Recombinant Desulfovibrio vulgaris Zinc resistance-associated protein homolog (DVU_3384)

What is DVU_3384 and what structural features suggest its function?

DVU_3384 is annotated as a zinc resistance-associated protein (ZraP) in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It has gained research interest due to its significant upregulation across multiple stress conditions, particularly under oxidative stress exposure. The protein contains several structural features of interest, including a cysteine residue in the C-terminal region and multiple histidine residues in the N-terminal region, both located within glycine-rich and presumably flexible regions that may facilitate metal binding activity .

Unlike the E. coli YjaI protein that has confirmed zinc binding motifs (N-terminal HRWHGRC and C-terminal HGGHGMW sequences), DVU_3384 shows low sequence similarity to experimentally validated metal-binding proteins. This raises important questions about its actual function and metal specificity, requiring further experimental validation to confirm its role in zinc resistance or other cellular processes .

Expression System Selection

When expressing DVU_3384, researchers should consider multiple expression systems based on experimental goals:

• E. coli-based expression: Most commonly used for initial attempts, with BL21(DE3) or BL21-Star strains recommended for high-level expression . Growth conditions should include:

  • Cultivation in Luria-Bertani broth with appropriate antibiotic selection

  • Growth to OD600 of 0.6-0.8 before induction

  • Induction with IPTG at concentrations of 0.5 mM

  • Incubation for 4 hours at 37°C for optimal protein production

• Yeast expression systems: For difficult-to-express proteins, S. cerevisiae provides notable advantages. According to comparative studies, S. cerevisiae has demonstrated success with challenging proteins that could not be produced in E. coli, particularly for proteins requiring eukaryotic folding machinery . Advantages include:

  • Better folding for complex proteins

  • Ability to handle proteins toxic to bacterial cells

  • Reduced formation of inclusion bodies

Purification Strategy

A systematic purification approach should include:

• Cell disruption using either microfluidizer processor or chemical methods for efficient lysis

• Affinity chromatography as the primary purification step (Ni-NTA for His-tagged constructs)

• Ion exchange chromatography as a secondary purification step

• Protein quantification using fluorometric assays such as Qubit

• Purity confirmation through SDS-PAGE with CBB-G250 staining

Special consideration should be given to potential metal cofactors during purification. If studying zinc binding, buffers should avoid chelating agents like EDTA that might strip essential metals.

What evidence supports DVU_3384's role in oxidative stress response?

Transcriptomic and proteomic data provide substantial evidence for DVU_3384's involvement in oxidative stress response:

• Upregulation during oxygen exposure: DVU_3384 was highly upregulated during both low oxygen exposure (0.1% O2) and air exposure experiments with D. vulgaris .

• Universal stress response: DVU_3384 has been characterized as "universally upregulated across multiple stress conditions monitored in D. vulgaris," suggesting it may function as a general stress response candidate rather than having specific zinc resistance functionality .

• Comparative expression data: The table below summarizes DVU_3384 expression changes under various stress conditions:

These findings strongly suggest DVU_3384 participates in a broader stress response mechanism rather than solely in zinc resistance pathways. This has important implications for experimental design, as researchers should examine DVU_3384 function under various stress conditions rather than focusing exclusively on metal resistance.

How can researchers address contradictory data regarding DVU_3384's metal binding properties?

Resolving contradictions regarding DVU_3384's purported zinc binding function requires a multifaceted experimental approach:

Direct Metal Binding Studies

• Isothermal titration calorimetry (ITC): Conduct binding assays with purified DVU_3384 and various metal ions (Zn2+, Cu2+, Fe2+) to determine binding specificity and affinity constants.

• Inductively coupled plasma mass spectrometry (ICP-MS): Analyze metal content of purified protein to identify co-purifying metals, which may indicate physiologically relevant binding partners.

• Differential scanning fluorimetry: Measure protein thermal stability in the presence of different metals to identify stabilizing interactions.

