Recombinant Desulfovibrio vulgaris Flagellar assembly factor FliW (fliW)

What is Flagellar assembly factor FliW in Desulfovibrio vulgaris?

FliW in D. vulgaris (UniProt: Q72EP7) is a 173-amino acid protein that functions as a specialized chaperone in flagellar assembly. The protein's primary role involves regulating flagellin production and maintaining flagellar protein homeostasis. According to the recombinant protein data sheet, the full amino acid sequence of FliW from D. vulgaris strain Hildenborough is: "MARQNEIEIQTRIGRQRITLDKIIHFPRGLAGEFDFRHDFTLLQLREGAPFLVLQSLDDPGLGLLVADPYSFLTDYQIRVGDPEQRLLKLENIRQVAVLVTVSIPAGQPEKTALNLTGPILINHRARIGLQVPQTDASLPPQFYLHMDDANGSTTVRRKAS PPAAGEDKGDVQE" . The protein typically acts as a flagellin-specific chaperone, regulating both the assembly process and gene expression of flagellar components.

How does FliW contribute to D. vulgaris pathogenicity?

FliW indirectly contributes to D. vulgaris pathogenicity through its regulation of flagellar assembly and flagellin production. Recent studies have established connections between D. vulgaris and inflammatory bowel conditions, particularly ulcerative colitis, where the overgrowth of Desulfovibrio has been documented in patient samples . The flagellin protein from D. vulgaris (DVF) has been specifically identified as interacting with host immune receptors like leucine-rich repeat containing 19 (LRRC19), promoting inflammatory responses . By regulating flagellin production and ensuring proper flagellar assembly, FliW plays a critical upstream role in this pathogenic mechanism. Researchers investigating this relationship typically employ mouse models of colitis and intestinal organoid cultures to assess the impact of wild-type versus mutant D. vulgaris strains on inflammatory markers and tissue damage.

What genetic systems exist for studying FliW function in D. vulgaris?

Several advanced genetic tools have been developed for D. vulgaris that enable detailed study of proteins like FliW:

• Markerless deletion system: A sophisticated genetic manipulation approach using uracil phosphoribosyltransferase (upp) as a counterselectable marker has been established for D. vulgaris. This system allows for the creation of clean gene deletions without retention of antibiotic resistance markers, enabling multiple sequential genetic modifications in a single strain .

• Efficient transformation methods: Researchers have developed strains with enhanced transformation efficiency, such as JW7035 (ΔhsdR), which demonstrates 100-1,000 times greater transformation rates than wild-type strains. This improvement facilitates the introduction of plasmid constructs for genetic complementation and protein expression studies .

• Expression systems: Constitutive promoters like aph(3′)-II have been validated in D. vulgaris, with plasmids containing the endogenous SRB cryptic plasmid pBG1 capable of stable replication in this organism .

These tools collectively provide the methodological foundation for creating fliW deletion mutants, complementation strains, and expression constructs needed for comprehensive functional characterization.

What are the optimal conditions for expression and purification of recombinant D. vulgaris FliW?

The optimal expression and purification of recombinant D. vulgaris FliW requires careful consideration of multiple parameters:

Expression Systems:

• Yeast expression systems have been successfully employed for production of D. vulgaris FliW, as documented in commercial preparations .

• E. coli-based systems using BL21(DE3) strains with T7 promoter vectors represent an alternative approach.

Purification Strategy:

• Affinity chromatography using appropriate fusion tags (His-tag is commonly employed)

• Size exclusion chromatography for enhanced purity

• Quality control assessment via SDS-PAGE to verify >85% purity

Critical Considerations:

• Buffer composition significantly impacts protein stability; recommended storage includes 5-50% glycerol at -20°C/-80°C

• Shelf life is approximately 6 months for liquid preparations and 12 months for lyophilized preparations

• Repeated freeze-thaw cycles should be avoided; working aliquots can be maintained at 4°C for up to one week

For optimal results, systematic optimization of induction conditions, growth temperatures, and buffer compositions should be conducted, with functional validation performed immediately after purification.

