Recombinant Chromobacterium violaceum UPF0597 protein CV_1824 (CV_1824)

What is the UPF0597 protein CV_1824 from Chromobacterium violaceum?

CV_1824 is a protein belonging to the UPF0597 family found in Chromobacterium violaceum (strain ATCC 12472 / DSM 30191 / JCM 1249 / NBRC 12614 / NCIMB 9131 / NCTC 9757). It is a 430-amino acid protein with a molecular mass of approximately 44.365 kDa . The protein is classified as a member of the UPF0597 family, a group of proteins with currently uncharacterized function (UPF stands for Uncharacterized Protein Family). While its specific function remains to be fully elucidated, studying this protein may provide insights into C. violaceum's biology and pathogenicity.

What is known about Chromobacterium violaceum and its significance in research?

Chromobacterium violaceum is a rod-shaped, Gram-negative, facultatively anaerobic bacterium with a cosmopolitan distribution. Despite relatively few reported human infections (approximately 160 cases globally), it can cause deadly septicemia and infections in multiple organs including the lungs, liver, brain, spleen, and lymphatic systems . The bacterium produces violacein, a purple pigment with antimicrobial properties that helps it compete with other bacteria in ecological niches. C. violaceum has gained significant research interest as a model organism for studying quorum sensing mechanisms, as evidenced by the increasing number of publications in the past decade . Its growing resistance to multiple antibiotics makes it an important subject for antimicrobial research.

How is the CV_1824 protein's structure characterized?

The CV_1824 protein consists of 430 amino acids with the following sequence:
MSEREVRLWPEFVKALKQEVVPALGCTEPISLALAAALAARELGKAPERIDAWVSANLMKNGMGVTVPGTGTVGLPIAAAVGALGGDPDAKLEVLKNLTVEQVAAGKQMLADGKVKLGVAAVPNILYAEACVWHGDECARVAIADAHTNVIKIELNGEVKLKREAADAKPVETYDLGDATARDVYDFAMRAPLDSIAFIHDAAVLNSALADEGMSGKYGLHIGATLQRQIEAGLLSEGLLSNILTRTTAASDARMGGATLPAMSNSGSGNQGIAATMPVVAVAEHVKADRETLIRALALSHLIAVYIHTRLPKLSALCAVTTASMGAAAGMAQLLNGGYPAVSMAISSMIGDLAGMICDGASNSCAMKVSTSAGSGYKAVLMALDGTRVTGNEGIVAHDVDVSIANLGKLATQGMAQTDTQILQIMMDKR

The structural characterization would typically involve X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-electron microscopy to determine its three-dimensional structure. Bioinformatic analyses using tools like SWISS-MODEL, Phyre2, or I-TASSER can predict the tertiary structure based on homology with known protein structures. Circular dichroism spectroscopy can provide information about secondary structure elements (α-helices, β-sheets, random coils).

What experimental designs are recommended for studying CV_1824 function?

When designing experiments to elucidate CV_1824 function, a multi-faceted approach is recommended:

• Gene Knockout Studies: Creating CV_1824 deletion mutants in C. violaceum and comparing phenotypes with wild-type strains. This requires:

  • Using CRISPR-Cas9 or homologous recombination techniques

  • Analyzing growth curves, biofilm formation, and virulence in infection models

  • Measuring violacein production and quorum sensing activities

• Protein-Protein Interaction Studies:

  • Yeast two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

  • Bacterial two-hybrid systems

  • Proximity-dependent biotin identification (BioID)

• Transcriptomic Analysis:

  • RNA-Seq comparing wild-type and knockout strains under various conditions

  • qRT-PCR validation of differentially expressed genes

The experimental design should follow a completely randomized design with appropriate biological and technical replicates (minimum n=3) to allow for robust statistical analysis using ANOVA and post-hoc tests .

How should recombinant CV_1824 protein expression be optimized?