Structural Approaches

• X-ray crystallography or NMR spectroscopy: Determine the three-dimensional structure of DVU_3384 with and without potential metal cofactors to identify binding sites and coordination geometry.

• Homology modeling: Create computational models based on proteins with confirmed metal binding properties to predict potential binding sites in DVU_3384.

Mutagenesis Studies

Conduct site-directed mutagenesis of putative metal-binding residues, particularly:

• The cysteine residue in the C-terminal region

• Histidine residues in the N-terminal region

Each mutant should be tested for:

• Metal binding capacity using the methods described above

• Functional complementation in knockout studies

• Ability to confer stress resistance

A systematic approach addressing all these aspects will help resolve the contradiction between DVU_3384's annotation as a zinc resistance protein and its apparent role in general stress response.

What experimental designs are appropriate for determining DVU_3384's physiological role in vivo?

Robust experimental design for studying DVU_3384's physiological role should include:

Genetic Manipulation Approaches

• Creation of knockout mutants: Generate precise deletion mutants of DVU_3384 in D. vulgaris using techniques like λ-Red recombination, CRISPR-Cas systems, or homologous recombination.

• Complementation studies: Reintroduce wild-type and mutant versions of DVU_3384 to confirm phenotypes are specifically due to loss of this gene.

• Expression under controlled promoters: Study the effects of DVU_3384 overexpression and controlled expression.

Phenotypic Characterization

Test knockout and complemented strains under various conditions:

• Metal stress: Growth in media with elevated concentrations of zinc and other metals

• Oxidative stress: Exposure to varying oxygen levels (0.1%, 1%, 21%) and oxidative agents (H2O2)

• General stressors: Heat shock, pH stress, osmotic stress

Omics Approaches

• Transcriptomics: Use RNA-seq to compare gene expression profiles between wild-type and knockout strains under stress conditions. This approach has revealed significant changes during oxygen exposure experiments .

• Proteomics: Identify changes in protein abundance and post-translational modifications.

• Metabolomics: Examine changes in metabolite profiles, particularly those related to metal homeostasis and oxidative stress.

Specific Experimental Controls

• Include established stress response mutants as positive controls (e.g., PerR regulon mutants which showed coordinated response to oxygen exposure )

• Perform time-course experiments to distinguish between direct and indirect effects

• Include other D. vulgaris stress response genes as controls, such as those identified in the σ54-dependent regulome

This comprehensive approach will provide multiple lines of evidence regarding DVU_3384's true physiological role, avoiding over-reliance on any single experimental technique.

How do recombination concerns apply to experiments involving DVU_3384?

When designing recombinant DNA experiments with DVU_3384, researchers must consider both experimental validity and biosafety guidelines:

Biosafety Guidelines

Most DVU_3384 experiments will fall under NIH Guidelines Section III-F (Exempt Experiments) or Section III-D-1-a if using Risk Group 2 organisms as hosts . Specifically:

• Exempt status applies when:

  • The experiments consist entirely of DNA from D. vulgaris when propagated only in D. vulgaris or a closely related strain (Section III-F-4)

  • Standard cloning vectors with less than 50% of a Risk Group 2 pathogen are used in non-conjugative E. coli (Appendix C-I)

• BSL-2 containment would be required if:

  • Inserting DVU_3384 into Risk Group 2 pathogens (Section III-D-1)

  • Using defective viral vectors for expression (Section III-D-1-a)

For most laboratory research with DVU_3384 in standard expression hosts, BSL-1 containment is generally sufficient, though institutional biosafety committee approval should always be obtained before beginning work.

What methods are available for studying protein-protein interactions involving DVU_3384?

Understanding DVU_3384's interaction partners is crucial for elucidating its cellular function. Several complementary approaches should be employed:

Affinity-Based Methods

• Co-immunoprecipitation (Co-IP): Using antibodies against DVU_3384 or an epitope tag to pull down protein complexes.

