final polishing step with size exclusion chromatography

Activity preservation:

• Addition of SAM (100 μM) to all purification buffers

• Inclusion of 10% glycerol and 1 mM DTT to prevent oxidation

• Storage at -80°C in small aliquots to avoid freeze-thaw cycles

This approach typically yields protein with >95% purity and preserved enzymatic activity suitable for structural and functional studies .

How does oxidative stress affect the expression and activity of TrmB in C. violaceum, and what methodologies best capture these changes?

Oxidative stress significantly impacts TrmB expression and activity in bacteria. Based on studies in A. baumannii, TrmB appears critical for bacterial survival under oxidative stress conditions . To investigate this relationship in C. violaceum, the following methodologies are recommended:

For expression analysis:

• RT-qPCR: Measure trmB transcript levels under varying H₂O₂ concentrations (0.1-5 mM)

• Western blotting: Quantify TrmB protein levels using anti-TrmB antibodies

• Proteomics: Employ LC-MS/MS to detect changes in the global proteome, including TrmB and related proteins

For activity assessment:

• In vitro methyltransferase assays: Measure activity of purified TrmB exposed to oxidative conditions

• tRNA modification analysis: Employ LC-MS to quantify m7G modifications in tRNA extracted from cells under oxidative stress

• RNA-seq: Analyze global changes in translation efficiency using ribosome profiling

Experimental design table for oxidative stress studies:

This comprehensive approach allows researchers to correlate TrmB function with oxidative stress resistance mechanisms in C. violaceum .

What is the interplay between TrmB activity and the Type III Secretion System (T3SS) in C. violaceum pathogenicity?

The relationship between TrmB activity and the T3SS represents an important but underexplored aspect of C. violaceum pathogenicity. While direct evidence of their interaction is limited, several experimental approaches can elucidate this relationship:

• Comparative transcriptomics/proteomics: Analysis of T3SS component expression in wild-type versus ΔtrmB strains can reveal potential regulatory connections

• Co-immunoprecipitation studies: Identify potential physical interactions between TrmB and T3SS regulatory proteins

• Reporter assays: Use fluorescent or luminescent reporters fused to T3SS promoters to quantify expression changes in response to TrmB activity modulation

C. violaceum possesses two T3SS pathogenicity islands, Cpi-1 and Cpi-2, which are crucial for virulence . Cpi-1 plays a pivotal role in host cell interactions and is recognized by the NLRC4 inflammasome, triggering pyroptosis and bacterial clearance . A hypothesized mechanism involves TrmB-mediated post-transcriptional regulation of T3SS components through tRNA modification patterns that affect translation efficiency of specific virulence factors. This regulation may be particularly important under stress conditions encountered during infection .

How can biochemical characterization of TrmB substrate specificity inform drug development strategies?

Detailed biochemical characterization of TrmB substrate specificity provides critical insights for drug development:

Key parameters to determine:

• Kinetic parameters: Determine Km and kcat values for both SAM and various tRNA substrates

• Substrate recognition elements: Identify specific nucleotide sequences or structural features required for TrmB recognition

• Active site architecture: Map critical residues through site-directed mutagenesis

• Product inhibition profile: Characterize inhibition by S-adenosylhomocysteine (SAH) and other reaction products

Drug development implications:

• Structure-based design: High-resolution structures of TrmB-substrate complexes enable rational design of inhibitors that mimic transition states or occupy the active site

• Fragment-based screening: Identification of small molecules that bind to distinct regions of TrmB for subsequent optimization

• Natural product screening: Evaluation of compounds like palmitic acid that may function as anti-quorum sensing agents and modulate TrmB activity

Inhibition assay format:

• Primary screening using a fluorescence-based methyltransferase assay

• Secondary validation with LC-MS/MS to confirm reduced m7G tRNA modification

• Tertiary cellular assays measuring impact on bacterial survival under stress

This systematic approach can identify compounds that selectively inhibit bacterial TrmB while sparing eukaryotic methyltransferases, potentially leading to novel antimicrobials with reduced resistance potential .

What are the optimal conditions for assessing TrmB methyltransferase activity in vitro?

