Recombinant Desulfovibrio vulgaris Protein TolB (tolB)

What is TolB protein and what is its significance in Desulfovibrio vulgaris?

TolB is a periplasmic protein in Desulfovibrio vulgaris that belongs to the Tol-Pal system, which is involved in maintaining outer membrane integrity in Gram-negative bacteria. In D. vulgaris Hildenborough, TolB (UniProt ID: Q726K4) consists of 442 amino acids with the functional expression region spanning residues 27-442 . The significance of TolB in D. vulgaris relates to its potential role in cell envelope biogenesis and maintenance, which is particularly important in this anaerobic, sulfate-reducing bacterium that faces various environmental stresses including metal toxicity and oxidative stress. Understanding TolB function contributes to our broader knowledge of how D. vulgaris maintains cellular integrity during environmental stress responses and bioremediation processes .

How does recombinant TolB protein differ from native TolB in Desulfovibrio vulgaris?

Recombinant Desulfovibrio vulgaris TolB protein is produced in E. coli expression systems rather than being isolated from D. vulgaris itself . This heterologous expression may result in structural differences including post-translational modifications. The recombinant protein typically has >85% purity as determined by SDS-PAGE and contains the full sequence of the mature protein . During recombinant production, tag sequences may be added to facilitate purification and detection. These modifications could potentially affect protein folding, activity, or interaction capabilities compared to the native protein. Researchers should consider these differences when designing experiments, especially when studying protein-protein interactions that might be influenced by subtle structural changes.

What are the optimal storage conditions for recombinant D. vulgaris TolB protein?

The shelf life and stability of recombinant D. vulgaris TolB protein depend on several factors including storage state, buffer ingredients, and storage temperature. For liquid formulations, the recommended shelf life is approximately 6 months when stored at -20°C/-80°C. Lyophilized (freeze-dried) forms demonstrate greater stability with a shelf life of 12 months at -20°C/-80°C . To maintain protein integrity, repeated freeze-thaw cycles should be avoided. For short-term work, aliquots can be stored at 4°C for up to one week . When reconstituting lyophilized protein, it is recommended to briefly centrifuge the vial, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and add 5-50% glycerol (final concentration) before aliquoting for long-term storage .

What is the recommended protocol for reconstituting lyophilized recombinant D. vulgaris TolB?

The recommended reconstitution protocol involves several critical steps to ensure optimal protein activity:

• Centrifugation: Briefly centrifuge the vial containing lyophilized protein to bring contents to the bottom.

• Reconstitution: Add deionized sterile water to achieve a final protein concentration of 0.1-1.0 mg/mL.

• Stabilization: Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) to prevent freeze damage during storage.

• Aliquoting: Divide the reconstituted protein into small working aliquots to minimize freeze-thaw cycles.

• Storage: Store the aliquots at -20°C or -80°C for long-term storage, or at 4°C for up to one week for immediate use .

This methodological approach ensures maximum retention of protein structure and function while minimizing degradation during storage and handling.

How can researchers verify the structural integrity and activity of recombinant TolB after reconstitution?

Verifying the structural integrity and activity of recombinant TolB after reconstitution involves multiple analytical approaches:

• SDS-PAGE analysis: To confirm the expected molecular weight (approximately 44 kDa) and purity (>85%) .

• Circular dichroism (CD) spectroscopy: To assess secondary structure elements and proper protein folding.

• Size exclusion chromatography: To detect potential aggregation or oligomerization.

• Functional binding assays: To verify interaction with known binding partners such as Pal protein.

• Thermal stability assays: Using differential scanning fluorimetry to determine if the protein maintains its expected melting temperature.

Researchers should particularly focus on comparing freshly reconstituted protein with samples stored under different conditions to establish optimal handling protocols specific to their experimental requirements.

What expression systems are optimal for producing functional recombinant D. vulgaris TolB?

• E. coli strain selection: BL21(DE3) strains are commonly used for recombinant protein expression due to their deficiency in lon and ompT proteases.

• Codon optimization: Adapting the D. vulgaris TolB gene sequence to E. coli codon usage can significantly improve expression levels.

• Temperature modulation: Lower induction temperatures (16-25°C) often produce more soluble protein than standard 37°C conditions.

• Induction parameters: IPTG concentration and induction timing should be optimized based on construct design.

• Fusion tags: Strategic selection of fusion tags (His, Strep, or FLAG tags) can improve solubility and facilitate purification .

When designing expression constructs, researchers should consider the region spanning amino acids 27-442, which represents the full mature protein sequence .

What is the relationship between TolB and biofilm formation in D. vulgaris?

The relationship between TolB and biofilm formation in D. vulgaris represents an important research frontier. D. vulgaris forms biofilms that contribute to microbially-induced corrosion, causing significant economic damage to metal infrastructure . These biofilms are characterized by cells embedded in a polymer matrix, making them extremely difficult to eliminate .

