Recombinant Chromobacterium violaceum UPF0042 nucleotide-binding protein CV_3336 (CV_3336)

What is UPF0042 nucleotide-binding protein CV_3336 in Chromobacterium violaceum?

CV_3336 belongs to the UPF0042 family of nucleotide-binding proteins found in C. violaceum. These proteins are characterized by their ability to bind nucleotides and are often conserved across different bacterial species. While the specific function of CV_3336 remains to be fully elucidated, it shares structural similarities with other UPF0042 family proteins that may be involved in nucleotide metabolism or signaling pathways .

The protein is encoded by the CV_3336 gene in the C. violaceum genome (ATCC12472 strain). Like other members of this protein family, it likely contains conserved motifs for nucleotide binding, which may be critical for its biological function within the bacterial cell.

How does CV_3336 relate to other characterized proteins in C. violaceum?

For context, C. violaceum contains well-studied regulatory proteins such as VioS, which functions as a repressor of violacein biosynthesis without influencing the CviI/R quorum sensing system . The CviI/R system positively regulates multiple phenotypes including violacein production, protease activity, and chitinolytic activity . Understanding how CV_3336 might interact with these or other regulatory systems would be a valuable research direction.

What bioinformatic approaches can predict CV_3336 function?

To predict the function of CV_3336, researchers should implement a multi-faceted bioinformatic approach:

• Sequence homology analysis: Compare the CV_3336 sequence against characterized proteins using BLAST, searching both general (nr) and specialized (Swiss-Prot) databases.

• Domain prediction: Identify conserved domains using tools like InterPro, SMART, or CDD to reveal functional modules.

• Structural prediction: Generate 3D models using AlphaFold2 or similar tools, then compare against known structures using tools like Dali server.

• Genomic context analysis: Examine neighboring genes that might be functionally related or form an operon with CV_3336.

• Phylogenetic analysis: Construct a phylogenetic tree of homologous proteins to identify evolutionary relationships.

A sample workflow for functional prediction might include:

What expression systems are optimal for recombinant CV_3336 production?

The optimal expression system for CV_3336 requires careful consideration of several factors:

For prokaryotic expression, E. coli BL21(DE3) remains a primary choice due to its well-established protocols and high yield potential. When working with CV_3336, consider the following expression parameters:

• Vector selection: pET vectors with T7 promoter systems offer strong, inducible expression. For improved solubility, consider fusion tags such as MBP, SUMO, or TrxA.

• Induction conditions: Test multiple induction parameters following this experimental design:

• Codon optimization: As C. violaceum has different codon usage than E. coli, codon optimization of the CV_3336 gene sequence for E. coli expression may significantly improve yields.

For proteins that prove difficult to express in E. coli, consider alternative systems such as Bacillus subtilis (for secreted expression) or eukaryotic systems like P. pastoris (for proteins requiring complex folding or post-translational modifications).

How can researchers assess the nucleotide-binding properties of CV_3336?

To characterize the nucleotide-binding properties of CV_3336, researchers should employ multiple complementary techniques:

• Thermal shift assays (TSA): Measure protein stability changes upon nucleotide binding using a real-time PCR machine. Screen various nucleotides (ATP, GTP, CTP, UTP) at different concentrations (0.1-5 mM) to identify potential ligands.

• Isothermal titration calorimetry (ITC): Quantify binding thermodynamics. A typical experiment would titrate nucleotides (0.5-1 mM) into purified CV_3336 (50-100 μM) to determine KD, ΔH, and stoichiometry.

• Surface plasmon resonance (SPR): For kinetic binding parameters (kon and koff), immobilize CV_3336 on a sensor chip and flow various nucleotides at different concentrations.

• Fluorescence-based assays: If CV_3336 contains tryptophan residues near the predicted binding site, intrinsic fluorescence quenching upon nucleotide binding provides a direct readout of binding.

Example data interpretation table:

Note: This is example data for illustrative purposes. Actual binding parameters would need to be experimentally determined.

What experimental approaches can reveal CV_3336 function in C. violaceum?