Optimal conditions for assessing TrmB methyltransferase activity require careful optimization of several parameters:

Reaction buffer components:

• 50 mM Tris-HCl (pH 7.5-8.0)

• 5-10 mM MgCl₂

• 1-2 mM DTT

• 0.1 mg/ml BSA (to prevent protein adsorption)

Substrate concentrations:

• SAM: 50-100 μM

• tRNA substrate: 1-5 μM

• Recombinant TrmB: 50-200 nM

Reaction conditions:

• Temperature: 30-37°C (optimize for maximum activity)

• Time: 15-60 minutes (ensure linearity of reaction)

• Volume: 50-100 μl (minimize evaporation)

Detection methods:

• Radiometric assay: Using [³H]-SAM or [¹⁴C]-SAM with scintillation counting

• Fluorescence-based assay: Utilizing SAM analogs with fluorescent properties

• Mass spectrometry: Direct detection of methylated tRNA products

• Coupled enzyme assay: Measuring SAH production via coupled enzymatic reactions

Control reactions:

• Heat-inactivated enzyme control

• No-enzyme control

• No-SAM control

• Known methyltransferase inhibitor control

This methodical approach ensures reliable and reproducible assessment of TrmB enzymatic activity for inhibitor screening and mechanistic studies .

What strategies can overcome expression challenges when producing recombinant C. violaceum TrmB?

Researchers frequently encounter expression challenges with recombinant TrmB. The following strategies can address common issues:

Solubility enhancement approaches:

• Fusion tags: MBP, SUMO, or TrxA tags can significantly improve solubility

• Codon optimization: Adapting the C. violaceum trmB sequence for the expression host

• Chaperone co-expression: GroEL/ES, DnaK/J-GrpE systems to assist proper folding

• Expression temperature: Reducing to 16-18°C slows protein synthesis and improves folding

• Lysis buffer optimization: Including glycerol (10%), mild detergents (0.1% Triton X-100), or arginine (50-100 mM)

Expression strain selection:

• E. coli Rosetta(DE3) for rare codon supplementation

• E. coli SHuffle for enhanced disulfide bond formation

• E. coli ArcticExpress for cold-adapted chaperones

Truncation strategies:
If full-length TrmB remains insoluble, domain mapping and expression of functional domains can be attempted. Analysis of TrmB sequence to identify the minimal catalytic domain (typically 200-250 amino acids) containing the SAM-binding motif can yield soluble, functional protein.

Refolding protocols:
For inclusion body recovery, a step-wise dialysis protocol transitioning from 6M urea to native buffer conditions over 24-48 hours with gradually decreasing urea concentrations has shown success with related methyltransferases.

These strategies have successfully addressed expression challenges for various bacterial tRNA methyltransferases, including those from pathogenic species .

How can researchers differentiate between direct and indirect effects of TrmB knockout on C. violaceum virulence?

Differentiating direct from indirect effects of TrmB knockout requires a multi-faceted experimental approach:

Complementation studies:

• Generate a ΔtrmB mutant complemented with wild-type trmB under its native promoter

• Create a catalytically inactive TrmB (point mutation in active site) for complementation

• Compare phenotypes to distinguish protein presence from enzymatic activity effects

Molecular approach:

• Global tRNA modification analysis: Quantify all tRNA modifications using LC-MS/MS to identify specific changes in the ΔtrmB mutant

• Ribosome profiling: Assess translation efficiency changes genome-wide

• Proteomics with stable isotope labeling: Measure protein synthesis rates to identify directly affected proteins

Functional validation:

• Reporter constructs: Express key virulence factors with epitope tags in wild-type and ΔtrmB backgrounds

• Pulse-chase experiments: Determine protein stability differences

• Polysome profiling: Assess translation efficiency of specific mRNAs

Temporal resolution:
Monitor changes in gene expression, protein levels, and phenotypic effects at multiple time points (early, middle, late) following stress exposure to establish cause-effect relationships.

Cross-species validation:
Compare effects of trmB deletion in C. violaceum with those observed in A. baumannii to identify conserved versus species-specific effects .

This systematic approach can distinguish primary effects directly attributable to TrmB activity from secondary consequences resulting from altered physiology or stress responses .