Optimizing recombinant CV_1824 expression requires systematic evaluation of multiple expression parameters:

• Expression System Selection:

  • Bacterial systems: E. coli BL21(DE3), Rosetta, or Arctic Express for difficult proteins

  • Eukaryotic systems: Yeast (P. pastoris), insect cells (Sf9, Hi5), or mammalian cells

• Vector Design:

  • Incorporate appropriate fusion tags (6xHis, GST, MBP) to aid solubility and purification

  • Consider codon optimization for the expression host

  • Include TEV or thrombin cleavage sites for tag removal

• Expression Conditions Matrix:

  Parameter	Variables to Test
  Temperature	16°C, 25°C, 30°C, 37°C
  Induction time	4h, 8h, 16h, 24h
  Inducer concentration	0.1mM, 0.5mM, 1.0mM IPTG
  Media	LB, TB, 2YT, M9, auto-induction
  Cell density at induction	OD600 0.4-0.6, 0.8-1.0, >1.5

• Solubility Enhancement:

  • Addition of solubility enhancers (sorbitol, glycerol, arginine)

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Testing various lysis buffers with different pH, salt concentrations, and additives

Statistical design of experiments (DoE) approaches should be employed to efficiently identify optimal conditions with minimal experiments .

What analytical techniques are most appropriate for characterizing CV_1824 protein function?

A comprehensive analytical approach should include:

• Structural Analysis:

  • X-ray crystallography or Cryo-EM for high-resolution structure determination

  • Circular dichroism for secondary structure assessment

  • Thermal shift assays to evaluate protein stability

  • Small-angle X-ray scattering (SAXS) for solution conformation

• Biochemical Assays:

  • Enzyme activity assays (if enzymatic function is suspected)

  • Binding assays using isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR)

  • Limited proteolysis to identify flexible regions

• Cellular Localization:

  • Immunofluorescence microscopy with anti-CV_1824 antibodies

  • Subcellular fractionation followed by western blotting

  • GFP-fusion protein localization studies

• Functional Genomics:

  • Transcriptome analysis (RNA-Seq) comparing wild-type and CV_1824 knockout strains

  • Chromatin immunoprecipitation sequencing (ChIP-Seq) if DNA-binding is suspected

Data from these techniques should be analyzed using appropriate statistical methods, including t-tests for pairwise comparisons and ANOVA for multiple conditions, with significance typically set at p<0.05 .

How can researchers effectively analyze the relationship between CV_1824 and virulence mechanisms?

To analyze the relationship between CV_1824 and virulence mechanisms, researchers should:

• Infection Models:

  • In vitro: Human and animal cell line infection assays measuring cellular invasion, cytotoxicity, and inflammatory responses

  • In vivo: Mouse infection models comparing wild-type and CV_1824 knockout strains

• Virulence Factor Analysis:

  • Measure production of known virulence factors (violacein, biofilm, OMVs) in wild-type vs. mutant strains

  • Analyze T3SS effector protein production and secretion in relation to CV_1824 expression levels

  • Assess quorum sensing activity using reporter assays for CviI/CviR system

• Host Response Studies:

  • Evaluate NLRC4 inflammasome activation and pyroptosis in response to wild-type vs. CV_1824 mutants

  • Measure cytokine production (particularly IL-18) and NK cell activation

  • Assess bacterial clearance in tissue-specific infection models

• Correlation Analysis:

  • Use multivariate statistical approaches to identify correlations between CV_1824 expression levels and various virulence metrics

  • Employ principal component analysis to identify patterns across multiple variables

Data should be analyzed using mixed-effects models to account for both fixed and random effects across experiments, with appropriate corrections for multiple comparisons .

How might CV_1824 interact with the quorum sensing system in C. violaceum?

