Recombinant Desulfovibrio vulgaris Flagellar P-ring protein (flgI)

Basic Research Questions

• What is the biological function of FlgI in Desulfovibrio vulgaris?

  FlgI is a critical component of the bacterial flagellar motor in D. vulgaris, forming the P-ring structure located in the peptidoglycan layer. The P-ring functions as a bushing that allows smooth rotation of the flagellar rod and is required for proper flagellar assembly and motility . Disruption of FlgI causes a motility defect because the flagellar construction terminates at the rod structure, preventing formation of the hook and filament . In anaerobic sulfate-reducing bacteria like D. vulgaris, flagellar motility plays an important role in seeking optimal environmental conditions for growth and metabolism.

• What expression systems have been successfully used for recombinant D. vulgaris FlgI production?

  Escherichia coli has been successfully used as an expression system for recombinant D. vulgaris FlgI . In particular, the protein can be produced with tags such as His6 for purification purposes. Additionally, functional expression of D. vulgaris genes in Desulfovibrio desulfuricans has been demonstrated for other proteins, suggesting this could be a viable alternative expression system for obtaining properly folded and functionally active FlgI . When expressing the protein, it's important to consider the potential need for proper formation of disulfide bonds, as these may contribute to protein stability .

• How can I verify the purity and integrity of recombinant FlgI preparations?

  The purity of recombinant FlgI can be verified using standard SDS-PAGE analysis, with the protein appearing at approximately 38-39 kDa . Western blotting using anti-FlgI antibodies provides additional confirmation of identity. For structural integrity assessment, circular dichroism spectroscopy can evaluate secondary structure elements. Functional integrity can be assessed through complementation studies in FlgI-deficient strains, examining restoration of motility or flagellar structure formation. Mass spectrometry is recommended for confirming the exact molecular weight and potential post-translational modifications .

Advanced Research Questions

• What mutagenesis strategies are most effective for studying functional regions of D. vulgaris FlgI?

  Based on studies with E. coli FlgI, systematic cysteine substitution mutagenesis has proven highly effective for identifying functional regions . This approach involves:

  1. Designing substitutions at every 10th residue throughout the protein

  2. Using PCR-based site-directed mutagenesis with appropriate primers

  3. Verifying mutations by DNA sequencing

  4. Expressing mutants in a ΔflgI background strain

  5. Assessing protein expression levels by immunoblotting

  6. Evaluating motility through swarm assays and microscopy

  7. Analyzing solvent accessibility of different residues using thiol-specific labeling reagents like methoxypolyethylene glycol 5000 maleimide

  This comprehensive approach allows classification of residues into functional categories: those critical for protein stability, those essential for motility, and those exposed on the protein surface .

• How can I investigate FlgI interactions with other flagellar components in D. vulgaris?

  To investigate FlgI interactions with other flagellar components in D. vulgaris, researchers should consider:

  1. Tagless protein complex isolation: Utilize the "tagless" process developed for system-wide isolation of native membrane protein complexes in D. vulgaris to identify interacting partners .

  2. Co-immunoprecipitation: Express FlgI with a tag (His6 or Strep-tag II) to enable pulldown experiments. This approach successfully identified interactions between FlgT and MotY in Vibrio species .

  3. Two-dimensional heteronuclear NMR: This technique effectively mapped interaction sites between cytochrome c553 and formate dehydrogenase in D. vulgaris and could be adapted for FlgI interaction studies .

  4. Bacterial two-hybrid system: Using a specialized system for membrane proteins can help identify protein-protein interactions in vivo.

  5. Crosslinking experiments: Chemical crosslinking followed by mass spectrometry analysis can capture transient or weak interactions in the native membrane environment .

• What are the key differences between FlgI in D. vulgaris compared to other bacterial species?

  FlgI serves as the P-ring component across flagellated bacteria, but several key differences exist between D. vulgaris and other species:

  1. Structural association: In Vibrio species, FlgI associates with additional components like FlgT to form part of the H ring structure, which reinforces flagellar attachment to the cell envelope. D. vulgaris lacks the FlgT component present in Vibrio .

  2. Conserved regions: The N-terminal region (residues 1-120) of FlgI is highly conserved across species and particularly important for protein stability and P-ring formation, but specific sequence variations occur between species .

  3. Disulfide bonding: While intramolecular disulfide bond formation in E. coli FlgI is not necessary for P-ring assembly, it protects against protein degradation. The importance of disulfide bonds may differ in D. vulgaris given its anaerobic lifestyle .

  4. Genetic context: The genomic organization around flgI varies between species, potentially affecting co-regulation with other flagellar genes .

• How can I study the role of FlgI in D. vulgaris motility through genetic approaches?

  To investigate FlgI's role in D. vulgaris motility through genetic approaches:

  1. Generate targeted gene deletions or insertions: Using techniques like the λ red recombinase system adapted for D. vulgaris, researchers can create ΔflgI mutants .

  2. Create random transposon mutant libraries: Random barcode transposon-site sequencing (RB-TnSeq) has been successfully applied in D. vulgaris and can identify the effects of FlgI disruption across many growth conditions .

  3. Complementation studies: Reintroducing wild-type or mutant FlgI using vectors like pVB1 to verify phenotype restoration .

  4. Reporter gene fusions: Construct translational fusions with reporters like β-glucuronidase (uidA) to study flgI expression under different conditions .

  5. Conditional expression systems: Develop systems for controlled expression to study the effects of FlgI depletion or overexpression.

