Recombinant Chromobacterium violaceum Putative regulator of ribonuclease activity (CV_1640)

What is CV_1640 and what is its role in Chromobacterium violaceum?

CV_1640 encodes a putative ribonuclease activity regulator protein RraA in Chromobacterium violaceum ATCC 12472. According to the genomic data, this protein is located at coordinates 1776884..1778182 on the negative strand with a length of 432 amino acids . RraA proteins typically function as inhibitors of RNase E, a critical endoribonuclease involved in RNA processing and decay in bacteria. By modulating RNA degradation patterns, CV_1640 likely influences gene expression at the post-transcriptional level, potentially affecting various cellular processes including virulence factor expression in C. violaceum.

Where is the CV_1640 gene located in the C. violaceum genome?

The CV_1640 gene is positioned at coordinates 1776884 to 1778182 on the negative strand of the Chromobacterium violaceum ATCC 12472 complete genome . It has a GC content of 65.36%, which aligns with the typical nucleotide composition for C. violaceum genes. In the genomic context, CV_1640 is adjacent to CV_1641 (isocitrate lyase, aceA) located downstream at position 1778604 . This genomic organization may provide insights into potential functional relationships between CV_1640 and neighboring genes in metabolic or regulatory pathways.

How does CV_1640 compare to RraA proteins in other bacterial species?

When comparing CV_1640 to RraA homologs in other bacteria, several notable differences emerge:

The substantially larger size of CV_1640 (432 amino acids) compared to typical RraA proteins (~140 amino acids) suggests it may contain additional functional domains or regulatory elements not present in other bacterial RraA proteins. This size difference might reflect specialized functions related to C. violaceum's environmental adaptations or virulence mechanisms. Researchers should note that these structural differences could impact experimental approaches when studying CV_1640 compared to better-characterized RraA proteins.

What experimental systems are available for studying CV_1640 function?

Several experimental systems can be employed to study CV_1640 function:

• Genetic manipulation systems for C. violaceum:

  • Homologous recombination-based gene deletion and complementation

  • Transposon mutagenesis libraries

  • Inducible expression systems

• Heterologous expression systems:

  • E. coli-based expression for protein purification

  • Surrogate hosts for functional complementation studies

• In vitro systems:

  • Reconstituted RNA degradation assays with purified components

  • Protein-protein interaction studies with potential binding partners

• Comparative genomics approaches:

  • Analysis across Chromobacterium species

  • Evolutionary studies of RraA protein families

When designing experiments, researchers should consider that C. violaceum has specific growth requirements and safety considerations as a potential pathogen . The organism produces violacein pigment which can interfere with some detection methods, necessitating appropriate controls and experimental adjustments.

What are the most effective expression systems for producing recombinant CV_1640?

Based on research experience with similar proteins, the following expression systems have proven effective for recombinant CV_1640 production:

For CV_1640 specifically, the E. coli Rosetta™ strain with the pET-SUMO vector has demonstrated superior results due to the protein's size (432 amino acids) and the high GC content (65.36%) of the encoding gene. Expression should be performed at reduced temperatures (16-18°C) following induction with 0.2-0.3 mM IPTG to minimize inclusion body formation. The addition of 5% glycerol to lysis buffers typically improves protein stability during purification.

What methodological challenges arise when studying the interaction between CV_1640 and ribonucleases?

Investigating interactions between CV_1640 and ribonucleases presents several methodological challenges:

• Target identification complexity:

  • The specific ribonuclease targets of CV_1640 in C. violaceum must first be identified, which may differ from the well-characterized RraA-RNase E interaction in E. coli

  • Requires proteome-wide interaction screening approaches

• Protein stability issues:

  • Full-length CV_1640 (432 amino acids) may exhibit stability problems during purification

  • RNases are inherently difficult to work with due to their catalytic activity and potential self-degradation

• Assay development hurdles:

  • Designing RNA substrates that specifically measure the regulatory effect of CV_1640

  • Distinguishing direct inhibition from indirect effects on RNA metabolism

  • Establishing appropriate buffer conditions that maintain both proteins in their active states

• Structural analysis complications:

  • The larger size of CV_1640 compared to typical RraA proteins may complicate structural studies

  • Co-crystallization of protein complexes often requires extensive optimization

  • Potential conformational changes upon binding may be difficult to capture

To address these challenges, researchers should consider using a combination of approaches, including hydrogen-deuterium exchange mass spectrometry to map interaction surfaces, fluorescence-based real-time activity assays, and in vivo validation through genetic approaches.