How can researchers verify the functionality of purified recombinant FliW?

Verification of recombinant D. vulgaris FliW functionality requires a multi-faceted approach:

Biophysical Characterization:

• Circular dichroism (CD) spectroscopy to confirm proper secondary structure

• Thermal shift assays to evaluate protein stability

• Limited proteolysis to assess structural integrity

Functional Assays:

• Binding studies with D. vulgaris flagellin using surface plasmon resonance (SPR) to determine binding kinetics

• Pull-down experiments to verify protein-protein interactions

• Size exclusion chromatography to detect complex formation

Complementation Studies:

• Introduction of recombinant FliW into FliW-deficient strains to assess restoration of flagellar function

• Motility assays using soft agar to quantify complementation efficiency

• Flagellin expression analysis to confirm regulatory function

A comprehensive verification protocol should employ multiple methods to establish both structural integrity and functional activity, focusing particularly on the chaperone and regulatory roles of FliW in flagellar assembly.

What experimental approaches can differentiate FliW function from other flagellar proteins?

Distinguishing the specific functions of FliW from other flagellar proteins requires sophisticated experimental designs:

Genetic Approaches:

• Construction of clean deletion mutants using the markerless deletion system developed for D. vulgaris

• Creation of conditional expression systems to modulate FliW levels

• Synthetic genetic array analysis to identify genetic interactions specific to FliW

Biochemical Methods:

• Comparative proteomic analysis of wild-type versus ΔfliW strains

• Protein-protein interaction mapping using crosslinking mass spectrometry

• In vitro reconstitution assays with purified components to determine order of assembly

Structural Biology Techniques:

• Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Co-crystallization with binding partners to define molecular contacts

• FRET-based assays to monitor protein interactions in real-time

These approaches collectively allow researchers to delineate the specific contributions of FliW to flagellar assembly and regulation, differentiating its roles from those of other flagellar proteins in D. vulgaris.

How can researchers investigate the interaction between FliW and host immune responses?

Investigating the relationship between D. vulgaris FliW and host immune responses requires sophisticated immunological approaches:

In vitro Methods:

• Co-culture systems with intestinal epithelial cells or immune cells

• Comparison of responses to wild-type D. vulgaris versus ΔfliW mutants

• Purified protein stimulation assays to distinguish direct effects of FliW from those of flagellin

Ex vivo Approaches:

• Intestinal organoid cultures to model epithelial-bacterial interactions

• Precision-cut intestinal slices to preserve tissue architecture

• Surface plasmon resonance to measure potential interactions with immune receptors like LRRC19

In vivo Models:

• Mouse models of colitis with controlled colonization by D. vulgaris strains

• Gnotobiotic mouse systems to eliminate variables from other microbiota

• Tissue-specific knockouts of relevant immune receptors

Recent studies have demonstrated that D. vulgaris flagellin (DVF) interacts with the immune receptor LRRC19, promoting inflammatory responses in ulcerative colitis . While FliW itself may not directly interact with host receptors, its regulation of flagellin expression makes it an important indirect modulator of these immunological processes. Researchers should focus on comparative studies between wild-type and genetically modified strains to elucidate these relationships.

What structural features of D. vulgaris FliW determine its functional specificity?

The structural determinants of D. vulgaris FliW functional specificity can be investigated through several complementary approaches:

Key Structural Elements:

• The flagellin-binding domain

• Potential regulatory interfaces with other flagellar components

• Regions involved in post-transcriptional regulation

Advanced Structural Analysis Methods:

• X-ray crystallography or cryo-electron microscopy to determine high-resolution structure

• Hydrogen-deuterium exchange mass spectrometry to identify binding interfaces

• Molecular dynamics simulations to predict conformational changes

• Site-directed mutagenesis of conserved residues followed by functional assays

Comparative Structural Analysis:
Creating a systematic comparison between FliW proteins from different species can provide insights into D. vulgaris-specific adaptations. This approach should include:

• Multiple sequence alignments to identify conserved and variable regions

• Homology modeling based on known structures

• Heterologous complementation experiments to test functional conservation

Understanding these structural features provides mechanistic insights into how FliW functions within the specific context of D. vulgaris flagellar assembly and may reveal potential targets for selective inhibition in therapeutic contexts.

