Recombinant Chromobacterium violaceum Darcynin (CV_4311)

What is Darcynin CV_4311 and what are its basic characteristics?

Darcynin CV_4311 is a protein encoded by the CV_4311 gene in Chromobacterium violaceum (strain ATCC 12472 / DSM 30191 / JCM 1249 / NBRC 12614 / NCIMB 9131 / NCTC 9757). It belongs to the darcynin protein family with a molecular mass of 9.29 kDa. The protein consists of 79 amino acids with the following sequence: MKPIYARYPEVRMRWFDAEAFSARCSDVAMFETESVPAFYYLIDALRDSPLMTEPYFEFVDIIPAVEDGFRDYDAQLAQ . Understanding these basic characteristics is essential for designing experiments involving recombinant expression and functional studies of this protein.

How does Darcynin relate to other proteins in Chromobacterium violaceum?

Darcynin belongs to a specific protein family within C. violaceum, which is a gram-negative bacterium found in soil and freshwater environments in tropical and subtropical regions . While the specific function of Darcynin has not been fully characterized, C. violaceum produces various bioactive compounds including the purple pigment violacein, which has been extensively studied for its antibiotic properties and role in virulence . Researchers should consider Darcynin within the broader context of C. violaceum's protein expression profile, particularly when investigating potential interactions with other bacterial proteins or metabolites.

What is the genomic context of the CV_4311 gene in C. violaceum?

The CV_4311 gene encoding Darcynin is part of the C. violaceum genome, which has been fully sequenced for strain ATCC 12472. When designing primers for cloning or analyzing gene expression, researchers should consider the genomic context, including potential promoter regions and adjacent genes that might be functionally related. Unlike the well-characterized vioABCDE operon responsible for violacein biosynthesis , the regulatory elements controlling CV_4311 expression remain less defined. For comprehensive genomic context analysis, researchers should utilize genome browsers and bioinformatic tools specific to bacterial genomics.

What are the optimal expression systems for recombinant Darcynin production?

For recombinant expression of Darcynin, researchers should consider several expression systems based on the protein's characteristics. As a relatively small protein (79 amino acids, 9.29 kDa) , Darcynin is amenable to expression in bacterial systems like E. coli. The methodology should include:

• Vector selection: pET-based vectors for high-level expression or pGEX vectors for GST-fusion proteins to enhance solubility

• Host strain optimization: BL21(DE3) or Rosetta strains for codon optimization

• Induction conditions: IPTG concentration (0.1-1.0 mM), temperature (16-37°C), and duration (4-24 hours)

• Fusion tags: His6, GST, or MBP tags to facilitate purification and potentially enhance solubility

Expression protocols similar to those used for other small bacterial proteins should be adapted based on Darcynin's specific amino acid composition and predicted structural properties.

What purification strategies yield the highest purity and functional integrity of recombinant Darcynin?

Purification of recombinant Darcynin requires a strategic approach that maintains protein functionality while achieving high purity. Based on methodologies used for similar small bacterial proteins, the following multi-step purification protocol is recommended:

• Initial capture: Affinity chromatography using the appropriate resin based on the fusion tag (Ni-NTA for His-tagged protein, glutathione-sepharose for GST-fusion)

• Intermediate purification: Ion exchange chromatography based on Darcynin's theoretical isoelectric point

• Polishing step: Size exclusion chromatography to separate monomeric Darcynin from aggregates

• Tag removal: If necessary, using specific proteases (TEV, thrombin, or Factor Xa) followed by a second affinity step

Researchers should monitor protein purity via SDS-PAGE at each step and confirm the identity using western blotting or mass spectrometry. Functional integrity should be assessed through activity assays appropriate to the protein's predicted function.

How can solubility issues with recombinant Darcynin be addressed?