TolB, as a periplasmic protein involved in outer membrane stability, likely plays a role in biofilm architecture and integrity. Potential mechanisms include:

• Cell-surface adhesion: TolB may influence initial attachment to surfaces by affecting outer membrane proteins involved in adhesion.

• Extracellular matrix production: Changes in outer membrane composition influenced by TolB could alter the composition and properties of the biofilm matrix.

• Cell-cell interactions: TolB may affect cell-cell communication necessary for coordinated biofilm development.

• Stress response within biofilms: TolB could contribute to maintaining cellular integrity under the oxygen gradients and metabolite concentrations typical in biofilms.

Research on biofilm dispersal agents, such as rhamnolipids from Pseudomonas aeruginosa that can disperse over 98% of D. vulgaris biofilms , could potentially be influenced by TolB function, though this relationship requires further investigation.

How can protein-protein interaction studies with TolB inform our understanding of D. vulgaris membrane biology?

Protein-protein interaction studies with TolB can provide crucial insights into D. vulgaris membrane biology through several approaches:

• Pull-down assays with tagged recombinant TolB can identify interaction partners in the D. vulgaris periplasm and membrane fractions.

• Bacterial two-hybrid systems can verify specific interactions with predicted partners.

• Cross-linking studies can capture transient interactions under different growth conditions.

A systematic approach might involve:

Understanding TolB interactions is particularly relevant given D. vulgaris' complex membrane biology, which includes mechanisms for surviving metal toxicity and functioning in anaerobic environments . The interactions may reveal how TolB contributes to maintaining membrane integrity during sulfate reduction and energy metabolism processes that are central to D. vulgaris physiology.

What domains and motifs are present in D. vulgaris TolB and how do they compare to TolB proteins in other bacteria?

D. vulgaris TolB (UniProt ID: Q726K4) consists of 442 amino acids with the functional region spanning residues 27-442 . The protein contains several conserved domains typical of TolB proteins:

• N-terminal domain: Contains the signal sequence (residues 1-26) for periplasmic targeting and secretion

• β-propeller domain: Forms a β-propeller structure that serves as a protein interaction platform

• C-terminal domain: Contains the binding site for Pal protein

The amino acid sequence of D. vulgaris TolB (GPVQVDIYGPGQGSLNLAMAAPLGPTPGTPVSGMGVKLNGFINENLSFLPFLRLVDQRAILGGTVMQGYKSP...) shares conserved regions with TolB proteins from other bacteria, but with adaptations that may reflect the unique environmental niche of this anaerobic, sulfate-reducing bacterium . Comparative analysis suggests potential functional adaptations in the binding interfaces that could influence protein-protein interactions specific to D. vulgaris membrane biology.

What insights can structural biology provide into D. vulgaris TolB function?

Structural biology approaches can provide significant insights into D. vulgaris TolB function through several techniques:

• X-ray crystallography: Determining the three-dimensional structure of D. vulgaris TolB would reveal binding pockets, surface electrostatics, and conformational features.

• Cryo-electron microscopy: Could visualize TolB in complex with interaction partners, providing insights into functional complexes.

• NMR spectroscopy: Might identify dynamic regions and conformational changes upon ligand binding.

Structural comparisons with TolB proteins from other bacteria could highlight D. vulgaris-specific adaptations that relate to its anaerobic lifestyle and role in sulfate reduction. Particular attention should be paid to potential metal-binding sites, given D. vulgaris' involvement in metal bioremediation and resistance to metal toxicity . Structural information could also inform the development of inhibitors targeting TolB as potential anti-biofilm agents to combat microbially-induced corrosion .

How does TolB contribute to D. vulgaris' metal reduction capabilities and bioremediation potential?

D. vulgaris is known for its ability to reduce toxic metals such as uranium and chromium, making it valuable for bioremediation applications . While the direct role of TolB in metal reduction has not been explicitly demonstrated, its function in maintaining outer membrane integrity likely contributes to metal resistance mechanisms.

The genome sequence of D. vulgaris reveals complex anaerobic respiration pathways involved in metal reduction . As a periplasmic protein maintaining membrane structure, TolB may influence:

• Transport of metals across the outer membrane

• Stability of membrane-bound metal reductases

• Protection against metal toxicity by maintaining periplasmic compartmentalization

• Biofilm formation capabilities that enhance metal reduction in environmental settings

Research has shown that dissimilatory sulfate reduction improves resistance of D. vulgaris to metal stress, particularly against silver and uranium . The sulfide produced during this process provides protection against metal toxicity. TolB's role in maintaining membrane integrity could be crucial for supporting these metal resistance mechanisms, potentially by ensuring proper localization and function of proteins involved in sulfate reduction pathways.

