Recombinant Chromobacterium violaceum Flagellar assembly factor FliW (fliW)

What is the primary function of FliW in Chromobacterium violaceum?

FliW in C. violaceum functions as a flagellar assembly factor that binds to flagellin protein. It plays a critical role in a partner switching mechanism where FliW binds flagellin while the global regulator CsrA binds flagellin mRNA to regulate its expression. This coordinated interaction helps synchronize flagellin production with the capacity of the Type III Secretion System (T3SS) to secrete flagellin, ensuring efficient flagellar assembly .

How does FliW interact with flagellin in C. violaceum?

FliW binds specifically to the N-terminus of flagellin, forming a binding interface distinct from that of the FliS chaperone which binds to the C-terminus. This spatial arrangement allows both proteins to bind simultaneously to flagellin, forming a heterotrimeric FliC-FliS-FliW complex. Small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments have confirmed this complex formation, which subsequently interacts with the export gate protein FlhA .

What is the relationship between FliW and bacterial motility in C. violaceum?

C. violaceum is motile with a single polar flagellum and one to four lateral flagella, all essential for bacterial movement. The FliW protein helps regulate flagellin availability for flagellar assembly, thereby directly influencing bacterial motility. Proper regulation of flagellar components is critical as C. violaceum uses its motility to navigate through soil and water environments where it naturally resides .

How is C. violaceum classified taxonomically and what are its key characteristics?

Chromobacterium violaceum is a beta proteobacterium that produces violet-colored colonies due to the pigment violacein. It is a Gram-negative, rod-shaped, facultative anaerobic bacterium that is non-sporing. C. violaceum is motile with flagella and pili, and is positive for oxidase and catalase. It is predominantly found in tropical and subtropical regions within soil and water ecosystems .

How does the partner switching mechanism between FliW and CsrA regulate flagellin production?

The partner switching mechanism involving FliW and CsrA functions as a post-transcriptional regulatory system. When FliW binds to flagellin protein, it is unavailable to interact with CsrA. Consequently, CsrA remains free to bind flagellin mRNA, repressing its translation. Conversely, when flagellin levels decrease, FliW is released and can bind to CsrA, preventing it from repressing flagellin mRNA. This creates a feedback loop that maintains appropriate flagellin levels for efficient flagellar assembly. Research approaches to study this mechanism include protein-protein interaction assays, RNA binding studies, and quantitative proteomics .

What is the structural basis for the FliW-flagellin interaction and how does it compare across bacterial species?

While the search results don't provide specific structural data for C. violaceum FliW, comparative structural biology approaches would be valuable for understanding the FliW-flagellin interaction. Researchers could use X-ray crystallography, cryo-EM, or NMR spectroscopy to determine the structure of recombinant C. violaceum FliW alone and in complex with flagellin fragments. Homology modeling based on related FliW proteins could predict binding interfaces, which could then be validated through site-directed mutagenesis and binding affinity measurements .

How is FliW expression coordinated with the virulence mechanisms of C. violaceum?

C. violaceum possesses multiple virulence mechanisms, including T3SS (divided into Cpi-1 and Cpi-2), quorum sensing systems (CviI/CviR), and production of the pigment violacein. The coordination between flagellar assembly (involving FliW) and these virulence systems likely involves complex regulatory networks. Research could explore whether the quorum sensing system that regulates violacein production and biofilm formation also influences FliW expression. Transcriptomic and proteomic analyses under various environmental conditions could reveal co-regulation patterns between flagellar genes and virulence factors .

How does the heterotrimeric FliC-FliS-FliW complex interact with the FlhA export gate?

The heterotrimeric complex formed by FliC (flagellin), FliS (chaperone), and FliW interacts with the export gate protein FlhA as part of the flagellar export process. Research indicates that FliS and FliW are likely released during flagellin export through the T3SS. Investigators could employ protein-protein interaction studies, including pull-down assays, surface plasmon resonance, and hydrogen-deuterium exchange mass spectrometry to characterize the binding kinetics and conformational changes during this interaction. Mutagenesis of key residues could identify the specific interfaces involved in complex formation and disassembly .