When encountering solubility challenges with recombinant Darcynin, researchers should implement a systematic approach to optimization:

• Solubility enhancement tags: Fusion with solubility-enhancing proteins (MBP, SUMO, Trx)

• Buffer optimization: Screen various buffers with different pH values (6.0-8.5), salt concentrations (50-500 mM NaCl), and additives (glycerol 5-15%, reducing agents)

• Expression conditions: Lower temperature (16°C) and reduced inducer concentration

• Co-expression with chaperones: GroEL/GroES, DnaK/DnaJ systems to assist proper folding

• Refolding protocols: If inclusion bodies form, develop a refolding strategy using gradual dialysis or on-column refolding techniques

For particularly challenging cases, consider orthogonal approaches such as cell-free expression systems or non-conventional hosts like Brevibacillus or Pseudomonas species that might better accommodate proteins from C. violaceum.

What are the predicted functional domains of Darcynin based on sequence analysis?

While specific functional information about Darcynin CV_4311 is limited in the available literature, sequence analysis and structural prediction approaches can provide valuable insights. Researchers should employ multiple bioinformatics tools to identify potential functional domains, including:

• Sequence homology analysis with other characterized proteins in the darcynin family

• Secondary structure prediction using algorithms like PSIPRED or JPred

• Motif scanning through databases like PROSITE, Pfam, or InterPro

• Ab initio structure prediction using tools like AlphaFold or I-TASSER

The resulting predictions should guide experimental approaches for functional characterization, including targeted mutagenesis of predicted active sites or interaction domains.

How does Darcynin potentially interact with other components in the C. violaceum cellular environment?

Understanding Darcynin's potential interactions requires a combination of computational predictions and experimental validation. Based on studies of other C. violaceum proteins, researchers should consider:

• Pull-down assays using tagged recombinant Darcynin to identify potential binding partners

• Bacterial two-hybrid systems to screen for protein-protein interactions

• Co-immunoprecipitation experiments if antibodies against Darcynin are available

• Cross-linking mass spectrometry to capture transient interactions

When interpreting results, consider the broader context of C. violaceum biology, particularly the quorum-sensing systems that regulate many cellular processes in this bacterium . Darcynin may function within pathways regulated by the CviI/R system or interact with components of violacein biosynthesis.

What experimental approaches can elucidate Darcynin's potential role in antimicrobial activity?

C. violaceum produces several compounds with antimicrobial properties, most notably violacein . To investigate whether Darcynin contributes to antimicrobial activity, researchers should consider these methodological approaches:

• Recombinant expression and purification of Darcynin for direct antimicrobial assays

• Minimum inhibitory concentration (MIC) determination against a panel of indicator organisms

• Time-kill kinetics to characterize the mode of action

• Membrane permeabilization assays using fluorescent probes

• Synergy testing with known antimicrobials including violacein

• Generation of CV_4311 knockout mutants in C. violaceum to assess changes in antimicrobial activity profiles

Results should be interpreted within the context of C. violaceum's established antimicrobial properties, considering potential synergistic effects with violacein or other bacterial components.

How can site-directed mutagenesis be applied to investigate structure-function relationships in Darcynin?

For systematic structure-function analysis of Darcynin, researchers should implement a comprehensive site-directed mutagenesis approach:

• Target selection: Based on bioinformatic analysis, prioritize conserved residues, predicted functional motifs, or charged/aromatic amino acids that may participate in interactions

• Mutagenesis strategy:

  • Alanine scanning of selected regions

  • Conservative substitutions to preserve chemical properties

  • Non-conservative substitutions to disrupt potential functions

• Functional assessment of mutants:

  • Expression and solubility analysis

  • Stability evaluation using thermal shift assays

  • Activity assays comparing wildtype and mutant proteins

• Structural validation using techniques like circular dichroism or, if possible, X-ray crystallography or NMR

This systematic approach allows researchers to map critical residues and domains to specific functions and can reveal mechanistic insights about Darcynin's biological role.

What comparative genomic approaches could reveal Darcynin homologs in other bacterial species?

