Recombinant Desulfovibrio vulgaris Exodeoxyribonuclease 7 small subunit (xseB)

What is the quaternary structure of the ExoVII complex in bacteria?

Research using cryo-electron microscopy has revealed that in Escherichia coli, the ExoVII complex has a stoichiometry of XseA₄·XseB₂₄, which is significantly different from previous assumptions about the complex composition. The structure shows that:

• XseB folds into a simple α-helical hairpin structure of only 80 residues

• Multiple XseB subunits associate with each XseA molecule

• The interaction between XseA and XseB forms a tandem pair of three-helix bundles that comprise the ExoVII neck region

• The XseB subunit closest to the XseA catalytic domain uses part of its sequence to complete the nuclease fold

While this structure was determined in E. coli, similar architectural principles likely apply to the D. vulgaris ExoVII complex, though species-specific differences may exist.

What are the optimal conditions for expressing recombinant D. vulgaris xseB in E. coli?

Based on protocols used for similar recombinant proteins, the following methodology is recommended for expression:

• Vector selection: Use a T7 promoter-based expression system similar to that used for flavodoxin expression

• Host strain: BL21(DE3) is the preferred E. coli strain for expression

• Growth conditions:

  • Culture in LB medium supplemented with appropriate antibiotic (typically ampicillin at 50-100 μg/ml)

  • Incubate at 37°C with shaking until OD600 reaches 0.5-0.7

  • Induce with IPTG (0.5-1.0 mM) and continue incubation for 4-6 hours

  • Alternatively, use overnight induction at lower temperatures (16-25°C) for potentially higher soluble protein yields

• Harvest: Collect cells by centrifugation at 6,000 rpm for 15 minutes

The expression level should be monitored by SDS-PAGE analysis of whole-cell lysates before and after induction.

What purification strategy yields the highest purity recombinant xseB protein?

A multi-step purification protocol is recommended:

• Cell lysis: Resuspend cell pellet in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, and protease inhibitors) and disrupt by sonication or French press

• Initial purification: If the recombinant protein contains a His-tag, use Ni-NTA affinity chromatography

• Secondary purification: Apply ion-exchange chromatography (IEX) using either cation exchange (as xseB has a basic pI) or anion exchange depending on the buffer pH

• Polishing step: Size exclusion chromatography to remove aggregates and ensure homogeneity

• Quality control: Assess purity by SDS-PAGE (target >85%) and verify identity by Western blotting or mass spectrometry

How can researchers effectively analyze the structural properties of recombinant xseB?

Several complementary approaches provide comprehensive structural characterization:

• Dynamic Light Scattering (DLS): Assess protein homogeneity, aggregation state, and approximate molecular weight in solution . Data analysis using software like DynaPro (Wyatt Technology) can determine hydrodynamic radius and polydispersity.

• Circular Dichroism (CD) Spectroscopy: Analyze secondary structure content, particularly important for confirming the α-helical hairpin structure of xseB.

• Limited Proteolysis: Identify stable domains and flexible regions by subjecting the protein to controlled enzymatic digestion followed by mass spectrometry analysis.

• Thermal Stability Assays: Techniques like differential scanning fluorimetry (DSF) can determine protein stability under various buffer conditions.

• X-ray Crystallography or NMR Spectroscopy: For high-resolution structural analysis, considering xseB's relatively small size (81 amino acids in D. vulgaris) .

• Cross-linking Experiments: Study the interaction interfaces between xseB subunits and with xseA by chemical cross-linking followed by mass spectrometry.

What methodologies can be used to study the interaction between xseB and xseA in D. vulgaris?

Several approaches can characterize the xseA-xseB interaction:

• Co-purification Assays: Express both proteins, with different tags, and perform pull-down experiments to verify complex formation.

• Surface Plasmon Resonance (SPR): Determine binding kinetics and affinity by immobilizing one protein (typically xseA) and flowing the other (xseB) at various concentrations.

• Isothermal Titration Calorimetry (ITC): Measure thermodynamic parameters of binding, including stoichiometry, which is particularly important given the XseA₄·XseB₂₄ complex observed in E. coli .