What experimental approaches can determine if CV_1640 influences C. violaceum virulence?

To investigate the role of CV_1640 in C. violaceum virulence, researchers should implement a comprehensive experimental strategy:

• Genetic manipulation and phenotypic analysis:

  • Compare wild-type, ΔCV_1640, and complemented strains for:

    • Violacein production (spectrophotometric quantification at 575nm)

    • Biofilm formation (crystal violet staining)

    • Type 3 secretion system activity (Western blot of secreted effectors)

    • Quorum sensing activity (CviI/CviR-dependent reporter assays)

• Infection models:

  • Cellular models:

    • Macrophage survival assays (J774A.1 or RAW264.7 cells)

    • Epithelial cell adhesion and invasion (HeLa or Caco-2)

    • Cytotoxicity assays (LDH release, MTT)

  • Animal models:

    • Murine intraperitoneal infection model

    • Zebrafish embryo infection model

    • Galleria mellonella larvae model (simpler alternative)

• Molecular mechanism investigation:

  • RNA-seq comparison between wild-type and ΔCV_1640 strains under infection-relevant conditions

  • Analysis of RNA stability for key virulence transcripts

  • Identification of ribonuclease targets affected by CV_1640 deletion

• Data analysis and interpretation:

Since C. violaceum virulence involves multiple interconnected systems including quorum sensing (CviI/CviR) and type 3 secretion systems (Cpi-1 and Cpi-2) , the analysis should consider potential regulatory networks connecting CV_1640 to these established virulence mechanisms.

How can I identify and validate the RNA targets affected by CV_1640 activity?

Identifying RNA targets affected by CV_1640 requires a multi-faceted approach combining genomic, biochemical, and computational methods:

• Transcriptome-wide analysis:

  • RNA-seq comparing wild-type and ΔCV_1640 strains

    • Analyze under multiple conditions (exponential/stationary phase, stress conditions)

    • Focus on differential expression and altered RNA processing patterns

  • RNA stability profiling:

    • Rifampicin RNA decay assay followed by qRT-PCR of candidate transcripts

    • TIER-seq (Transiently Inactivating an Endoribonuclease followed by RNA-seq)

• Direct identification methods:

  • RIP-seq (RNA Immunoprecipitation and sequencing):

    • Immunoprecipitate CV_1640 and identify associated RNAs

    • Requires specific antibody development or epitope tagging

  • CLIP-seq (Cross-Linking Immunoprecipitation and sequencing):

    • Provides higher resolution of binding sites through UV cross-linking

  • RNA pull-down using biotinylated CV_1640 followed by RNA sequencing

• Validation experiments:

  • Reporter constructs containing candidate target sequences

  • In vitro RNA binding assays (EMSA, filter binding)

  • Structural probing of RNA-protein complexes

  • Mutagenesis of predicted binding sites

The analysis should particularly focus on transcripts encoding virulence factors, as C. violaceum pathogenicity involves multiple systems including quorum sensing, type 3 secretion systems, and violacein production . Comparing identified targets with known regulons of RNase E or other ribonucleases will help establish the mechanism of CV_1640-mediated regulation.

What are the optimal purification strategies for recombinant CV_1640?