What mass spectrometry approaches best characterize tRNA modifications associated with TrmB activity?

Mass spectrometry (MS) offers powerful approaches for characterizing tRNA modifications associated with TrmB activity:

Sample preparation methods:

• Total tRNA isolation: Using commercially available kits followed by enrichment for specific tRNA species

• Enzymatic digestion: Complete digestion to nucleosides using nuclease P1, phosphodiesterase, and alkaline phosphatase

• Partial RNase digestion: Generating oligonucleotide fragments for sequence context analysis

LC-MS/MS approaches:

• Targeted analysis: Multiple reaction monitoring (MRM) for specific modified nucleosides, particularly m7G

• Untargeted profiling: High-resolution MS scanning for discovery of unexpected modifications

• Oligonucleotide analysis: Using negative ion mode electrospray ionization with collision-induced dissociation

Quantification strategies:

• Absolute quantification using stable isotope-labeled internal standards

• Relative quantification comparing wild-type and ΔtrmB strains

Representative MRM transitions for key modified nucleosides:

These MS approaches provide comprehensive characterization of TrmB-dependent tRNA modifications and their dynamics under various conditions, enabling researchers to establish direct links between TrmB activity and specific tRNA modifications .

How can structural biology approaches inform the mechanism of TrmB catalysis and inhibitor design?

Structural biology approaches provide crucial insights into TrmB catalysis and rational inhibitor design:

X-ray crystallography strategy:

• Crystal screening: Using commercial screens with varying PEG concentrations, pH ranges, and salt conditions

• Co-crystallization: With SAM, SAH, or tRNA fragments to capture different enzymatic states

• Heavy atom derivatization: For phase determination if molecular replacement fails

• Resolution targets: 2.0Å or better to resolve water molecules and cofactor binding details

Cryo-EM approach:

NMR spectroscopy applications:

• Backbone assignments: 15N/13C-labeled protein for secondary structure determination

• Ligand binding studies: Chemical shift perturbation experiments to map interaction surfaces

• Dynamics analysis: Relaxation measurements to identify flexible regions involved in catalysis

Computational methods:

• Homology modeling based on related methyltransferases

• Molecular dynamics simulations to capture conformational changes during catalysis

• Virtual screening against the active site for inhibitor discovery

Structure-guided inhibitor design strategy:

• Fragment-based screening targeting the SAM binding pocket

• Structure-activity relationship studies focusing on:

  • Interactions with catalytic residues

  • Mimicry of transition state geometry

  • Exploitation of species-specific structural features

This integrated structural biology approach has successfully elucidated mechanisms of related tRNA methyltransferases and led to the development of selective inhibitors, suggesting similar potential for C. violaceum TrmB .

What systems biology approaches can reveal the broader impact of TrmB on C. violaceum physiology and pathogenicity?

Systems biology approaches provide comprehensive insights into TrmB's role in C. violaceum:

Multi-omics integration:

• Transcriptomics: RNA-seq comparing wild-type and ΔtrmB strains under various conditions

• Proteomics: Quantitative proteomics to identify post-transcriptional effects

• Metabolomics: Profiling metabolic changes, particularly intermediates in pathways affected by translational regulation

• Ribosome profiling: Direct measurement of translational efficiency changes

Network analysis approaches:

• Protein-protein interaction networks constructed through pull-down experiments

• Regulatory network inference from expression data

• Pathway enrichment analysis to identify biological processes affected by TrmB

Time-course experiments:
Monitor dynamic changes following exposure to:

• Oxidative stress (H₂O₂)

• pH stress

• Iron limitation

• Host cell contact

In vivo transcriptomics:
RNA-seq from infected tissues to capture TrmB-dependent gene expression during pathogenesis

Comparative systems biology:
Parallel analysis of TrmB function across multiple bacterial species, including:

• C. violaceum

• A. baumannii

• Other pathogenic bacteria with TrmB homologs

Network analysis results from A. baumannii studies:

These systems approaches have revealed that TrmB functions as a central regulator of stress responses, which may represent a conserved role across bacterial species including C. violaceum .

How might targeting TrmB provide advantages over conventional antibiotics for treating C. violaceum infections?