• Electrophoretic Mobility Shift Assay (EMSA): If studying DNA-protein interactions, EMSA can verify if the xseA-xseB complex binds DNA differently than individual subunits .

• DNA Affinity Assays: For identifying specific DNA-protein interactions and characterizing binding sites .

• Analytical Ultracentrifugation: Determine stoichiometry and complex size in solution.

• Cryo-electron Microscopy: For visualization of the entire complex architecture, as performed for the E. coli ExoVII complex .

How can the nuclease activity of reconstituted ExoVII containing D. vulgaris xseB be assayed?

The functional activity of reconstituted ExoVII can be assessed using several approaches:

• Single-stranded DNA Degradation Assay:

  • Incubate the enzyme with labeled ssDNA (e.g., 5'-radiolabeled or fluorescently labeled oligonucleotides)

  • Analyze the degradation products by denaturing gel electrophoresis

  • Quantify the extent of degradation by measuring band intensity

• Real-time Nuclease Activity Assays:

  • Use fluorescence-based assays with quenched fluorescent substrates

  • Monitor the increase in fluorescence as DNA is degraded in real-time

  • Determine kinetic parameters (Km, Vmax) under various conditions

• Circular DNA Nicking Assay:

  • Incubate the enzyme with supercoiled plasmid DNA

  • Analyze the conversion to nicked and linear forms by agarose gel electrophoresis

  • Quantify the relative proportions of different DNA forms

Optimal assay conditions typically include:

• Buffer: 20-50 mM Tris-HCl (pH 7.5-8.5)

• Salt: 50-100 mM NaCl or KCl

• Divalent cations: 1-10 mM MgCl₂ or MnCl₂

• Reducing agent: 1-5 mM DTT or β-mercaptoethanol

• Temperature: 30-37°C

What is known about the DNA substrate specificity of ExoVII containing D. vulgaris xseB?

While specific data for D. vulgaris ExoVII is limited, research on ExoVII from other bacteria provides insights that likely apply:

• Substrate Preference:

  • Primary activity on single-stranded DNA

  • Limited activity on double-stranded DNA with single-stranded regions

  • No activity on fully base-paired double-stranded DNA

• Direction of Degradation:

  • ExoVII typically shows bidirectional exonuclease activity (from both 5' and 3' ends)

  • The rate of degradation may differ for 5' versus 3' ends

• Length Requirements:

  • Minimum substrate length requirements should be determined experimentally

  • Typically requires single-stranded regions of at least 4-6 nucleotides

To experimentally determine substrate specificity for D. vulgaris ExoVII, researchers should:

• Test various DNA structures (ssDNA, dsDNA with overhangs, nicked DNA, gapped DNA)

• Vary substrate lengths and sequences

• Compare degradation rates under identical reaction conditions

How can researchers generate and characterize xseB deletion mutants in D. vulgaris?

Creating and analyzing xseB deletion mutants involves several steps:

• Mutant Construction:

  • Shuttle plasmid method: Construct a deletion cassette in a plasmid like pG1

  • Design primers to amplify regions flanking the xseB gene

  • Include appropriate restriction sites for cloning

  • Introduce the construct into D. vulgaris through electroporation or conjugation

• Verification of Mutants:

  • PCR analysis with primers flanking the deletion site

  • Sequencing to confirm the precise deletion

  • Western blotting to verify absence of the xseB protein

• Phenotypic Analysis:

  • Growth rate measurements under standard and stress conditions

  • DNA repair capacity following exposure to DNA-damaging agents

  • Mutation frequency assays (e.g., frequency of rifampicin-resistant mutants)

  • Comparative genome analysis to identify potential suppressor mutations

For complementation studies, the entire coding sequence of xseB can be amplified and cloned into an appropriate expression vector like pTCV-P Tet .

What experimental approaches can determine the physiological role of xseB in D. vulgaris?