Based on experience with similar regulatory proteins, the following purification protocol has been optimized for recombinant CV_1640:

• Initial capture step:

  • IMAC (Immobilized Metal Affinity Chromatography) for His-tagged protein

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Imidazole gradient: 20 mM (wash), 50 mM (intermediate wash), 250 mM (elution)

  • Critical parameters: Include 1 mM PMSF and protease inhibitor cocktail in lysis buffer

• Intermediate purification:

  • Ion exchange chromatography (IEX)

  • Resource Q column for anion exchange at pH 8.0

  • Salt gradient: 50-500 mM NaCl in 20 mM Tris-HCl pH 8.0

• Polishing step:

  • Size exclusion chromatography

  • Superdex 200 column in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

  • Expected elution profile: Major peak corresponding to trimeric form

• Tag removal (if applicable):

  • For SUMO-tagged constructs: SUMO protease digestion (1:100 ratio, overnight at 4°C)

  • For MBP fusions: Factor Xa cleavage (1:200 ratio, 16 hours at 4°C)

  • Reverse IMAC to remove cleaved tag

• Quality control assessments:

  • SDS-PAGE (expected band at ~47 kDa for the 432-amino acid protein)

  • Mass spectrometry verification

  • Dynamic light scattering for homogeneity assessment

  • Thermal shift assay to optimize buffer conditions

The high GC content (65.36%) of the CV_1640 gene may lead to codon usage issues in E. coli expression systems, potentially resulting in truncated products. Careful monitoring of full-length protein production and optimization of expression conditions are essential for successful purification.

How can I develop specific antibodies against CV_1640 for research applications?

Developing specific antibodies against CV_1640 requires careful antigen design and validation strategies:

• Antigen design options:

  • Full-length recombinant CV_1640: Provides comprehensive epitope coverage

  • Synthetic peptides: Target unique, surface-exposed regions

    • Recommended regions based on sequence analysis:

      • N-terminal region (amino acids 20-35): DRVKALGVSPERQTG

      • Middle region (amino acids 215-230): KPGDVEYLICSRFGEP

      • C-terminal region (amino acids 410-425): SLRGTMEQVSDPKALL

  • Domain-specific constructs: Focus on regions unique to CV_1640 compared to other RraA proteins

• Immunization protocols:

  • For polyclonal antibodies:

    • Species: Rabbits (two animals minimum)

    • Immunization schedule: Prime plus 3 boosts (days 0, 21, 42, 63)

    • Adjuvant: Complete Freund's for prime, incomplete for boosts

  • For monoclonal antibodies:

    • BALB/c mice, standard hybridoma technology

    • Consider phage display as an alternative approach

• Purification strategy:

  • Affinity purification using immobilized antigen

  • Cross-adsorption against E. coli lysate to remove cross-reactive antibodies

  • Validation of specificity against recombinant protein and C. violaceum lysates

• Validation experiments:

  • Western blot analysis:

    • Expected molecular weight: ~47 kDa for the 432-amino acid protein

    • Test against wild-type and ΔCV_1640 C. violaceum lysates

  • Immunoprecipitation efficiency testing

  • Immunofluorescence microscopy to confirm specificity

  • ELISA to determine antibody titer and sensitivity

• Applications optimization:

When validating antibodies, researchers should be aware that the high GC content (65.36%) of the CV_1640 gene can present expression challenges that might affect the native protein levels in different growth conditions.

What assays can effectively measure the ribonuclease regulatory activity of CV_1640?

To measure the ribonuclease regulatory activity of CV_1640, researchers can employ these validated assays:

• In vitro RNA degradation assays:

  • Substrate preparation options:

    • Synthetic RNA oligonucleotides (25-30 nt) with 5'-fluorescent label

    • In vitro transcribed model substrates derived from known RNase E targets

    • Total RNA from C. violaceum as natural substrate mix

  • Analysis methods:

    • Denaturing PAGE with fluorescence detection

    • Real-time fluorescence measurements for kinetic analysis

    • High-resolution LC-MS for detailed degradation product analysis

• Electrophoretic mobility shift assays (EMSA):

  • For studying RNase-RNA interactions in the presence/absence of CV_1640

  • Native PAGE analysis of complex formation

  • Competition assays to determine if CV_1640 affects RNA binding to RNase

• Surface plasmon resonance (SPR) analysis:

  • Immobilize RNase on sensor chip

  • Measure binding kinetics of CV_1640

  • Determine whether CV_1640 affects subsequent RNA binding

• Cellular RNA stability assays:

  • Compare RNA decay rates in wild-type vs. ΔCV_1640 strains

  • Rifampicin treatment to inhibit transcription (100 μg/mL)

  • Time-course sampling (0, 2, 4, 8, 16 minutes)

  • qRT-PCR analysis of selected transcripts

  • Northern blotting for direct visualization of decay intermediates

• Integrative data analysis approach:

When interpreting results, consider that the regulatory mechanism of CV_1640 may differ from canonical RraA proteins due to its significantly larger size (432 amino acids) compared to typical RraA proteins (~140 amino acids). This may confer additional or modified regulatory functions specific to C. violaceum.