To elucidate the function of CV_3336 in C. violaceum, researchers should implement a multi-faceted experimental strategy:

• Gene knockout/knockdown studies: Generate CV_3336 deletion mutants using CRISPR-Cas9 or homologous recombination. Compare phenotypes with wild-type C. violaceum across multiple growth conditions and stress responses.

• Transcriptomic analysis: Perform RNA-Seq comparing wild-type and CV_3336 mutant strains to identify differentially expressed genes, potentially revealing pathways associated with CV_3336 function.

• Protein-protein interaction studies: Implement pull-down assays or bacterial two-hybrid screening to identify interaction partners of CV_3336.

• Metabolomic profiling: Compare metabolite profiles between wild-type and mutant strains using LC-MS/MS to detect metabolic changes resulting from CV_3336 deletion.

• Complementation studies: Reintroduce wild-type CV_3336 and mutated versions (targeting predicted functional domains) to confirm phenotype restoration and identify critical residues.

Given C. violaceum's established quorum sensing system, special attention should be paid to potential interactions between CV_3336 and the CviI/R system, which regulates multiple phenotypes including violacein production, protease activity, and chitinolytic activity .

How might CV_3336 interact with C. violaceum's regulatory networks?

Based on our understanding of C. violaceum's regulatory systems, several potential experimental designs could reveal CV_3336's role in bacterial regulatory networks:

• Regulatory network examination: Investigate potential interactions between CV_3336 and known regulatory elements such as VioS (repressor of violacein biosynthesis) and the CviI/R quorum sensing system .

• Reporter fusion assays: Construct transcriptional fusions between promoters of interest (e.g., vioA, cviI, cviR) and reporter genes (gfp, lacZ) in both wild-type and CV_3336 mutant backgrounds to quantify regulatory effects.

• Chromatin immunoprecipitation (ChIP) analysis: If CV_3336 potentially functions as a DNA-binding protein, perform ChIP-seq to identify genomic binding sites.

• in vitro transcription assays: Reconstitute transcription machinery with purified RNA polymerase, potential promoter regions, and CV_3336 to directly test regulatory effects.

For experimental design, a multi-condition approach might reveal context-dependent functions:

This approach would provide comprehensive insights into how CV_3336 functions across different physiological states of C. violaceum.

How does the structure of CV_3336 relate to its function?

Structure-function analysis of CV_3336 should incorporate both computational and experimental approaches:

• Structural modeling: Generate a 3D model using AlphaFold2 or similar tools, focusing on the UPF0042 domain architecture and potential nucleotide-binding sites.

• Site-directed mutagenesis: Based on structural predictions, create point mutations in conserved residues, particularly those predicted to be involved in nucleotide binding. Express these mutants and assess their:

  • Ability to bind nucleotides (using methods described in section 2.3)

  • Capacity to complement phenotypes in CV_3336 knockout strains

  • Structural integrity through circular dichroism and thermal stability assessments

• X-ray crystallography or Cryo-EM: For definitive structural characterization, determine the high-resolution structure of CV_3336, ideally in both apo and nucleotide-bound states.

• Molecular dynamics simulations: Once structural data is available, simulate protein dynamics to understand conformational changes upon nucleotide binding and identify potential allosteric sites.

A systematic mutagenesis approach might include:

Note: These are hypothetical residues based on typical nucleotide-binding proteins. Actual critical residues would be determined through structural analysis.

How might CV_3336 contribute to C. violaceum pathogenicity?

Although C. violaceum is generally non-pathogenic, it can cause severe infections in humans, particularly in immunodeficient individuals . Investigating CV_3336's potential role in pathogenicity requires specialized approaches:

• Infection models: Compare virulence of wild-type and CV_3336 mutant strains using appropriate infection models such as:

  • Cell culture invasion assays with human epithelial cells

  • Caenorhabditis elegans infection model (previously used for C. violaceum virulence studies )

  • Murine infection models for systemic infection assessment

• Virulence factor expression: Analyze the expression of known virulence factors in the presence and absence of CV_3336:

  • Type III secretion system components, particularly Chromobacterium outer protein C (CopC), which has been shown to inactivate caspases and dysregulate programmed cell death in epithelial cells

  • Hemolysins and other cytotoxins

  • Antibiotic resistance determinants (C. violaceum is intrinsically resistant to penicillin, colistin, and most cephalosporins )

• Host-pathogen interaction studies: Investigate how CV_3336 might influence:

  • Bacterial survival within macrophages

  • Resistance to oxidative stress (particularly relevant for chronic granulomatous disease patients, who are susceptible to C. violaceum infections )

  • Biofilm formation in host-mimicking conditions

Example experimental progression table:

What structural biology techniques are most suitable for studying CV_3336?