How might TolB function relate to the energy metabolism of D. vulgaris under different growth conditions?

The relationship between TolB function and D. vulgaris energy metabolism represents an important research question. Transcriptomic analysis has revealed that D. vulgaris significantly adjusts its gene expression in response to different electron donors and growth phases . While TolB-specific expression data is limited in the provided search results, we can infer potential relationships:

D. vulgaris employs different hydrogenases depending on the electron donor available. In lactate-based media, HynBA-1 appears to function as the primary periplasmic hydrogenase, while in formate-based media, multiple periplasmic hydrogenases including HynBA-1 and Hyd carry out this role . The Tol-Pal system (including TolB) likely plays a role in maintaining the integrity of the cell envelope under these different metabolic conditions.

A comparison of gene expression patterns in D. vulgaris under different growth conditions reveals:

As a periplasmic protein involved in membrane maintenance, TolB function likely adapts to these metabolic shifts, potentially through regulated expression or post-translational modifications that influence its interaction with other membrane components.

What experimental approaches are most effective for studying TolB's role in D. vulgaris biofilm formation and dispersal?

Studying TolB's role in D. vulgaris biofilm formation and dispersal requires multi-faceted experimental approaches:

• Gene deletion/complementation studies:

  • Creating ΔtolB knockout mutants to assess effects on biofilm formation

  • Complementation with wild-type and mutated tolB to identify critical residues

  • Quantification of biofilm formation using crystal violet staining or confocal microscopy

• Protein localization during biofilm development:

  • Fluorescently tagged TolB to track localization during biofilm formation

  • Immunogold labeling with anti-TolB antibodies for transmission electron microscopy

  • Fractionation of biofilm cells to determine TolB distribution between membrane and extracellular matrix

• Interaction studies within biofilms:

  • In vivo cross-linking to capture TolB interaction partners specifically in biofilm state

  • Comparative proteomics between planktonic and biofilm states to identify differential TolB interactions

• Biofilm dispersal assays:

  • Testing if known biofilm dispersal agents like rhamnolipids from P. aeruginosa (which can disperse >98% of D. vulgaris biofilms ) affect TolB localization or function

  • Determining if TolB overexpression or mutation affects susceptibility to dispersal agents

These approaches would provide comprehensive insights into how TolB contributes to the formation and maintenance of D. vulgaris biofilms, which are implicated in microbially-induced corrosion of metal infrastructure in oil wells and drilling equipment .

What computational approaches can predict TolB's functional interactions within the D. vulgaris proteome?

Several computational approaches can predict TolB's functional interactions within the D. vulgaris proteome:

• Protein-protein interaction network analysis:

  • Construction of interaction networks based on genomic context, co-expression data, and homology to known interactions

  • Identification of functional modules containing TolB through clustering algorithms

  • Prediction of novel interactions through network topology analysis

• Molecular dynamics simulations:

  • Modeling of TolB structure and dynamics in the periplasmic environment

  • Simulation of potential interactions with other periplasmic and membrane proteins

  • Prediction of binding affinities and interaction interfaces

• Genomic context analysis:

  • Identification of conserved gene neighborhoods around tolB

  • Analysis of co-evolutionary patterns with potential interaction partners

  • Prediction of functional associations through gene fusion events

These computational predictions should be integrated with experimental data from transcriptomic and proteomic studies. For example, the global transcriptomic analysis of D. vulgaris under different growth conditions provides a foundation for identifying co-expressed genes that might functionally interact with TolB.

How can advanced imaging techniques contribute to understanding TolB localization and dynamics in D. vulgaris cells?

Advanced imaging techniques offer powerful approaches to understand TolB localization and dynamics in D. vulgaris:

• Super-resolution microscopy (STORM, PALM):

  • Visualization of TolB distribution with nanometer precision

  • Tracking of dynamic changes in localization under different growth conditions

  • Co-localization studies with other membrane components

• Cryo-electron tomography:

  • 3D visualization of TolB in the native cellular context

  • Analysis of spatial relationships with the cell envelope components

  • Structural insights into TolB-containing complexes in situ

• FRAP (Fluorescence Recovery After Photobleaching):

  • Measurement of TolB mobility within the periplasm

  • Assessment of potential binding to immobile structures

  • Comparison of dynamics under different growth conditions or stresses

• Single-molecule tracking:

  • Real-time monitoring of individual TolB molecules

  • Determination of diffusion coefficients and binding kinetics

  • Identification of potential confinement zones within the cell

These techniques would be particularly valuable for understanding how TolB distribution and dynamics change during biofilm formation, which is relevant to D. vulgaris' role in microbially-induced corrosion . Additionally, imaging studies could reveal how TolB localization responds to environmental stresses such as metal exposure, providing insights into its role in metal resistance mechanisms .