To identify and characterize Darcynin homologs across bacterial species, researchers should implement a multi-faceted comparative genomics approach:

• Sequence-based searches:

  • BLAST searches against comprehensive databases (NCBI nr, UniProt)

  • PSI-BLAST for distant homologs

  • HMM-based searches using darcynin family profiles

• Phylogenetic analysis:

  • Multiple sequence alignment of identified homologs

  • Construction of phylogenetic trees to visualize evolutionary relationships

  • Reconciliation with species trees to identify potential horizontal gene transfer events

• Synteny analysis:

  • Examination of gene neighborhoods across species

  • Identification of conserved operonic structures

• Structural comparisons:

  • Analysis of predicted secondary structures

  • Comparison of conserved domains and motifs

This comprehensive approach can reveal evolutionary patterns and potentially identify functional homologs in clinically relevant or environmentally important bacterial species.

What high-throughput screening approaches can identify potential Darcynin inhibitors or activators?

For researchers interested in identifying molecules that modulate Darcynin activity, several high-throughput screening (HTS) methodologies can be implemented:

• Activity-based screens:

  • Development of a robust biochemical assay reflecting Darcynin's function

  • Adaptation to microplate format for automated screening

  • Primary screening of diverse compound libraries (natural products, synthetic libraries)

• Binding-based screens:

  • Thermal shift assays to detect ligand-induced stability changes

  • Surface plasmon resonance for direct binding detection

  • Fluorescence polarization assays if Darcynin can be fluorescently labeled

• Cell-based screens:

  • Reporter systems in recombinant bacteria expressing Darcynin

  • Phenotypic screens measuring changes in relevant cellular processes

• In silico screening:

  • Structure-based virtual screening if Darcynin structure is available

  • Pharmacophore-based approaches using known interacting molecules

Hit validation should include dose-response analysis, mechanism of action studies, and selectivity profiling against related proteins.

How can researchers address the challenge of potential contamination with violacein when working with C. violaceum extracts?

When working with native Darcynin from C. violaceum extracts, contamination with violacein presents a significant challenge due to its intense purple pigmentation and potential interference with assays . Researchers should implement these methodological solutions:

• Separation strategies:

  • Differential solubility exploitation (violacein is poorly soluble in aqueous buffers)

  • Sequential extraction procedures targeting different cellular compartments

  • Chromatographic separation optimized for violacein removal

• Genetic approaches:

  • Use of violacein-deficient C. violaceum strains (vioABCDE mutants)

  • Controlled expression systems to minimize violacein production

• Analytical considerations:

  • Selection of detection wavelengths that minimize violacein interference

  • Internal controls to account for violacein background

  • Mass spectrometry-based approaches for specific detection of Darcynin

These methodologies ensure more accurate characterization of Darcynin without interference from violacein's strong chromophoric properties.

What strategies can overcome the challenge of potential endotoxin contamination in recombinant Darcynin preparations?

For applications requiring endotoxin-free Darcynin preparations, especially in immunological studies or potential therapeutic investigations, researchers should implement a comprehensive endotoxin management strategy:

• Prevention during expression:

  • Use of endotoxin-free growth media and reagents

  • Selection of low-endotoxin E. coli strains (ClearColi)

  • Consideration of gram-positive expression hosts as alternatives

• Removal during purification:

  • Triton X-114 phase separation

  • Polymyxin B-based affinity chromatography

  • Ion exchange chromatography under conditions that separate proteins from endotoxins

  • Ultrafiltration with specialized membranes

• Validation:

  • LAL (Limulus Amebocyte Lysate) testing to confirm endotoxin levels

  • Endotoxin-specific enzymatic assays

  • Cell-based assays to confirm lack of endotoxin effects

A typical endotoxin removal workflow should achieve levels below 0.1 EU/mg protein for research applications and even lower thresholds for potential therapeutic applications.

What analytical methods are most appropriate for confirming the structural integrity of recombinant Darcynin?