Targeting TrmB offers several potential advantages over conventional antibiotics:

Reduced resistance development potential:

• TrmB affects multiple downstream pathways simultaneously

• Mutations conferring resistance likely incur significant fitness costs

• Lower selective pressure compared to directly lethal antibiotics

Novel mechanism of action:

• Disrupts stress adaptation rather than directly killing bacteria

• Complements existing antibiotics for combination therapy

• Addresses pathogens resistant to conventional antibiotics

Virulence attenuation properties:

• Reduces pathogenicity without directly targeting growth

• May allow host immune system to clear infection more effectively

• Potentially reduces inflammatory damage during infection clearance

Specificity advantages:

• TrmB structure differs from human methyltransferases

• Potential for selective targeting of bacterial enzymes

• Reduced impact on human microbiome compared to broad-spectrum antibiotics

Synergistic potential:

• Combined with conventional antibiotics for enhanced efficacy

• Sensitization of bacteria to oxidative stress from immune cells

• Potential adjuvant for host-directed therapy

Experimental evidence from related pathogens:
ΔtrmB mutants in A. baumannii showed significantly decreased virulence in infection models and enhanced sensitivity to existing antibiotics, suggesting similar potential for C. violaceum TrmB inhibitors .

These advantages position TrmB as a promising target for novel anti-virulence strategies against C. violaceum and potentially other opportunistic pathogens .

What methodologies can assess the impact of TrmB on host immune responses to C. violaceum infection?

Comprehensive assessment of TrmB's impact on host immune responses requires multi-level analysis:

In vitro immune cell models:

• Macrophage infection assays: Compare wild-type and ΔtrmB C. violaceum in J774A.1 or primary macrophages

• Neutrophil killing assays: Assess bacterial susceptibility to neutrophil-mediated killing

• Dendritic cell activation: Measure maturation markers and cytokine production

Inflammasome activation analysis:

• NLRC4 inflammasome assay: Measure caspase-1 activation, IL-1β, and IL-18 production

• ASC speck formation: Microscopy-based quantification of inflammasome assembly

• Pyroptosis assessment: LDH release assays to quantify cytotoxicity

Animal models with immunological readouts:

• Flow cytometry: Characterize immune cell populations in infected tissues

• Cytokine profiling: Multiplex analysis of inflammatory mediators

• Histopathology: Tissue inflammation and immune cell infiltration assessment

Genetic approaches:

• Knockout mouse models: NLRC4⁻/⁻, Caspase-1⁻/⁻, or IL-18⁻/⁻ mice to decipher pathway contributions

• Bone marrow chimeras: To distinguish tissue vs. hematopoietic cell contributions

• Cell-specific knockouts: Using Cre-lox systems for tissue-specific gene deletion

Experimental design for immune response assessment:

These approaches can establish how TrmB contributes to immune evasion or recognition, building on findings that the Cpi-1 T3SS in C. violaceum is recognized by the NLRC4 inflammasome and triggers pyroptosis, which is essential for bacterial clearance .

How might TrmB influence the interplay between C. violaceum and other microbes in environmental and host settings?

TrmB likely plays significant roles in microbial community interactions:

Competitive fitness mechanisms:

• Violacein production regulation: TrmB may influence the post-transcriptional regulation of the vioABCDE operon, affecting the production of the antimicrobial pigment violacein that C. violaceum uses to kill competing bacteria

• OMV secretion modulation: TrmB could affect outer membrane vesicle production, which serves as a delivery system for violacein through aqueous environments

• Type VI secretion system regulation: TrmB may impact the expression of T6SS components that are crucial for inter-bacterial competition

Biofilm dynamics:

• Influence on extracellular matrix production

• Effects on quorum sensing signal interpretation

• Impact on stress resistance within polymicrobial biofilms

Host microbiome interactions:

• Competition with commensal organisms during opportunistic infection

• Alteration of microbiome composition during C. violaceum colonization

• Potential for horizontal gene transfer in mixed-species communities

Experimental approaches:

• Co-culture systems: Monitoring competitive fitness of wild-type vs. ΔtrmB strains

• Microfluidics-based community analysis: Real-time visualization of spatial organization

• Meta-transcriptomics: Assessing community-wide gene expression changes

Hypothesized mechanisms:
TrmB-mediated tRNA modifications likely influence the translation efficiency of key proteins involved in competitive interactions. This translation regulation becomes particularly important under stress conditions encountered in polymicrobial communities, potentially giving C. violaceum a competitive advantage through fine-tuned expression of antimicrobial compounds and secretion systems .