  • Requires: Specific antibodies or tagged version of DVU_3384

  • Advantages: Can capture native complexes

  • Limitations: Transient interactions may be missed

• Pull-down assays: Using purified recombinant DVU_3384 as bait.

  • Protocol should include crosslinking steps to capture transient interactions

  • Consider both aerobic and anaerobic conditions as D. vulgaris is an anaerobe

• Bacterial two-hybrid systems: For screening potential interaction partners.

  • Advantages: Can identify novel interactions in a cellular context

  • Limitations: May produce false positives/negatives

Mass Spectrometry-Based Approaches

• Affinity purification-mass spectrometry (AP-MS): Combines affinity purification with sensitive MS detection.

• Cross-linking MS (XL-MS): Uses chemical cross-linkers to stabilize interactions before MS analysis.

  • Particularly useful for capturing transient interactions

  • Can provide structural information about interaction interfaces

• Proximity-dependent labeling: Methods like BioID or APEX2 to identify proteins in close proximity to DVU_3384 in vivo.

Structural and Biophysical Methods

• Surface plasmon resonance (SPR) or bio-layer interferometry (BLI): To measure binding kinetics of purified proteins.

• Microscale thermophoresis (MST): For detecting interactions with minimal sample consumption.

• NMR spectroscopy: For detailed characterization of interaction interfaces.

Special Considerations for DVU_3384

Based on its potential role in stress response , interaction studies should be performed under:

• Normal growth conditions

• Oxidative stress conditions

• Metal stress conditions (particularly with zinc)

This will help determine whether DVU_3384's interaction partners change during stress response, providing insight into its functional mechanisms.

How should researchers address the challenges of studying proteins from anaerobic organisms like D. vulgaris?

Working with proteins from D. vulgaris, an anaerobic sulfate-reducing bacterium, presents unique challenges that require specialized approaches:

Oxygen Sensitivity Considerations

• Protein stability: DVU_3384 may be sensitive to oxidation given D. vulgaris' anaerobic nature. Research has shown significant changes in D. vulgaris protein expression under even low (0.1%) oxygen conditions . Strategies to address this include:

  • Working in anaerobic chambers when possible

  • Including reducing agents (DTT, β-mercaptoethanol) in buffers

  • Rapid purification protocols to minimize oxygen exposure

  • Comparing protein function under aerobic versus anaerobic conditions

• Functional assays: Activity measurements should be performed under anaerobic conditions to accurately reflect the protein's native environment.

Expression Challenges

• Heterologous expression optimization: While E. coli remains the most common expression host, researchers should consider:

  • Growth under microaerobic conditions

  • Use of specialized E. coli strains adapted for expression of oxygen-sensitive proteins

  • Yeast-based expression as an alternative (S. cerevisiae has shown success with challenging proteins)

• Context-dependent folding: D. vulgaris proteins may require specific chaperones or redox conditions for proper folding. Consider co-expression with D. vulgaris chaperones or expression in closely related organisms.

Experimental Design Modifications

Compared to standard aerobic experiments, studies with D. vulgaris proteins require:

• Modified growth conditions: When studying DVU_3384 in its native context, growth conditions must reflect D. vulgaris' anaerobic lifestyle. Studies have used controlled oxygen exposure (0.1% O2) for experimental purposes .

• Appropriate controls: Include closely related proteins from both anaerobic and aerobic organisms as controls.

• Careful interpretation of stress response data: As shown in comparative studies, DVU_3384 responds differently to low oxygen (0.1% O2) versus air exposure , indicating the importance of precise control over experimental conditions.

These considerations are essential for obtaining physiologically relevant results when studying DVU_3384 and other proteins from anaerobic organisms.

Genetic Controls

• Empty vector controls: When expressing recombinant DVU_3384, include parallel experiments with the same expression vector lacking the DVU_3384 gene.