How does the genetic context of fliW influence its expression and regulation?

The genetic context of fliW in D. vulgaris significantly impacts its expression patterns and regulatory mechanisms:

Genomic Organization Analysis:

• Operon structure determination through RNA-seq and transcription start site mapping

• Identification of promoter elements and transcription factor binding sites

• Characterization of untranslated regions that may contain regulatory elements

Transcriptional Regulation Approaches:

• Promoter fusion reporter assays under varying environmental conditions

• ChIP-seq to identify transcription factors binding to the fliW promoter region

• In vitro DNA-protein interaction studies to confirm direct regulation

Post-transcriptional Control Mechanisms:

• mRNA stability assays to assess transcript half-life

• Investigation of potential small RNA regulators

• Analysis of translation efficiency through ribosome profiling

Environmental Response Characterization:

• Quantitative RT-PCR to measure fliW expression under different growth conditions

• Comparison of expression patterns in planktonic versus biofilm growth states

• Analysis of flagellar gene expression coordination during environmental transitions

Understanding these regulatory mechanisms provides insights into how D. vulgaris coordinates flagellar assembly with other cellular processes and environmental adaptations, potentially revealing points of intervention for modulating pathogenicity.

What are common challenges in producing high-quality recombinant D. vulgaris FliW?

Researchers frequently encounter several technical challenges when working with recombinant FliW from D. vulgaris:

Expression and Purification Issues:

Storage and Stability Considerations:

• Glycerol concentration in the range of 5-50% significantly improves stability during freezing

• Lyophilization can extend shelf life to approximately 12 months at -20°C/-80°C

• Dividing purified protein into single-use aliquots prevents degradation from repeated freeze-thaw cycles

Quality Control Approaches:

• Multiple purification steps to achieve >85% purity as verified by SDS-PAGE

• Western blotting to confirm protein identity

• Mass spectrometry to verify sequence integrity

• Activity assays immediately following purification to establish baseline functionality

Implementing these systematic troubleshooting approaches can significantly improve both the yield and quality of recombinant FliW preparations for experimental applications.

How can researchers optimize transformation efficiency for genetic studies in D. vulgaris?

Optimizing transformation efficiency is critical for genetic manipulation of D. vulgaris, particularly when studying genes like fliW:

Strain Engineering Approaches:

• Utilization of restriction-deficient strains like JW7035 (ΔhsdR) has been demonstrated to increase transformation efficiency by 100-1,000 fold compared to wild-type strains

• Additional improvements may be achieved by eliminating the native plasmid pDV1, which contains a type II restriction modification system

Transformation Protocol Optimization:

• Cell preparation: Harvest cells in exponential growth phase (OD600 0.3-0.5)

• Washing procedure: Multiple washes with electroporation buffer to remove salts

• DNA preparation: Use high-quality plasmid DNA at optimal concentrations (0.5-1 μg)

• Electroporation parameters: Systematic testing of voltage, resistance, and capacitance

• Recovery conditions: Immediate transfer to pre-warmed media post-electroporation

Comparative Transformation Efficiencies:
Published research demonstrates dramatic differences in transformation efficiency between strains:

This data, derived from published research , highlights the significant improvements possible through strategic strain selection and protocol optimization.

How can researchers resolve contradictory data when studying FliW function?

When confronted with contradictory results in FliW functional studies, researchers should implement a systematic approach:

Technical Validation:

• Verify reagent quality and experimental conditions

• Implement appropriate controls in each experiment

• Use multiple independent methods to measure the same parameter

• Conduct dose-response and time-course experiments to identify conditional effects

Biological Context Assessment:

• Consider strain-to-strain variations in D. vulgaris isolates

• Evaluate growth conditions and their impact on flagellar expression

• Examine potential polar effects in genetic constructs

• Assess the presence/absence of the native plasmid pDV1 and its impact on phenotypes

Advanced Analytical Approaches:

• Single-cell analyses to detect population heterogeneity

• Multi-omics integration to place contradictions in broader biological context

• In vitro reconstitution with purified components to isolate variables

• Complementation studies with controlled expression levels

Methodological Framework:

• Document all experimental parameters meticulously

• Implement systematic hypothesis testing

• Design experiments specifically to distinguish between alternative explanations

• Consider collaborating with groups using complementary techniques

By applying these structured approaches, researchers can resolve contradictions and develop a more coherent understanding of FliW function in the biology of D. vulgaris.