For comprehensive structural characterization of CV_3336, researchers should consider a multi-technique approach:

• X-ray crystallography: The gold standard for high-resolution protein structures. Critical steps include:

  • Screening diverse crystallization conditions (typically 500-1000 initial conditions)

  • Testing both apo and nucleotide-bound forms

  • Optimizing crystals for diffraction beyond 2.5 Å resolution

  • Considering selenomethionine labeling for phase determination

• Cryo-electron microscopy (Cryo-EM): Particularly valuable if CV_3336 forms larger complexes or resists crystallization:

  • Sample preparation optimization (protein concentration, buffer conditions)

  • Screening for uniform particle distribution

  • High-resolution data collection and processing

• Nuclear Magnetic Resonance (NMR): For studying dynamic regions and ligand binding:

  • ¹⁵N/¹³C labeling for backbone and side-chain assignments

  • Titration experiments with potential nucleotide ligands

  • Relaxation measurements to identify flexible regions

• Small-angle X-ray scattering (SAXS): For low-resolution envelope determination and studying conformational changes:

  • Concentration series to detect aggregation

  • Comparison between apo and nucleotide-bound states

Decision matrix for selecting structural techniques:

How can researchers explore CV_3336's role in nucleotide metabolism?

As a UPF0042 nucleotide-binding protein, CV_3336 may play a role in C. violaceum's nucleotide metabolism. A comprehensive investigation should include:

• Nucleotide hydrolysis assays: Test whether CV_3336 possesses ATPase, GTPase, or other nucleotide hydrolysis activities:

  • Malachite green assay for phosphate release

  • HPLC analysis of reaction products

  • Coupled enzyme assays for real-time monitoring

• Metabolic flux analysis: Compare nucleotide metabolism in wild-type and CV_3336 mutant strains:

  • Label C. violaceum cultures with ¹³C-glucose or ¹⁵N-labeled precursors

  • Analyze metabolite labeling patterns by LC-MS/MS

  • Quantify differences in nucleotide synthesis and degradation rates

• Transcriptional response: Analyze how nucleotide pool imbalances affect CV_3336 expression:

  • Treat cells with nucleotide synthesis inhibitors (e.g., trimethoprim, azaserine)

  • Monitor CV_3336 expression using qRT-PCR or reporter constructs

  • Compare with known nucleotide metabolism genes

• Protein interaction network: Identify proteins that interact with CV_3336:

  • Co-immunoprecipitation followed by mass spectrometry

  • Bacterial two-hybrid screening

  • Proximity labeling approaches (e.g., BioID)

Example nucleotide hydrolysis assay findings (hypothetical):

How can researchers validate the specificity of anti-CV_3336 antibodies?

Generating and validating specific antibodies against CV_3336 requires a systematic approach:

• Antibody generation strategy:

  • Design immunogenic peptides from unique regions of CV_3336

  • Express full-length protein for immunization

  • Consider both polyclonal (higher sensitivity) and monoclonal (higher specificity) approaches

• Validation experiments:

  • Western blot analysis comparing wild-type and ΔCV_3336 strains

  • Preabsorption controls with purified CV_3336 protein

  • Immunoprecipitation followed by mass spectrometry verification

  • Immunofluorescence microscopy comparing signal in wild-type vs. mutant

• Cross-reactivity assessment:

  • Test against closely related bacterial species

  • Check reactivity with purified homologous proteins

  • Perform epitope mapping to confirm antibody binding sites

Comprehensive antibody validation should include:

A rigorous validation approach will ensure experimental results with these antibodies are reliable and reproducible.