To elucidate the physiological function:

• Comparative Growth Analysis:

  • Culture wild-type and ΔxseB strains under various conditions

  • Test different carbon and energy sources

  • Analyze growth under stress conditions (oxidative stress, DNA damage, temperature, pH)

• Transcriptomic Analysis:

  • RNA-Seq to compare gene expression patterns between wild-type and mutant

  • Identify compensatory responses and affected pathways

  • RT-PCR and Northern blotting to validate specific expression changes

• Mutation Rate and Spectrum Analysis:

  • Determine spontaneous mutation frequencies using resistance to antibiotics like rifampicin

  • Analyze the types of mutations that occur in the absence of xseB

  • Compare mutation spectra after exposure to different DNA-damaging agents

• DNA Damage Response:

  • Measure survival after exposure to DNA-damaging agents

  • Analyze DNA repair kinetics using techniques like comet assay

  • Examine recombination frequencies

• Protein Interaction Network:

  • Identify interaction partners through co-immunoprecipitation or bacterial two-hybrid assays

  • Determine if xseB interacts with proteins beyond the ExoVII complex

How can genome-wide approaches be used to study xseB function in the context of DNA repair pathways in D. vulgaris?

Genome-wide methods provide comprehensive insights into xseB function:

• Transposon Mutagenesis Screens:

  • Create a random barcode transposon-site sequencing (RB-TnSeq) library in wild-type and ΔxseB backgrounds

  • Compare fitness effects of genome-wide mutations in both backgrounds

  • Identify synthetic lethal or synthetic sick interactions that suggest functional relationships

• ChIP-Seq Analysis:

  • Perform chromatin immunoprecipitation with antibodies against tagged xseB

  • Map genome-wide binding locations to identify where ExoVII functions

  • Analyze binding patterns before and after DNA damage

• Synthetic Genetic Array (SGA) Analysis:

  • Systematically combine ΔxseB with other mutations to identify genetic interactions

  • Construct a genetic interaction map to position xseB within cellular pathways

• Comparative Genomics:

  • Analyze conservation and co-evolution patterns of xseB across diverse bacteria

  • Identify correlated gene gains/losses that suggest functional relationships

• High-throughput Phenotyping:

  • Screen ΔxseB against libraries of chemicals or stressors

  • Identify specific conditions where xseB is particularly important

What experimental designs would be most effective for resolving contradictory findings about xseB function?

When faced with contradictory findings, systematic experimental approaches are crucial:

• Strain Background Verification:

  • Sequence verification of all strains to identify potential laboratory-driven evolution

  • Documentation of strain lineage and maintenance conditions

  • Cross-laboratory validation using standardized strains

• Multi-method Validation:

  • Apply complementary techniques to address the same question

  • For protein-protein interactions: combine in vitro (e.g., ITC, SPR) and in vivo techniques (bacterial two-hybrid, FRET)

  • For functional roles: unite genetic, biochemical, and physiological approaches

• Controlled Variable Experiments:

  • Systematically vary one parameter at a time (pH, temperature, salt concentration, etc.)

  • Determine the specific conditions under which contradictory results occur

  • Create a comprehensive parameter space map of xseB function

• Quantitative Analysis of Confounding Factors:

  • Design factorial experiments to analyze interactions between variables

  • Use statistical approaches to identify significant factors and interactions

  • Control for random and systematic errors through appropriate replication and controls

• Advanced Genetic Approaches:

  • Create point mutants that separate different functions of xseB

  • Use domain swapping between xseB from different organisms

  • Apply conditional expression or degradation systems to study acute versus chronic effects

Example experimental design for resolving contradictory DNA binding results:

What strategies are recommended for developing antibodies against D. vulgaris xseB for research applications?