• Complementation controls: For knockout studies, include:

  • Wild-type D. vulgaris

  • Clean deletion mutant (ΔDVU_3384)

  • Complemented strain (ΔDVU_3384 + DVU_3384)

  • Point mutant complementation (e.g., mutating putative metal-binding residues)

• Related gene controls: Include experiments with other stress response proteins identified in D. vulgaris, such as those in the PerR regulon which showed coordinated response to oxygen exposure .

Biochemical Controls

• Protein purity controls:

  • SDS-PAGE analysis to confirm absence of contaminating proteins

  • Western blot verification of identity

  • Mass spectrometry analysis to confirm sequence

• Metal binding specificity controls:

  • Test multiple metals beyond zinc (Fe2+, Cu2+, Mn2+, Ni2+)

  • Include known metal-binding proteins as positive controls

  • Use proteins with mutated binding sites as negative controls

• Activity assays:

  • Include both positive controls (known active proteins) and negative controls (heat-denatured proteins)

  • Test activity under both aerobic and anaerobic conditions

Environmental Controls

• Stress condition controls:

  • Precise control of oxygen levels (as seen in the 0.1% O2 versus air exposure experiments)

  • Consistent metal concentrations across experiments

  • Temperature and pH standardization

• Time-course measurements:

  • Sample at multiple time points (e.g., 15, 30, 60, 120 minutes) as done in oxygen exposure studies

  • This helps distinguish between direct and indirect effects

Data Analysis Controls

• Statistical validation:

  • Minimum of three biological replicates for all experiments

  • Appropriate statistical tests with correction for multiple comparisons

  • Power analysis to determine adequate sample sizes

• Normalization methods:

  • Use multiple reference genes for qRT-PCR

  • Apply appropriate normalization for proteomics data

Following these control guidelines will strengthen the validity and reproducibility of DVU_3384 research findings.

How can researchers integrate multi-omics data to understand DVU_3384 function in the context of cellular networks?

Integrating multi-omics data provides a comprehensive view of DVU_3384's role within cellular networks:

Data Integration Approaches

• Correlation network analysis: Identify genes/proteins with expression patterns similar to DVU_3384 across multiple stress conditions. Previous studies have shown DVU_3384 is upregulated during various stress responses , suggesting it functions within broader stress response networks.

• Pathway enrichment analysis: Determine whether genes differentially expressed in DVU_3384 knockout strains belong to specific metabolic or regulatory pathways.

• Protein-protein interaction networks: Combine experimentally determined interactions with predicted interactions to place DVU_3384 in a functional context.

• Regulatory network reconstruction: Identify potential transcription factors controlling DVU_3384 expression. Research has identified DVU_3384 as potentially regulated by σ54-dependent regulators .

Multi-Omics Experimental Design

To generate comprehensive datasets for integration:

• Transcriptomics:

  • RNA-seq comparing wild-type and ΔDVU_3384 strains under multiple conditions

  • Time-course experiments to capture dynamic responses

  • Analysis of gene clusters with similar expression patterns

• Proteomics:

  • Quantitative proteomics to identify changes in protein abundance

  • Phosphoproteomics to identify signaling changes

  • Protein-protein interaction studies using AP-MS

• Metabolomics:

  • Targeted analysis of metal homeostasis metabolites

  • Global metabolic profiling under stress conditions

Computational Tools for Data Integration

• Computational tools for multi-omics integration:

  • Weighted gene co-expression network analysis (WGCNA)

  • Multi-omics factor analysis (MOFA)

  • Network-based data integration approaches

• Visualization methods:

  • Cytoscape for network visualization

  • Pathway maps highlighting multi-omics data

Case Study Example

The table below outlines a multi-omics integration approach for DVU_3384:

By integrating these diverse data types, researchers can develop testable hypotheses about DVU_3384's function within the broader cellular stress response networks of D. vulgaris.