  Technique	Advantages	Challenges	Applications
  Targeted gene deletion	Precise modification	Requires homologous recombination	Functional analysis
  RB-TnSeq	High-throughput phenotyping	Complex data analysis	Fitness under multiple conditions
  Complementation	Confirms gene function	Potential expression artifacts	Structure-function studies
  Reporter fusions	Studies gene expression	May affect protein function	Regulatory analysis

• What are the most effective methods for isolating intact flagellar basal bodies containing FlgI from D. vulgaris?

  Isolating intact flagellar basal bodies containing FlgI from D. vulgaris requires specialized techniques:

  1. Cell growth optimization: Grow D. vulgaris under conditions that promote flagellar expression, typically in anaerobic media with lactate as carbon source and sulfate as electron acceptor .

  2. Cell harvesting and lysis: Carefully harvest cells and perform gentle lysis to preserve fragile structures. Methods using lysozyme treatment have been shown to preserve membrane association of protein complexes in D. vulgaris better than mechanical disruption methods like French press .

  3. Differential centrifugation: Perform sequential centrifugation steps to separate cellular components.

  4. Density gradient ultracentrifugation: Use cesium chloride or sucrose gradients to purify basal body structures.

  5. Detergent treatment: Carefully optimize detergent concentrations to solubilize membrane components while preserving protein-protein interactions.

  6. Quality control: Verify basal body integrity through electron microscopy and immunoblotting for key components including FlgI .

  The purified basal bodies can be analyzed by SDS-PAGE and stained to identify components, with further confirmation through N-terminal amino acid sequencing or mass spectrometry .

• How does environmental sulfide exposure affect FlgI expression and function in D. vulgaris?

  As an anaerobic sulfate-reducing bacterium, D. vulgaris is regularly exposed to sulfide, which may impact FlgI expression and function:

  1. Redox regulation: Sulfide can influence the redox state of proteins containing cysteine residues. Similar to DsrC in D. vulgaris, which undergoes redox-dependent conformational changes involving disulfide bond formation, FlgI function may be modulated by environmental sulfide levels through effects on disulfide bonding .

  2. Gene expression effects: Systematic studies using RB-TnSeq in D. vulgaris have enabled measurement of gene fitness contributions under various stress conditions. Similar approaches could reveal how sulfide exposure affects FlgI expression .

  3. Structural modifications: Sulfide might induce post-translational modifications affecting FlgI stability or interactions. Techniques like redox state-specific gel-shift assays (similar to those developed for DsrC) could monitor these changes .

  4. Metabolic integration: Sulfide production is central to D. vulgaris metabolism, and flagellar activity may be coordinated with energy generation. Studying FlgI under varying sulfate reduction conditions could reveal functional linkages between motility and energy metabolism.

  To investigate these effects, researchers should monitor FlgI expression, redox state, and flagellar assembly under controlled sulfide concentrations using techniques like qRT-PCR, redox-sensitive protein labeling, and electron microscopy.

• What techniques can be employed to study the interaction between the FlgI P-ring and the peptidoglycan layer in D. vulgaris?

  Studying the interaction between the FlgI P-ring and the peptidoglycan layer in D. vulgaris requires specialized approaches:

  1. Cryo-electron tomography: This technique can visualize the native arrangement of the flagellar basal body within the cell envelope, showing how the P-ring interfaces with the peptidoglycan layer.

  2. Peptidoglycan binding assays: Purified recombinant FlgI can be tested for direct binding to isolated peptidoglycan using co-sedimentation or surface plasmon resonance.

  3. Protein crosslinking in situ: Chemical crosslinkers that target specific functional groups can be used to capture interactions between FlgI and peptidoglycan components before cell lysis.

  4. Domain mapping: Creating truncated versions of FlgI can help identify specific regions involved in peptidoglycan interaction, similar to the approach used for "chopped" rubrerythrin in D. vulgaris .

  5. Fluorescence microscopy: Fluorescently tagged FlgI combined with peptidoglycan-specific dyes can reveal co-localization patterns.

  6. Comparative analysis: Comparing the behavior of D. vulgaris FlgI with orthologs from other bacteria that have different peptidoglycan compositions can provide insights into adaptation mechanisms .

• How can I design experiments to understand the role of FlgI in D. vulgaris pathogenicity or inflammation in host systems?

  Recent research indicates that D. vulgaris may contribute to gut inflammation , making it important to understand how flagellar components like FlgI might influence host-microbe interactions:

  1. In vitro cell culture models: Expose intestinal epithelial cell lines to purified recombinant FlgI and measure inflammatory markers (IL-1β, TNF-α, IL-10) to determine if FlgI directly triggers inflammatory responses.

  2. Mutant comparison studies: Compare wild-type D. vulgaris with ΔflgI mutants in their ability to:

     • Adhere to epithelial cells

     • Induce pro-inflammatory cytokine production

     • Activate pattern recognition receptors like TLR5 (which recognizes flagellin)

     • Stimulate immune cell recruitment

  3. Animal models: Use DSS-induced colitis models as described in the search results to compare colonization and inflammatory potential of wild-type versus ΔflgI D. vulgaris strains.

  4. Ex vivo tissue studies: Expose intestinal tissue samples to different D. vulgaris strains and measure markers of inflammation and tissue damage.

  5. Immune recognition analysis: Determine whether FlgI is recognized by host immune system components using immunoprecipitation with host immune factors.

  These approaches would help delineate whether FlgI contributes to pathogenicity through structural roles in bacterial motility or through direct immunogenic effects.