Development of specific antibodies against xseB involves several steps:

• Antigen Preparation:

  • Use purified recombinant xseB protein as the immunogen

  • Consider both the full-length protein and synthetic peptides corresponding to unique, exposed regions

  • Ensure protein purity >85% by SDS-PAGE analysis

• Immunization Protocol:

  • For polyclonal antibodies: immunize rabbits or mice with recombinant xseB with appropriate adjuvants

  • For monoclonal antibodies: immunize mice followed by hybridoma generation

  • Typical immunization schedule: initial immunization followed by 3-4 booster injections at 2-3 week intervals

• Antibody Validation:

  • Western blotting against recombinant protein and D. vulgaris cell lysates

  • Immunoprecipitation to verify native protein recognition

  • Immunofluorescence to confirm specificity in fixed cells

  • Testing against ΔxseB mutant strains as negative controls

• Antibody Characterization:

  • Determine titer by indirect ELISA (expect values around 1:3200 after complete immunization)

  • Assess sensitivity using serial dilutions of purified protein

  • Evaluate cross-reactivity with xseB from related species

  • Confirm functional applications (Western blot, IP, ChIP, immunofluorescence)

How can researchers optimize detection methods for studying xseB expression and localization in D. vulgaris?

Optimized detection methods include:

• Western Blotting:

  • Sample preparation: Use cell lysis buffers containing protease inhibitors

  • Gel percentage: 15-18% SDS-PAGE gels for optimal resolution of the small xseB protein

  • Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

  • Blocking: 5% non-fat milk or BSA in TBST

  • Antibody dilution: Optimize based on antibody titer (typically 1:1000 to 1:5000)

  • Detection: Use high-sensitivity ECL or fluorescent secondary antibodies

• Immunofluorescence Microscopy:

  • Cell fixation: 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100

  • Blocking: 3% BSA in PBS for 1 hour

  • Antibody dilution: Primary (1:100 to 1:500), secondary (1:500 to 1:2000)

  • Counterstaining: DAPI for nucleoid visualization

  • Controls: Include ΔxseB mutant and secondary-only controls

• Chromatin Immunoprecipitation (ChIP):

  • Crosslinking: 1% formaldehyde for 10-20 minutes

  • Sonication: Optimize to generate 200-500 bp DNA fragments

  • Immunoprecipitation: Use protein A/G beads with anti-xseB antibody

  • Controls: Include IgG control and input samples

  • Analysis: qPCR for specific loci or ChIP-seq for genome-wide binding

• Flow Cytometry:

  • Cell preparation: Fix with 2% paraformaldehyde, permeabilize with 0.1% Triton X-100

  • Staining: Use fluorophore-conjugated antibodies against xseB

  • Controls: Include unstained, secondary-only, and ΔxseB controls

  • Analysis: Quantify expression levels and population heterogeneity

How does D. vulgaris xseB compare structurally and functionally to homologs in other bacterial species?

Comparative analysis reveals important insights:

Comparative analysis approach:

• Multiple sequence alignment of xseB sequences

• Structural modeling based on available structures

• Phylogenetic analysis to trace evolutionary relationships

• Experimental comparison of recombinant proteins from different species

What insights can be gained from studying xseB in the context of sulfate-reducing bacteria metabolism and evolution?

Studying xseB in sulfate-reducing bacteria provides unique perspectives:

• Adaptations to Anaerobic Environment:

  • Potential modifications to DNA repair systems in anaerobic organisms

  • Possible interactions with sulfur metabolism pathways

  • Adaptations to deal with specific DNA damage types prevalent in anaerobic environments

• Evolutionary Considerations:

  • Conservation patterns specific to sulfate-reducing bacteria

  • Co-evolution with other DNA repair and recombination systems

  • Horizontal gene transfer events that may have influenced xseB evolution

• Metabolic Integration:

  • Potential connections between DNA repair and energy metabolism

  • Regulation of xseB in response to metabolic state

  • Links to stress responses specific to sulfate-reducing lifestyle

• Biofilm Formation:

  • Potential role in biofilm development, as DNA repair systems are often important in biofilm contexts

  • Links to extracellular DNA metabolism

  • Connections to surface adhesion mechanisms reported in D. vulgaris

Research approaches:

• Comparative genomics across diverse sulfate-reducing bacteria

• Transcriptomic analysis under various metabolic conditions

• Phenotypic analysis of ΔxseB mutants under conditions relevant to sulfate reducers

• Evolution experiments under selective pressures specific to anaerobic environments