Comprehensive structural characterization of recombinant Darcynin requires multiple complementary analytical approaches:

• Primary structure confirmation:

  • Mass spectrometry (LC-MS/MS) for accurate mass determination and sequence coverage

  • N-terminal sequencing to confirm the correct start site

  • Peptide mapping following enzymatic digestion

• Secondary structure analysis:

  • Circular dichroism (CD) spectroscopy to estimate α-helix, β-sheet, and random coil content

  • Fourier-transform infrared (FTIR) spectroscopy as a complementary approach

• Tertiary structure assessment:

  • Intrinsic tryptophan fluorescence to probe the folding environment

  • Dynamic light scattering to assess homogeneity and aggregation state

  • Limited proteolysis to identify stable domains and flexible regions

• Higher-order structure (if applicable):

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

  • Analytical ultracentrifugation for oligomerization state determination

Each method provides distinct and complementary information, and researchers should select appropriate techniques based on available instrumentation and specific research questions.

How might Darcynin expression be regulated in the context of C. violaceum quorum sensing systems?

C. violaceum utilizes a sophisticated quorum sensing (QS) system based on the CviI/R proteins, which regulates various phenotypes including violacein production . Investigating potential QS regulation of Darcynin expression requires a multi-faceted approach:

• Promoter analysis:

  • Bioinformatic identification of potential QS-responsive elements upstream of CV_4311

  • Construction of reporter fusions (e.g., CV_4311 promoter::lacZ or ::gfp)

  • Measurement of promoter activity in wildtype vs. QS mutants (ΔcviI, ΔcviR)

• Transcriptional analysis:

  • RT-qPCR to measure CV_4311 mRNA levels under various conditions

  • RNA-seq comparison of wildtype vs. QS mutants

  • Temporal expression analysis during bacterial growth phases

• Protein expression correlation:

  • Western blot or mass spectrometry-based proteomics

  • Comparison of Darcynin levels in response to exogenous AHLs

  • Analysis in vioS mutants to examine potential co-regulation with violacein

These approaches can reveal whether Darcynin belongs to the QS regulon and potentially shares regulatory mechanisms with the violacein biosynthetic pathway.

What is the potential role of Darcynin in C. violaceum pathogenicity?

Although C. violaceum rarely causes human infections, it can lead to severe and often fatal disease with high mortality rates . Investigating Darcynin's potential contribution to pathogenicity requires:

• Virulence model systems:

  • Construction of CV_4311 deletion mutants

  • Complementation studies to confirm phenotypes

  • Comparison of virulence in appropriate infection models

• Host-interaction studies:

  • Macrophage infection assays (survival, cytokine production)

  • Neutrophil interaction (phagocytosis, killing, NET formation)

  • Epithelial cell adhesion and invasion assays

• Potential virulence mechanisms:

  • Assessment of cytotoxicity against various cell types

  • Evaluation of immunomodulatory properties

  • Investigation of potential antimicrobial resistance contributions

When studying potential virulence roles, researchers should be mindful of C. violaceum's pathogenicity profile, including septicemia and abscess formation in visceral organs following skin inoculation .

What are the comparative expression yields of Darcynin across different recombinant systems?

The following data table summarizes predicted expression yields based on similar proteins from C. violaceum in various expression systems:

Note: Actual yields may vary based on specific constructs, expression conditions, and optimization efforts. This table provides a starting point for system selection based on specific research requirements.

What are the predicted structural properties of Darcynin based on bioinformatic analysis?

This predictive structural analysis provides a foundation for experimental characterization and suggests Darcynin may function as a cytoplasmic protein with possible roles in protein-protein interactions rather than enzymatic activity.

How does Darcynin compare to other characterized proteins from C. violaceum?

This comparative analysis places Darcynin in the broader context of C. violaceum proteins:

This comparative framework helps researchers place Darcynin studies within the broader context of C. violaceum biology and suggests potential functional relationships to investigate.