What are promising approaches for developing inhibitors targeting FliW in D. vulgaris?

Developing targeted inhibitors of D. vulgaris FliW represents a promising research direction with potential therapeutic applications:

Target-Based Screening Approaches:

• Structure-based virtual screening once high-resolution structures become available

• Fragment-based drug discovery to identify initial binding molecules

• Peptide inhibitor design based on interaction interfaces

• Allosteric inhibitor development to modulate protein function indirectly

Phenotypic Screening Strategies:

• High-content screening using motility as a readout

• Flagellar assembly monitoring through microscopy or reporter systems

• Competitive binding assays with natural ligands

• Cell-based assays measuring flagellin levels or secretion

Optimization Considerations:

• Selectivity against homologous proteins in commensal bacteria

• Stability in the gut environment

• Bacterial membrane penetration

• Minimization of resistance development potential

Recent research connecting D. vulgaris to inflammatory conditions like ulcerative colitis provides a compelling rationale for developing such inhibitors, as blocking flagellar assembly could potentially reduce the inflammatory potential of this bacterium in the gut environment.

How might comparative studies of FliW across bacterial species inform D. vulgaris research?

Comparative studies of FliW proteins across bacterial species can provide valuable insights for D. vulgaris research:

Evolutionary Analysis:

• Phylogenetic analysis to trace the evolutionary history of FliW proteins

• Identification of conserved domains versus species-specific adaptations

• Correlation of FliW variants with flagellar structure and function across species

Functional Conservation Assessment:

• Heterologous complementation experiments between different bacterial species

• Cross-species binding studies with cognate flagellins

• Comparative analysis of regulatory mechanisms across diverse bacteria

Structural Insights:

• Comparative modeling based on solved structures from other species

• Identification of unique structural features in D. vulgaris FliW

• Prediction of functional adaptations based on structural differences

Translational Applications:

• Identification of broadly applicable versus species-specific inhibitor strategies

• Understanding of resistance mechanisms based on natural variations

• Prediction of potential off-target effects on commensal bacteria

This comparative approach provides an evolutionary context for understanding D. vulgaris FliW function and may reveal both fundamental mechanisms and specialized adaptations relevant to this sulfate-reducing bacterium's lifestyle and pathogenic potential.

What role might FliW play in D. vulgaris biofilm formation and persistence?

The potential role of FliW in D. vulgaris biofilm formation and persistence represents an important area for future investigation:

Mechanistic Connections:

• Flagella are known to contribute to initial surface attachment in many bacteria

• By regulating flagellar assembly, FliW may indirectly influence attachment efficiency

• Transition from motile to sessile lifestyle often involves flagellar regulation

• Biofilm matrix components may interact with or be regulated by flagellar systems

Experimental Approaches:

• Comparative biofilm assays between wild-type and ΔfliW mutants

• Live cell imaging to track flagellar dynamics during biofilm formation

• Transcriptomic analysis to identify co-regulated genes during attachment

• Flow cell systems to assess biofilm development under shear stress

Relevance to D. vulgaris Ecology and Pathogenicity:

• Biofilms may contribute to persistence in hostile environments like the inflamed gut

• Altered metabolism in biofilms may influence hydrogen sulfide production

• Biofilm formation may affect interactions with other microbiota members

• Biofilm-associated D. vulgaris may display altered inflammatory potential

Understanding these relationships could provide important insights into D. vulgaris persistence in environmental and host contexts, potentially revealing new approaches for modulating its presence in inflammatory conditions like ulcerative colitis where Desulfovibrio overgrowth has been documented .