What are the optimal conditions for expressing recombinant C. violaceum FliW in heterologous systems?

For recombinant expression of C. violaceum FliW, researchers should consider the following protocol:

• Expression System Selection:

  • E. coli BL21(DE3) is recommended for initial expression trials

  • Consider C. violaceum's GC content (~64.83%) when optimizing codon usage for the expression host

• Expression Conditions:

  • Induce at OD₆₀₀ 0.6-0.8 with 0.1-0.5 mM IPTG

  • Lower induction temperature (16-25°C) may improve solubility

  • Cultivate in rich media (e.g., LB or 2xYT) supplemented with appropriate antibiotics

• Protein Solubility Enhancement:

  • Test fusion tags (His, GST, MBP) to improve solubility

  • Screen various buffer conditions (pH 6.0-8.0, 50-200 mM NaCl)

  • Consider co-expression with chaperones if solubility is problematic

What purification strategy is most effective for obtaining high-purity recombinant FliW?

A multi-step purification approach is recommended for obtaining high-purity recombinant FliW:

• Initial Capture:

  • Affinity chromatography using His-tag (IMAC) or GST-tag

  • Recommended binding buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

• Intermediate Purification:

  • Ion exchange chromatography (considering FliW's predicted pI)

  • Protease cleavage to remove fusion tags if necessary

  • Size exclusion chromatography to separate monomeric from aggregated forms

• Quality Control:

  • SDS-PAGE and Western blotting to confirm purity and identity

  • Dynamic light scattering to assess homogeneity

  • Mass spectrometry to verify intact mass and post-translational modifications

How can researchers effectively study the interaction between FliW and flagellin in vitro?

To study FliW-flagellin interactions, the following methodological approaches are recommended:

• Protein-Protein Interaction Assays:

  • Surface Plasmon Resonance (SPR) to determine binding kinetics (kon, koff) and affinity (KD)

  • Isothermal Titration Calorimetry (ITC) to measure thermodynamic parameters (ΔH, ΔS, ΔG)

  • Microscale Thermophoresis (MST) for rapid screening of binding conditions

• Structural Studies:

  • HDX-MS to map binding interfaces and conformational changes

  • SAXS to characterize complex formation in solution

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

• Functional Assays:

  • Pull-down assays using recombinant proteins to validate direct interactions

  • Fluorescence-based assays (FRET) to monitor complex formation in real-time

  • Analytical ultracentrifugation to determine stoichiometry and complex stability

What methods are recommended for studying the role of FliW in C. violaceum flagellar assembly in vivo?

For in vivo studies of FliW function in C. violaceum:

• Genetic Manipulation:

  • Gene knockout/knockdown strategies using homologous recombination

  • CRISPR-Cas9 system for precise genome editing

  • Complementation with wild-type or mutant alleles to confirm phenotypes

• Phenotypic Characterization:

  • Motility assays (swimming and swarming) on semi-solid agar

  • Electron microscopy to visualize flagellar structure

  • High-speed video microscopy to quantify swimming behavior

• Molecular Monitoring:

  • Fluorescent protein fusions to track subcellular localization

  • Quantitative RT-PCR to measure gene expression changes

  • Proteomic analysis to detect alterations in the flagellar protein assembly

How should researchers interpret changes in flagellar assembly when FliW is mutated or deleted?