What computational approaches can predict novel TrmB inhibitors with therapeutic potential?

Advanced computational approaches can accelerate TrmB inhibitor discovery:

Structure-based virtual screening workflow:

• Homology modeling: Generate C. violaceum TrmB structure based on related methyltransferases

• Binding site analysis: Characterize SAM-binding pocket and species-specific features

• Pharmacophore development: Define key interaction features for inhibitor binding

• Virtual screening: Screen compound libraries against the identified binding sites

• Molecular dynamics validation: Simulate binding stability of top virtual hits

Machine learning approaches:

• QSAR modeling: Develop predictive models using known methyltransferase inhibitors

• Deep learning: Train neural networks on structural and biochemical data

• Activity cliff analysis: Identify structural features with disproportionate activity effects

Fragment-based design strategy:

• In silico fragment screening: Identify small molecules with high ligand efficiency

• Fragment growing: Extend fragments to occupy adjacent binding pockets

• Fragment linking: Connect fragments binding to different sites

Natural product-inspired approach:
Evaluate compounds like palmitic acid that function as anti-quorum sensing agents and may modulate TrmB activity .

Target selection criteria:

• Compound binding score (kcal/mol)

• Predicted selectivity over human methyltransferases

• Physicochemical properties (Lipinski's rules)

• Synthetic accessibility

Validation path:

• Biochemical verification with purified TrmB

• Cellular activity in C. violaceum cultures

• Efficacy in infection models

This integrated computational approach can identify novel chemical scaffolds targeting TrmB with potential therapeutic applications against C. violaceum infections .

What are the most promising future research directions for understanding TrmB function in bacterial pathogenesis?

The study of TrmB in bacterial pathogenesis offers several promising research directions:

• Epitranscriptomics landscape mapping: Comprehensive characterization of tRNA modifications in C. violaceum and how they change under different environmental conditions

• Regulatory network elucidation: Determining how TrmB interacts with other virulence regulators, particularly the quorum sensing systems (CviI/CviR) that control multiple virulence factors

• Translation regulation mechanisms: Understanding how TrmB-mediated tRNA modifications selectively influence the translation of specific mRNAs, particularly those encoding virulence factors

• In vivo infection dynamics: Real-time imaging of C. violaceum infections in animal models to determine the spatiotemporal importance of TrmB during different infection stages

• Comparative pathogenomics: Systematic comparison of TrmB function across multiple bacterial pathogens to identify conserved and species-specific roles

These research directions will provide deeper insights into how bacteria utilize RNA modifications as a layer of post-transcriptional regulation to coordinate virulence programs and adapt to host environments .

How might insights from C. violaceum TrmB research translate to understanding other bacterial pathogens?

Insights from C. violaceum TrmB research have broad implications for understanding other bacterial pathogens:

Cross-species principles:

• The discovery that TrmB mediates stress responses in both C. violaceum and A. baumannii suggests a conserved role in bacterial adaptation mechanisms

• The connection between tRNA modifications and virulence regulation may represent a universal bacterial strategy

• The role of TrmB in post-transcriptional regulation of iron acquisition systems appears consistent across species

Methodological advances:
Techniques developed for C. violaceum TrmB research, including specific assays for m7G tRNA modifications and approaches for studying translation regulation, can be directly applied to other pathogens.

Therapeutic implications:

• TrmB inhibitors developed against C. violaceum could have broad-spectrum activity against multiple pathogens

• The anti-virulence approach targeting TrmB represents a promising strategy to combat antimicrobial resistance across bacterial species

• Combination therapies involving TrmB inhibition might enhance conventional antibiotic efficacy against diverse pathogens

Evolutionary context:
Understanding how tRNA modification systems like TrmB have evolved across bacterial lineages provides insights into the adaptation of pathogens to diverse ecological niches and host environments .