The relationship between CV_1824 and the CviI/CviR quorum sensing system can be investigated through:

• Gene Expression Analysis:

  • qRT-PCR to determine if CV_1824 expression changes in response to AHL signal molecules

  • RNA-Seq to identify co-regulated genes in the quorum sensing regulon

  • Promoter-reporter assays to examine if CV_1824 expression is directly regulated by CviR

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation experiments to detect physical interaction between CV_1824 and CviR or other quorum sensing components

  • Bacterial two-hybrid assays to confirm direct protein interactions

  • FRET or BRET assays to detect interactions in living cells

• Functional Analysis:

  • Measurement of violacein production in CV_1824 knockout vs. wild-type strains

  • Biofilm formation assays to determine if CV_1824 affects this quorum-regulated process

  • Chromatin immunoprecipitation to identify if CviR binds to the CV_1824 promoter region

Since the CviI/CviR system regulates multiple virulence factors including biofilm formation and violacein biosynthesis, understanding CV_1824's role in this network could provide insights into novel therapeutic approaches targeting bacterial communication .

What is the potential role of CV_1824 in outer membrane vesicle formation?

To investigate CV_1824's role in OMV formation:

• Structural Analysis:

  • Determine if CV_1824 contains transmembrane domains or membrane association motifs

  • Bioinformatic comparison with known OMV-associated proteins

• OMV Isolation and Characterization:

  • Compare OMV production between wild-type and CV_1824 knockout strains using ultracentrifugation and nanoparticle tracking analysis

  • Analyze protein and lipid composition of OMVs from both strains using proteomics and lipidomics

  • Electron microscopy to assess morphological differences in OMVs

• OMV Functional Studies:

  • Compare antimicrobial activity of OMVs from wild-type and mutant strains

  • Assess violacein content in OMVs using spectrophotometric and HPLC analyses

  • Evaluate OMV-mediated DNA and protein transfer capabilities

Given that C. violaceum uses OMVs to deliver violacein to competing bacteria and that OMV secretion is controlled by the quorum sensing system, CV_1824 might play a role in this process, potentially as part of the VacJ/Yrb system that modulates OMV secretion .

How should contradictory results in CV_1824 functional studies be approached?

When encountering contradictory results:

• Systematic Verification:

  • Repeat experiments with additional biological and technical replicates

  • Vary experimental conditions to identify context-dependent effects

  • Use alternative methodologies to measure the same parameters

• Statistical Reassessment:

  • Apply more rigorous statistical tests (e.g., non-parametric methods for non-normal data)

  • Consider Bayesian approaches to incorporate prior knowledge

  • Use bootstrap or permutation tests when parametric assumptions are violated

• Reconciliation Strategies:

  • Develop testable hypotheses that could explain the apparent contradictions

  • Consider strain-specific or condition-specific effects

  • Investigate possible post-translational modifications or alternative splicing

  • Examine if protein complex formation affects function in context-dependent ways

• Meta-analysis Approach:

  • Systematically compare methodologies, strains, and conditions across studies

  • Identify patterns that might explain variable results

  • Construct a decision tree to guide future experimental design

A well-designed factorial experiment can help identify interaction effects between variables that might explain contradictory results .

What are the major challenges in determining the physiological relevance of CV_1824 in C. violaceum pathogenesis?

Key challenges include:

• Model System Limitations:

  • Human infections are rare, making clinical correlations difficult

  • Animal models may not fully recapitulate human infection dynamics

  • In vitro systems lack the complexity of host-pathogen interactions

• Functional Redundancy:

  • Multiple proteins may compensate for CV_1824 absence

  • Knockout phenotypes might be subtle or context-dependent

  • Genetic compensation mechanisms may mask the protein's true role

• Environmental Variability:

  • C. violaceum inhabits diverse ecological niches with varying selection pressures

  • Laboratory conditions may not reflect natural environments

  • Strain variations might affect the relevance of findings across isolates

• Methodological Approaches:

  Challenge	Potential Solution
  Low protein expression	Optimize codons, use stronger promoters
  Protein instability	Test various buffer conditions, add stabilizing agents
  Lack of functional assays	Develop high-throughput screening approaches
  Low infection rate in models	Consider alternative infection routes or sensitizing hosts

Addressing these challenges requires a multidisciplinary approach combining molecular biology, structural biology, immunology, and computational biology techniques.