When analyzing the effects of FliW mutations or deletion on flagellar assembly:

• Direct Effects Assessment:

  • Quantify flagellin levels in cellular fractions (cytoplasmic, membrane, secreted)

  • Measure flagellar length and number using electron microscopy

  • Evaluate flagellar gene expression changes through transcriptomics

• Functional Consequences Analysis:

  • Correlate swimming speed with flagellar structural changes

  • Assess biofilm formation capacity, as altered motility may impact attachment

  • Examine changes in virulence using infection models

• Molecular Mechanism Interpretation:

  • Monitor CsrA activity on flagellin mRNA in wild-type versus mutant backgrounds

  • Investigate compensatory mechanisms that may arise in FliW mutants

  • Analyze the formation of the heterotrimeric FliC-FliS-FliW complex

What statistical approaches are most appropriate for analyzing FliW-flagellin binding data?

For rigorous analysis of FliW-flagellin binding data:

• Binding Kinetics Analysis:

  • Use non-linear regression models to fit SPR or ITC data

  • Apply Scatchard or Hill plots to assess cooperativity

  • Implement global fitting approaches for multiple datasets

• Comparative Statistics:

  • ANOVA with post-hoc tests for comparing multiple binding conditions

  • Student's t-test (paired or unpaired) for two-condition comparisons

  • Non-parametric tests (Mann-Whitney, Kruskal-Wallis) for non-normally distributed data

• Data Visualization:

  • Create dose-response curves with 95% confidence intervals

  • Use residual plots to assess goodness of fit

  • Generate Bland-Altman plots to compare different binding assay methods

How can researchers distinguish direct FliW effects from indirect consequences in flagellar assembly studies?

To differentiate between direct and indirect effects of FliW on flagellar assembly:

• Temporal Analysis:

  • Conduct time-course experiments to identify primary versus secondary effects

  • Use pulse-chase labeling to track protein synthesis and deployment

  • Implement inducible expression systems for controlled FliW depletion/restoration

• Pathway Dissection:

  • Use epistasis analysis with mutations in other flagellar genes

  • Create point mutations that specifically disrupt individual protein interactions

  • Employ chemical genetics with small molecule inhibitors of specific pathway steps

• Systems Biology Approaches:

  • Integrate transcriptomic, proteomic, and metabolomic data

  • Construct network models of flagellar assembly regulations

  • Use mathematical modeling to predict direct versus cascade effects

What is the relationship between FliW function and C. violaceum pathogenicity in host infection models?

To investigate the relationship between FliW and C. violaceum pathogenicity:

What emerging technologies could advance our understanding of FliW function in C. violaceum?

Several cutting-edge technologies offer promising approaches for deeper insights into FliW function:

• Single-molecule techniques:

  • Single-molecule FRET to observe FliW-flagellin interactions in real-time

  • Optical tweezers to measure forces involved in protein-protein binding

  • Super-resolution microscopy to visualize flagellar assembly dynamics

• Systems-level approaches:

  • Genome-wide CRISPRi screens to identify genetic interactions with FliW

  • Proteome-wide thermal shift assays to detect FliW-dependent protein stability changes

  • Cross-linking mass spectrometry to map the complete FliW interaction network

• Computational methods:

  • Molecular dynamics simulations to predict conformational changes upon binding

  • Machine learning algorithms to identify patterns in large-scale FliW-related datasets

  • Integrative modeling combining low and high-resolution structural data

How might the study of FliW contribute to broader understanding of bacterial pathogenesis?

Understanding FliW function has implications beyond flagellar assembly:

• Virulence regulation mechanisms:

  • Elucidation of how post-transcriptional regulation via FliW-CsrA coordinates with quorum sensing systems

  • Insights into synchronization between motility and virulence factor expression

  • Understanding of niche adaptation strategies through motility regulation

• Host-pathogen interactions:

  • Mechanisms of flagellin recognition by host NAIP-NLRC4 inflammasomes

  • Role of flagellar regulation in immune evasion strategies

  • Contribution to bacterial persistence in various host environments

• Therapeutic targets:

  • Potential for anti-virulence strategies targeting flagellar assembly without selecting for resistance

  • Design of specific inhibitors for the FliW-flagellin interaction

  • Development of attenuated strains for vaccine candidates