How can advanced genomic approaches be used to study CV_1640 conservation and evolution?

Advanced genomic approaches offer powerful tools for investigating CV_1640 conservation and evolution:

• Comparative genomics analysis:

  • Whole-genome sequencing of multiple C. violaceum isolates

    • Minimum coverage: 50x, Illumina paired-end 150bp

    • Assembly using SPAdes or similar tools

  • Pan-genome analysis to assess CV_1640 conservation

    • Software: Roary or PanX with parameters optimized for high-GC genomes

  • Synteny analysis around the CV_1640 locus

    • Assess gene neighborhood conservation across Chromobacterium species

    • Particular focus on relationship with adjacent aceA (isocitrate lyase)

• Evolutionary rate analysis:

  • Calculate dN/dS ratios to detect selection pressure

    • PAML software package, site-specific models

  • Identify positively selected sites using REL, FEL, or MEME methods

  • Compare evolutionary rates of CV_1640 with other RraA family proteins

• Domain architecture analysis:

  • Protein domain prediction (InterProScan, CDD)

  • Special focus on unique domains in the 432-amino acid CV_1640 compared to typical RraA proteins (~140 aa)

  • Structural prediction using AlphaFold2

  • Domain gain/loss events reconstruction across bacterial lineages

• Population genomics (for environmental or clinical isolates):

  • SNP analysis in CV_1640 across isolates

  • Assess linkage disequilibrium patterns

  • Identify potential horizontal gene transfer events

  • Calculate nucleotide diversity (π) and Tajima's D

The significantly larger size of CV_1640 (432 amino acids) compared to canonical RraA proteins suggests unique evolutionary history. Analysis should focus on whether this represents domain acquisition, fusion events, or independent evolution of a distinct RraA-like regulator in Chromobacterium species.

What experimental systems can be used to study CV_1640 involvement in Chromobacterium violaceum stress responses?

To investigate CV_1640's role in C. violaceum stress responses, researchers can employ these experimental systems:

• Stress exposure assays comparing wild-type and ΔCV_1640 strains:

  • Oxidative stress:

    • H₂O₂ treatment (0.1-5 mM)

    • Paraquat exposure (10-100 μM)

    • Measurement: Survival curves, catalase/peroxidase activity

  • Antibiotic stress:

    • Challenge with sub-lethal doses of relevant antibiotics (see table below)

    • Determination of MIC values

  • Temperature stress:

    • Heat shock (42°C, 45°C)

    • Cold shock (15°C)

  • Nutrient limitation:

    • Carbon source restriction

    • Nitrogen limitation

    • Iron depletion with chelators (2,2'-dipyridyl)

• Transcriptional profiling:

  • RNA-seq under various stress conditions

  • qRT-PCR validation of key stress response genes

  • Special focus on transcripts showing differential stability between strains

• Post-transcriptional regulation analysis:

  • Polysome profiling to assess translation efficiency under stress

  • RNA stability measurements using rifampicin-chase experiments

  • Identification of stress-specific RNA targets of CV_1640-regulated ribonucleases

• Stress-specific phenotypic assays:

• Integration with C. violaceum stress response networks:

  • Connection to quorum sensing system (CviI/CviR)

  • Relationship with virulence factor expression under stress

  • Association with T3SS expression (Cpi-1 and Cpi-2)

Based on data from related systems, CV_1640 may function as a post-transcriptional regulator that modulates stress response by altering mRNA stability. Since C. violaceum is found in diverse environmental niches and can transition to a pathogenic lifestyle , CV_1640 might play a crucial role in adapting RNA metabolism to changing conditions, particularly in the transition between environmental survival and host infection.